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Sample records for antibody fab2 fragment

  1. Method for the preparation of bispecific F(ab')2mu fragments from mouse monoclonal antibodies of the immunoglobulin M class and characterization of the fragments.

    PubMed

    Morimoto, K; Inouye, K

    1999-04-22

    Bispecific F(ab')2mu fragments (Bs F(ab')2mu) binding simultaneously both sialyl Lewis A antigen (SLA) and human carcinoembryonic antigen (CEA) were prepared by disulfide bond exchange between F(ab')2mu fragments derived from IgM monoclonal antibodies (mAbs) against SLA and CEA, and were purified to homogeneity in a one-step procedure of hydrophobic interaction HPLC. The final yield of Bs F(ab')2mu from F(ab')2mu fragments was 70-78%, and the purity was higher than 98%. The immunoreactivities of the Bs F(ab')2mu fragments against SLA and CEA were almost the same as those of the respective parental F(ab')2mu fragments. The dissociation constant (0.17 microM) of the Bs F(ab')2mu for CEA was in good agreement with that of the parental F(ab')2mu fragments. Although the number of applications of IgM mAbs is restricted because of the large molecular mass and low solubility, Bs F(ab')2mu might, nevertheless, be a useful tool for immunotherapy and immunodiagnosis.

  2. Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

    PubMed Central

    Koetter, Ina; Schwab, Matthias; Fritz, Peter; Kimmel, Martin; Alscher, M. Dominik; Braun, Niko

    2013-01-01

    Background Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. Methods Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. Results Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. Conclusion This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug

  3. Metastatic colorectal cancer: Radioimmunoscintigraphy with a stabilized In-111-labeled F(ab')2 fragment of an anti-CEA monoclonal antibody

    SciTech Connect

    Lamki, L.M.; Patt, Y.Z.; Rosenblum, M.G.; Shanken, L.J.; Thompson, L.B.; Schweighardt, S.A.; Frincke, J.M.; Murray, J.L. )

    1990-01-01

    Metastatic colorectal cancer was detected with stabilized F(ab')2 fragments of ZCE-025, an anti-carcino-embryonic antigen (CEA) monoclonal antibody (MoAb). The fragments were prepared by cross-linking Fab' with a bifunctional cross-linking agent, bis-(maleimido)methyl ether. The authors labeled 2 mg of ZCE-025 with 5 mCi (185 MBq) of indium-111 and injected the material intravenously, either alone or with unlabeled F(ab')2, into 16 patients. Lesion detection, pharmacokinetics, and relative body distribution were evaluated and compared with those of the intact immunoglobulin (IgG1) antibody. Stabilized F(ab')2 fragments were more useful than the intact antibody in detection of lesions: Overall sensitivity of F(ab')2 fragments for all the patients was 79.4%, whereas overall sensitivity of the intact IgG1 antibody was 32%. This anti-CEA-stabilized F(ab')2 fragment may be a powerful diagnostic tool that can achieve higher sensitivity at smaller protein doses than the intact IgG1 antibody.

  4. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

    PubMed Central

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-01-01

    alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model. PMID:2476813

  5. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

    PubMed

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-09-01

    alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model.

  6. Comparative tissue distribution in mice of the alpha-emitter 211At and 131I as labels of a monoclonal antibody and F(ab')2 fragment.

    PubMed

    Garg, P K; Harrison, C L; Zalutsky, M R

    1990-06-15

    Because it decays by the emission of short-range, high-energy alpha-particles, the radiohalogen 211At might be a particularly useful nuclide for some types of radioimmunotherapy. However, no suitable gamma-emitting nuclide of astatine exists which would permit either imaging prior to therapy to obtain radiation dosimetry estimates or performing experiments in paired-label format. Since iodine is the halogen above astatine in the periodic table, we investigated whether the in vivo distribution of 131I could be used to mimic the biodistribution of 211At. In this study, the N-succinimidyl 3-(trialkylstannyl)benzoate method was used to label C110 IgG, an antibody directed against carcinoembryonic antigen, and its (Fab')2 fragment with 211At and 131I. Paired-label experiments were performed in normal mice comparing the tissue distribution of 211At- versus 131I-labeled C110 IgG and F(ab')2 as well as [211At]astatide versus [131I]iodide and m-[211At]astatobenzoic acid versus m-[131I]iodobenzoic acid, potential catabolites of proteins radiohalogenated via the N-succinimidyl 3-(trialkylstannyl)benzoate method. With the exception of thyroid, retention of astatide in tissues was higher than that of iodide; and, with the halobenzoic acids, uptake of 211At was higher than 135I in thyroid, stomach, and spleen. Use of the N-succinimidyl 3-(trialkylstannyl)benzoate method to label C110 IgG with 211At and 131I resulted in similar distributions of the two nuclides. In contrast, loss of 211At from the F(ab')2 fragment was considerably more rapid than 131I, suggesting that different astatination methods may be required for use with F(ab')2 fragments. PMID:2340501

  7. Preparation of F(ab')2 fragments of immunoglobulin G.

    PubMed

    Killion, J J; Holtgrewe, E M

    1983-11-01

    We describe a simple protocol for the preparation of F(ab')2 fragments of immunoglobulin G, based upon the known Fc- binding properties of protein A-Sepharose. The fragment preparations of xenogeneic and allogeneic anti-IgG were noncytotoxic to intact target cells, and were able to block the cytotoxicity of intact antibody. This method should therefore be useful for functional studies not requiring biochemical homogeneity.

  8. Imaging of human leukemic T-cell xenografts in nude mice by radiolabeled monoclonal antibodies and F(ab')2 fragments

    SciTech Connect

    Vacca, A.; Buchegger, F.; Carrel, S.; Mach, J.P.

    1988-01-01

    Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (/sup 131/I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.

  9. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    SciTech Connect

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.; Kudryk, B.J.; Subramanian, G.; Ritter-Hrncirik, C.A.; Witanowski, L.S.; Tillapaugh-Fay, G.; Urrutia, E.; Zapf-Longo, C.

    1988-07-01

    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with /sup 131/I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with /sup 131/I-labeled F(ab')2 and Fab and /sup 111/In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with /sup 111/In and its better physical characteristics for imaging, compared to /sup 131/I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging.

  10. Immunoscintigraphy of adenocarcinomas by means of radiolabeled F(ab')2 fragments of an anti-carcinoembryonic antigen monoclonal antibody: A multicenter study

    SciTech Connect

    Siccardi, A.G.; Buraggi, G.L.; Callegaro, L.; Colella, A.C.; De Filippi, P.G.; Galli, G.; Mariani, G.; Masi, R.; Palumbo, R.; Riva, P. )

    1989-06-01

    F(ab')2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody F023C5, determined to be more suitable than intact IgG and Fab fragments for immunoscintigraphy, were labeled with 131I or conjugated to DTPA for instant 111In-labeling, and administered i.v. (2-3 mCi/0.5 mg) to 509 patients in 11 nuclear medicine departments: 284 patients had gastrointestinal adenocarcinomas, 204 had nongastrointestinal adenocarcinomas and 21 were control; serum CEA was elevated in 169 patients, normal in 115, and not determined in 225. The following results were obtained: (a) no adverse reactions; (b) tumor imaging in 324 patients (in particular, in 81.5% CEA-seropositive and in 69.0% CEA-seronegative patients); (c) no significant difference in sensitivity among the results of the 11 departments; (d) no significant difference in overall sensitivity between 131I-and 111In-labeled immunoradiopharmaceuticals; (e) the fraction of documented lesions imaged was 73.3% in CEA-seropositive and 53.7% in CEA-seronegative patients; (f) the detection of liver metastases was hampered, particularly when using the 111In-labeled reagent, by nonspecific radioactivity uptake; (g) the major cause of negative immunoscintigraphy results was a lack of CEA in tumor lesions, as documented by immunohistochemistry; (h) lesion size is also important since the sensitivity was 64% for lesions up to 2 cm in diameter and 84% for larger lesions; (i) many unexpected radiolocalizations were recorded. Most were identified as occult tumor lesions. In 35 patients, this finding contributed to the early detection of tumor recurrences.

  11. Radioimmunodetection of lymph node invasion in prostatic cancer. The use of iodine 123 (123I)-labeled monoclonal anti-prostatic acid phosphatase (PAP) 227 A F(ab')2 antibody fragments in vivo.

    PubMed

    Leroy, M; Teillac, P; Rain, J D; Saccavini, J C; Le Duc, A; Najean, Y

    1989-07-01

    The therapeutic indications in prostatic cancer depend on the regional and distant extension of the cancer and are difficult to assess before lymphadenectomy. Radioimmunodetection of lymph node involvement with monoclonal anti-prostatic acid phosphatase (PAP) antibodies can be proposed as a noninvasive alternative to lymphadenectomy. Fifteen patients with various stages of histologically proven prostatic cancer were examined by immunolymphoscintigraphy (ILS) before treatment to detect lymph node metastases. These patients had Stage A (n = 7), Stage B (n = 3), Stage C (n = 2), and Stage D (n = 3) tumors. They received between 100 and 400 micrograms of monoclonal antibody 227 A in the form of F(ab')2 fragments labeled with iodine 123 (123I). The antibody was injected directly into the periprostatic area. ILS images were obtained after 1, 3, 6, and 24 hours. Three days later, each patient underwent a lymphadenectomy for histologic examination. The results of the histologic examination and ILS were compared. In ten patients, the examination did not show any images capable of being interpreted as lymphadenopathy and histologic examination confirmed the integrity of the nodes examined. In five cases, scintigraphy suggested the presence of lymph node invasion by prostatic cancer and this was confirmed by histologic examination in three of the five cases. Overall, in terms of lymphadenopathy, this examination had a sensitivity of 100% and a specificity of 83%. Therefore, ILS appears to be capable of detecting lymph node metastases in prostatic cancer.

  12. Immunoglobulin fragments, F(ab')2, that are cytotoxic to enzyme-treated cells.

    PubMed

    Holtgrewe, E M; Killion, J J

    1984-07-01

    Bivalent immunoglobulin fragments of IgG, F(ab')2, prepared from normal murine sera were found to be cytotoxic to neuraminidase-treated cells. The fragments were cytotoxic to both allogenic and syngeneic targets (with respect to the source of the sera), suggesting that the antigen bound by the F(ab')2 is not related to the major histocompatibility locus of mice (H-2).

  13. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    PubMed

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  14. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    PubMed

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  15. Cooperative mixtures of bispecific F(ab')2 antibodies for delivering saporin to lymphoma in vitro and in vivo

    SciTech Connect

    French, R.R.; Courtenay, A.E.; Ingamells, S.; Stevenson, G.T.; Glennie, M.J. )

    1991-05-01

    We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of (3H)leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins could provide an important new strategy in immunotherapy.

  16. Effect of postponed treatment with an anti-tumour necrosis factor (TNF) F(ab')2 fragment on endotoxin-induced cytokine and neutrophil responses in chimpanzees.

    PubMed

    van der Poll, T; Levi, M; ten Cate, H; Jansen, J; Biemond, B J; Haagmans, B L; Eerenberg, A; van Deventer, S J; Hack, C E; ten Cate, J W

    1995-04-01

    TNF is considered to be an intermediate factor in endotoxin-induced release of other cytokines and endotoxin-induced neutrophil degranulation. Little is known about the effect of postponed treatment with anti-TNF in primate endotoxin models. To assess the effect of delayed treatment with anti-TNF in endotoxaemia, six healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In three of these animals the administration of endotoxin was followed after 30 min by a bolus i.v. injection of the anti-TNF F(ab')2 fragment MAK 195F (0.1 mg/kg). Post-treatment with MAK 195F completely prevented the appearance of TNF activity in serum elicited by endotoxin, and markedly reduced the rises in the serum concentrations of IL-6 and IL-8. In addition, the endotoxin-induced increases in the type I and type II soluble TNF receptors were also profoundly inhibited by MAK 195F, suggesting that TNF is involved in the release of its own soluble receptors in endotoxaemia. Neutrophilic leucocytosis was not affected by MAK 195F. In contrast, MAK 195F did significantly abrogate neutrophil degranulation, as measured by the plasma concentrations of lactoferrin. These results indicate that treatment with anti-TNF 30 min after the administration of endotoxin is still effective in attenuating the induction of the cytokine network and of neutrophil degranulation.

  17. Biodistribution and tumor localization of 111In-labeled unmodified and modified F(ab')2 fragments of human monoclonal IgM (16.88) in a nude mouse model.

    PubMed

    Quadri, S M; Siddiqui, A; Klein, J L; Vriesendorp, H M

    1995-05-01

    Unmodified F(ab')2 and modified Fab'-BMH-Fab' fragments of human monoclonal IgM (16.88) were compared for biodistribution and tumor localization in nude mice bearing LS-174T human colon carcinoma xenografts. Although both unmodified and modified fragments of IgM cleared rapidly from the blood, the radioactivity retentions for each fragment in liver and kidney were significantly different. Kidney uptake of the modified fragment was about 4-fold lower than kidney uptake of the unmodified fragment. Radioactivity uptake in liver was 2-4-fold higher for the modified fragment. Lower liver and higher kidney uptake of unmodified fragments reflected the labile disulfide linkage of F(ab')2 in their hinge region and the subsequent behavior of the Fab' fragments resulting from the reduction of the disulfide linkage. Higher liver and lower kidney retention of modified fragments, on the other hand, resulted from the different cleavage mechanism of the stable thioether linkage. Tumor targeting was similar for unmodified and modified fragments at approx. 4% of injected dose per gram. These results indicate that the changes in fragment linkage chemistry may provide different pharmacokinetic patterns in vivo and improve the therapeutic application of radiolabeled fragments in human patients.

  18. PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract.

    PubMed

    Koussoroplis, Salome Juliette; Paulissen, Geneviève; Tyteca, Donatienne; Goldansaz, Hadi; Todoroff, Julie; Barilly, Céline; Uyttenhove, Catherine; Van Snick, Jacques; Cataldo, Didier; Vanbever, Rita

    2014-08-10

    Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4h post-delivery, whereas the clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' contents in the lungs had decreased to 10 and 14% of the dose initially deposited, respectively. However, this value was 40% for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways. PMID:24845126

  19. Preclinical evaluation of 67Cu-labeled intact and fragmented anti-colon carcinoma monoclonal antibody MAb35.

    PubMed

    Smith, A; Alberto, R; Blaeuenstein, P; Novak-Hofer, I; Maecke, H R; Schubiger, P A

    1993-12-01

    The anti-carcinoembryonic antigen murine monoclonal antibody MAb35 and its F(ab')2 fragment were labeled with 131I or the potential therapeutic nuclide 67Cu. In vivo distribution patterns were compared in nude mice bearing human tumor xenografts by coinjection of the 131I- and 67Cu-labeled materials, thereby minimizing variations due to xenograft and host animal. The results showed that the 67Cu-labeled intact MAb35 achieved twice the percentage of injected dose/g tumor when compared to its 131I-labeled counterpart, without significant impairment of the wholebody distribution pattern. However, this effect was not evident in the case of F(ab')2, where high uptake of 67Cu was found in the kidney without any enhancement of accumulation in the target xenografts. To investigate the underlying causes of the different distribution patterns observed, iodine labeling was also performed using a more stable linkage, and the results indicated that the observed differences cannot be explained by simple deiodination of conventionally labeled preparations. We conclude that the intact form of the 67Cu-labeled antibody may be superior to the F(ab')2 fragment for use in our intended clinical studies. Our continuing work on the processing of radiometal-labeled F(ab')2 fragments, at the systemic and cellular level, will hopefully lead to a strategy to circumvent the problem of high kidney accumulation.

  20. Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

    PubMed

    Duska, L R; Hamblin, M R; Bamberg, M P; Hasan, T

    1997-01-01

    The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer. PMID:9062404

  1. Peptide linkers lead to modification of liver metabolism and improved tumor targeting of copper-67-labeled antibody fragments.

    PubMed

    Novak-Hofer, I; Zimmermann, K; Schubiger, P A

    2001-12-01

    In order to determine if tumor/nontarget tissue ratios of 67Cu-labeled antibody fragments can be improved, modifying the DO3A copper chelate with tripeptide linkers was investigated. The peptide-linked chelates 1,4,7,10-tetraazacyclodecane-N,N',N",N"'-tetraacetate (DOTA)-triglycyl-L-p-isothiocyanato-phenylalanine (DOTA-R1-NCS), DOTA-glycyl-phenylalanyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R2-NCS), DOTA-glycyl-prolyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R3-NCS) and DOTA-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R4-NCS) were synthesized and coupled to F(ab')2 fragments of anti-colon carcinoma mAb35. In vitro, the 67Cu-labeled antibody fragments were fully immunoreactive and stable in human serum. In vivo in nude mice bearing human colon carcinoma xenografts the conjugates R1 and R3 showed improved tumor uptake and lower levels of radioactivity in the liver compared with the other conjugates. Biodistributions of the DOTA-R2-F(ab')2 showed at early time points after injection higher levels of radioactivity in the liver, lower levels of activity persisting in the blood and lower accumulation of activity in the tumor. When liver homogenates were analyzed 30 min post injection by SDS-PAGE or FPLC gel chromatography, it was found that radioactivity was released more slowly from the triglycine (R1)-F(ab')2 than from the immunoconjugates with the R2 or the R4 linker. The main radioactive metabolites were protein bands at 66 kD, 31 kD and low molecular weight fragments. The results show that the rate of cleavage of the copper complex from F(ab')2 fragments in vivo can be influenced by the amino acid sequence close to the complex, with significant consequences on biodistributions.

  2. Stabilization of antibody fragments in adverse environments.

    PubMed

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  3. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  4. Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma.

    PubMed

    Carrel, F; Amstutz, H; Novak-Hofer, I; Schubiger, P A

    1997-08-01

    Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (Kd approximately 10(-9) M) to target cells in vitro but formed aggregates at 37 degrees C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent "refolding" could be achieved. In contrast, chCE7- Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (Kd approximately 10(-10) M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab')2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab')2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the

  5. Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins.

    PubMed

    Derocq, J M; Casellas, P; Laurent, G; Ravel, S; Vidal, H; Jansen, F

    1988-10-15

    The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.

  6. Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens

    SciTech Connect

    Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.

    1987-08-01

    Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with /sup 125/I. The /sup 125/I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography.

  7. The role of the antibody Fc region in rapid expulsion of Trichinella spiralis in suckling rats.

    PubMed Central

    Carlisle, M S; McGregor, D D; Appleton, J A

    1991-01-01

    When an IgG2c monoclonal antibody specific for Trichinella spiralis muscle stage larvae was cleaved with pepsin to yield F(ab')2 fragments, the latter retained their capacity to cause mucus entrapment and rapid expulsion of larvae from the intestines of suckling rats. When fed to pups, the F(ab')2 fragments of this antibody and the F(ab')2 fragments of a similarly prepared IgG2a antibody caused mucus entrapment of muscle larvae (ML), demonstrating that trapping is not dependent upon the Fc region of the antibody molecule. Despite the fact that these two antibodies had similar specificities and that their F(ab')2 fragments caused larval entrapment in mucus, F(ab')2 fragments of the IgG2a antibody failed to protect rat pups. Fragments of the IgG2c antibody caused rapid expulsion when injected into pups, but the distribution of larvae was dramatically different from when the fragments were delivered orally. These results indicate that entrapment of T. spiralis in mucus is not in itself the cause of the expulsion. The more likely possibility is that antibody impedes a function of Trichinella spiralis that is related to the capacity of the parasite to reside in its epithelial niche. PMID:1769702

  8. The role of the antibody Fc region in rapid expulsion of Trichinella spiralis in suckling rats.

    PubMed

    Carlisle, M S; McGregor, D D; Appleton, J A

    1991-11-01

    When an IgG2c monoclonal antibody specific for Trichinella spiralis muscle stage larvae was cleaved with pepsin to yield F(ab')2 fragments, the latter retained their capacity to cause mucus entrapment and rapid expulsion of larvae from the intestines of suckling rats. When fed to pups, the F(ab')2 fragments of this antibody and the F(ab')2 fragments of a similarly prepared IgG2a antibody caused mucus entrapment of muscle larvae (ML), demonstrating that trapping is not dependent upon the Fc region of the antibody molecule. Despite the fact that these two antibodies had similar specificities and that their F(ab')2 fragments caused larval entrapment in mucus, F(ab')2 fragments of the IgG2a antibody failed to protect rat pups. Fragments of the IgG2c antibody caused rapid expulsion when injected into pups, but the distribution of larvae was dramatically different from when the fragments were delivered orally. These results indicate that entrapment of T. spiralis in mucus is not in itself the cause of the expulsion. The more likely possibility is that antibody impedes a function of Trichinella spiralis that is related to the capacity of the parasite to reside in its epithelial niche.

  9. Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

    PubMed

    Koussoroplis, Salome-Juliette; Heywood, Sam; Uyttenhove, Catherine; Barilly, Céline; Van Snick, Jacques; Vanbever, Rita

    2013-09-15

    The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies. PMID:23850622

  10. A Simple Model System to Demonstrate Antibody Structure and Functions.

    ERIC Educational Resources Information Center

    O'Kennedy, Richard

    1991-01-01

    A model that can be used to show the arrangement of light and heavy chains, disulfide linkages, domains, and subclass variations in antibodies is given. It can be constructed and modified to illustrate Fab, F(ab')2, and Fc fragments, single domain and bifunctional antibodies, and labeling of antibodies. (Author)

  11. Immunoscintigraphy of colorectal carcinoma with F (ab')2 fragments of anti-CEA monoclonal antibody

    SciTech Connect

    Buraggi, G.; Callegaro, L.; Turrin, A.; Gennari, L.; Bombardieri, E.; Mariani, G.; Deleide, G.; Dovis, M.; Gasparini, M.; Doci, R.

    1987-01-01

    A monoclonal antibody to carcinoembryonic antigen (CEA) (F023C5), belonging to IgG1 class, was obtained by cell fusion technique. Preliminary screening on different tissues was performed with immunoperoxidase staining, which showed good specificity of the antibody for gastric and colorectal carcinomas. F(ab')2 fragments were subsequently prepared and labeled with /sup 131/I and /sup 111/In. After immunoreactivity check the radiopharmaceuticals were injected intravenously. Sixteen patients with 22 primary or secondary localizations of colorectal carcinoma were studied following the recommendations of the ethical Committee of the Istituto Nazionale Tumori, Milan, Italy. Serial scans were performed after injection of the two radioactive reagents. In vivo pharmacokinetics of the compound was studied. Radioactivity level in surgical specimens was measured, and immunostaining was performed. All tumors were found to express the antigen. Eleven out of 12 tumor localizations of the gastrointestinal tract and three out of ten liver metastases were imaged. Specificity of tumor uptake was assessed by simultaneous injection of an irrelevant antibody.

  12. Alternative downstream processes for production of antibodies and antibody fragments.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke

    2014-11-01

    Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. PMID:24859179

  13. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

    PubMed

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  14. Cloning, bacterial expression and crystallization of Fv antibody fragments

    NASA Astrophysics Data System (ADS)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  15. Considerations in producing preferentially reduced half-antibody fragments.

    PubMed

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity. PMID:26779832

  16. Development trends for generation of single-chain antibody fragments.

    PubMed

    Farajnia, Safar; Ahmadzadeh, Vahideh; Tanomand, Asghar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2014-10-01

    Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.

  17. Engineered Antibody Fragments for Immunodiagnosis of Papaya ringspot virus.

    PubMed

    Maheshwari, Yogita; Verma, H N; Jain, R K; Mandal, Bikash

    2015-07-01

    The present study was undertaken to clone and express the genes encoding antibody to the recombinant coat protein (rCP) of Papaya ringspot virus (PRSV) and to assess the engineered antibody for the detection of PRSV. A 33-kDa rCP of PRSV, which was produced in Escherichia coli, generated PRSV specific antibody in immunized mouse. The heavy and light chain variable domain genes (VH and VL) of 351 and 360 nucleotides, respectively, were cloned from the mRNA isolated from the spleen of the immunized mouse with rCP of PRSV. The VH and VL belong to the family IgG1 and kappa chain, respectively, and contained the framework regions and complementarity determining regions. The VH and VL genes were individually used to develop the expression constructs in pET28a (+) vector and 14-kDa proteins were obtained in E. coli. The amount of purified VH and VL proteins was 3-4 mg/l of bacterial culture. Both the antibody fragments recognized PRSV in the crude sap; however, the VL antibody fragment showed higher affinity to PRSV. The mixture of VH and VL detected PRSV as effectively as polyclonal antibody. The recombinant antibody fragments mixture detected PRSV in the field samples with 100 % accuracy in dot immunobinding assay (DIBA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of the detection of PRSV using antibody fragments was 1.0 and 10.0 ng in DIBA and ELISA, respectively. The results showed successful isolation of functional single-domain antibody encoding genes to PRSV directly from the immunized spleen cells of mouse. This study for the first time demonstrates application of bacterial expressed recombinant antibody fragments in immunodiagnosis of PRSV.

  18. Generation of recombinant antibody fragments for membrane protein crystallization.

    PubMed

    Mir, Syed H; Escher, Claudia; Kao, Wei-Chun; Birth, Dominic; Wirth, Christophe; Hunte, Carola

    2015-01-01

    Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.

  19. The Biochemical Properties of Antibodies and Their Fragments.

    PubMed

    Hnasko, Robert M

    2015-01-01

    Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

  20. Biotechnological applications of recombinant single-domain antibody fragments

    PubMed Central

    2011-01-01

    Background Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications. Results The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic. Conclusions Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments. PMID:21658216

  1. Demonstration of anti-idiotypic antibodies directed against IgM rheumatoid factor in the serum of rheumatoid arthritis patients.

    PubMed Central

    Hancock, W K; Barnett, E V

    1989-01-01

    We have identified the presence of anti-idiotypic activity against IgMRF in the sera of RA patients. Only patients seropositive for IgMRF had significant levels of anti-idiotypic activity, while seronegative patients and normal volunteers did not. When this anti-idiotypic activity was affinity-purified from a single RA patient, two separate binding activities were identified. IgG antibodies were pepsin-digested to F(ab')2 fragments before affinity-purification to remove the Fc portion capable of binding to IgMRF. Anti-idiotypic F(ab')2 fragments of IgG were eluted from an IgMRF-Sepharose 4B column. These F(ab')2 bound preferentially to IgMRF bearing an idiotype recognized by the anti-idiotypic murine monoclonal 17.109. A second anti-idiotypic F(ab')2 was affinity purified using rabbit anti-human Fc antibody bound to Sepharose 4B. These eluted antibodies behaved as the internal image of IgG, binding five out of seven IgMRF's tested. The binding of both anti-idiotypic F(ab')2 was inhibited with human IgG. The presence of both IgMRF and anti-idiotypic antibodies directed against it in the sera of RA patients suggests that anti-idiotypic antibodies alone are not capable of inhibiting the production of rheumatoid factor. PMID:2702773

  2. In vivo Imaging with Antibodies and Engineered Fragments

    PubMed Central

    Freise, Amanda C.; Wu, Anna M.

    2015-01-01

    Antibodies have clearly demonstrated their utility as therapeutics, providing highly selective and effective drugs to treat diseases in oncology, hematology, cardiology, immunology and autoimmunity, and infectious diseases. More recently, a pressing need for equally specific and targeted imaging agents for assessing disease in vivo, in preclinical models and patients, has emerged. This review summarizes strategies for developing and optimizing antibodies as targeted probes for use in non-invasive imaging using radioactive, optical, magnetic resonance, and ultrasound approaches. Recent advances in engineered antibody fragments and scaffolds, conjugation and labeling methods, and multimodality probes are highlighted. Importantly, antibody-based imaging probes are seeing new applications in detection and quantitation of cell surface biomarkers, imaging specific responses to targeted therapies, and monitoring immune responses in oncology and other diseases. Antibody-based imaging will provide essential tools to facilitate the transition to truly precision medicine. PMID:25934435

  3. Spectral characteristics of fluorescence and circular dichroism of aflatoxin B1 reaction with its anti-idiotypic antibody

    NASA Astrophysics Data System (ADS)

    Liu, Aiping; Yang, Hongxiu; Wang, Xiaohong; Chen, Fusheng

    2012-11-01

    Aflatoxin B1 (AFB1) is a toxic secondary metabolite and sensitive methods for its analysis have been developed. In our lab, a number of works have been carried out, including exploitation of detection methods and production of anti-idiotypic antibody (Ab2) against Fab fragment of anti-AFB1 antibody (Ab1). In this paper, Ab2 was generated upon the immunization of mice with F(ab')2 fragment, which was specific to AFB1 and obtained by pepsin digestion of Ab1. The characteristics of Ab2 was primarily investigated by indirect competitive enzyme-linked immunosorbent assay (icELISA), which indicated that Ab2, might bear an internal image of antigen AFB1 and was able to combine to F(ab')2 in competition with AFB1, and the concentration of Ab2 to cause 50% inhibition of binding (IC50) was 131.8 μg/mL. In addition, fluorescence and circular dichroism studies were designed to explore the mutual relationship among AFB1, F(ab')2 and Ab2. The fluorescence spectroscopy implied that both AFB1 and Ab2 act as a quencher upon F(ab')2, and the Ab2 could compete with AFB1 when both of Ab2 and AFB1 reacted with F(ab')2. The circular dichroism (CD) spectrum suggested that both the binding of Ab2 and AFB1 on F(ab')2 brought secondary conformation change of F(ab')2, especially in the changes of α helix and β sheet. The research performed would provide unique insight into the comprehension of interaction among AFB1, F(ab')2 and Ab2 as well as offer structural information for substitution researches of toxic antigen like AFB1.

  4. Fast antibody fragment motion: flexible linkers act as entropic spring.

    PubMed

    Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.

  5. Fast antibody fragment motion: flexible linkers act as entropic spring

    DOE PAGES

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unboundmore » state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. In conclusion, the Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.« less

  6. Fast antibody fragment motion: flexible linkers act as entropic spring.

    PubMed

    Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

  7. Fast antibody fragment motion: flexible linkers act as entropic spring

    PubMed Central

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

  8. Influence of molecular size on tissue distribution of antibody fragments

    PubMed Central

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C.; Lavé, Thierry; Shah, Dhaval K.

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. PMID:26496429

  9. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.

    PubMed

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia; Murray, Christopher J

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

  10. Fluorescent labeling of antibody fragments using split GFP.

    PubMed

    Ferrara, Fortunato; Listwan, Pawel; Waldo, Geoffrey S; Bradbury, Andrew R M

    2011-01-01

    Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems. PMID:21998685

  11. Polyreactivity of natural antibodies: exchange by HL-fragments.

    PubMed

    Sedykh, M A; Buneva, V N; Nevinsky, G A

    2013-12-01

    The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.

  12. Comparative study on the ability of IgG and F(ab')2 antivenoms to neutralize lethal and myotoxic effects induced by Micrurus nigrocinctus (coral snake) venom.

    PubMed

    León, G; Stiles, B; Alape, A; Rojas, G; Gutiérrez, J M

    1999-08-01

    A comparative study was performed on the ability of IgG and F(ab')2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(ab')2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(ab')2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus alpha-neurotoxins to acetylcholine receptor in vitro. The IgG and F(ab')2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(ab')2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(ab')2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.

  13. Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum.

    PubMed

    Liesi, P; Hager, G; Dodt, H U; Seppälä, I; Zieglgänsberger, W

    1995-02-01

    Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. PMID:7745613

  14. Chemical macrocyclization of peptides fused to antibody Fc fragments.

    PubMed

    Angelini, Alessandro; Diderich, Philippe; Morales-Sanfrutos, Julia; Thurnheer, Sarah; Hacker, David; Menin, Laure; Heinis, Christian

    2012-09-19

    To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.

  15. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

    SciTech Connect

    Cheng, F.M.; Hansen, E.B.; Taylor, C.R.; Epstein, A.L. )

    1991-02-06

    Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed (1) a linear uptake that increased over time without saturation in tumor spheroids and (2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

  16. Anticomplementary Activity of Horse IgG and F(ab')2 Antivenoms

    PubMed Central

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.

    2014-01-01

    Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs. PMID:24445201

  17. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Brient, Bruce W.; Nisonoff, Alfred

    1970-01-01

    Rabbit anti-idiotypic antibodies were prepared by injection of specifically purified anti-p-azobenzoate antibodies (D) from individual donor rabbits. Benzoate derivatives were found to be strong inhibitors of the reactions of D with anti-D antisera. There was a close correlation between the combining affinities of the benzoate derivatives used and their effectiveness as inhibitors. Compounds tested that are chemically unrelated to benzoate were ineffective. The results indicate either that the combining site of anti-benzoate antibody is part of an important idiotypic determinant, which is sterically blocked by hapten, or that the hapten induces a conformational change which alters idiotypic determinants not involving the active site. Such conformational changes, if they occur, must be restricted since hapten has little effect on the reactions of F(ab')2 fragments of anti-benzoate antibodies with antisera directed to rabbit fragment Fab and no detectable effect on reactions with antibodies directed to allotypic determinants. PMID:4097134

  18. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    SciTech Connect

    Quinn, T.P.

    2003-12-31

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

  19. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  20. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    PubMed

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  1. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  2. Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide.

    PubMed

    Griest, R E; Jeffrey, P D; Taylor, G L; Rees, A R

    1992-01-01

    The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen. PMID:1731084

  3. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  4. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

    PubMed Central

    Kirsch, Martina Inga; Hülseweh, Birgit; Nacke, Christoph; Rülker, Torsten; Schirrmann, Thomas; Marschall, Hans-Jürgen; Hust, Michael; Dübel, Stefan

    2008-01-01

    Background Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak. PMID:18764933

  5. Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

    SciTech Connect

    Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P. )

    1989-09-01

    Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb.

  6. Labeling of monoclonal antibodies with rhenium-186 using the MAG3 chelate for radioimmunotherapy of cancer: a technical protocol.

    PubMed

    Visser, G W; Gerretsen, M; Herscheid, J D; Snow, G B; van Dongen, G

    1993-11-01

    A detailed technical protocol is provided for reproducible and aseptical production of stable 186Re-monoclonal antibody conjugates. Labeled Mab E48 IgG and its F(ab')2 fragment which are promising candidates for radioimmunotherapy of squamous cell carcinoma of the head and neck were used for evaluation. S-benzoylmercaptoacetyltriglycine (S-benzoyl-MAG3) was used as a precursor. Rhenium-186-MAG3 was prepared via a unique solid-phase synthesis, after which known strategies for esterification and conjugation to Mab IgG/F(ab')2 were applied. The biodistribution of 186Re-E48 F(ab')2 in tumor-bearing nude mice was found to be comparable to that of analogously labeled 99mTc-E48 F(ab')2 or 131I-E48 F(ab')2, indicating that the intrinsic behavior of the antibody remains preserved when using this labeling technique. Radiolytic decomposition of 186Re-E48 IgG/F(ab')2 solutions of 10 mCi.ml-1 was effectively reduced by the antioxidant ascorbic acid. Upon increase of the Re-MAG3 molar amount, a conjugation of seven to eight Re-MAG3 molecules per Mab molecule was generally the maximum ratio that could chemically be obtained. Such a ratio did not impair the immunoreactivity or alter the in vivo biodistribution characteristics of the immunoconjugate, making this labeling procedure suitable for general clinical application. PMID:8229241

  7. The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

    PubMed Central

    Margni, R A; Leoni, J; Bazzurro, M

    1977-01-01

    The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions. Images Figure 1 PMID:415968

  8. Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

    PubMed

    Medecigo, M; Manoutcharian, K; Vasilevko, V; Govezensky, T; Munguia, M E; Becerril, B; Luz-Madrigal, A; Vaca, L; Cribbs, D H; Gevorkian, G

    2010-06-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

  9. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    PubMed Central

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  10. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  11. Assessment of the effect of triton X-114 on the physicochemical properties of an antibody fragment.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2014-01-01

    The effect of Triton X-114 on the physicochemical properties of a single-chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid-liquid extraction procedures.

  12. Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

    SciTech Connect

    Mohd, S.B.

    1987-01-01

    In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab')/sub 2/ and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab')/sub 2/ and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of /sup 75/Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab')/sub 2/ and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma.

  13. Stirred batch crystallization of a therapeutic antibody fragment.

    PubMed

    Hebel, Dirk; Huber, Sabine; Stanislawski, Bernd; Hekmat, Dariusch

    2013-07-20

    Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

  14. Myocardial Infarct Imaging of Antibodies to Canine Cardiac Myosin with Indium-111-Diethylenetriamine Pentaacetic Acid

    NASA Astrophysics Data System (ADS)

    Khaw, Ban An; Fallon, John T.; Strauss, H. William; Haber, Edgar

    1980-07-01

    Antibodies, by virtue of marked selectivity and affinity, may lend themselves to identification of structures of unique antigenic specificity in vivo. In experimental myocardial infarction in dogs, F(ab')2 fragments of antibodies to cardiac myosin that had been labeled with iodine-131 were shown to localize within the lesion. Because the energy characteristics of iodine isotopes are not ideal for imaging with a gamma camera, a new method for labeling antibody fragments with divalent or polyvalent radionuclides was developed. A bifunctional chelating agent, diethylenetriamine pentaacetic acid was covalently coupled, by an amide bond, to Fab fragments of antibodies to canine cardiac myosin. A stable chelate was then formed with indium-111, a nuclide that has appropriate half-life and energy characteristics for gamma imaging. Antibodies treated in this way retain their antigen-binding activity and are useful in locating myocardial infarcts in vivo.

  15. Myocardial infarct imaging of antibodies to canine cardiac myosin with indium-111-diethylenetriamine pentaacetic acid.

    PubMed

    Khaw, B A; Fallon, F T; Strauss, H W; Haber, E

    1980-07-11

    Antibodies, by virtue of marked selectivity and affinity, may lend themselves to identification of structures of unique antigenic specificity in vivo. In experimental myocardial infarction in dogs, F(ab')2 fragments of antibodies to cardiac myosin that had been labeled with iodine-131 were shown to localize within the lesion. Because the energy characteristics of iodine isotopes are not ideal for imaging with a gamma camera, a new method for labeling antibody fragments with divalent or polyvalent radionuclides was developed. A bifunctional chelating agent, diethylenetriamine pentaacetic acid was covalently coupled, by an amide bond, to Fab fragments of antibodies to canine cardiac myosin. A stable chelate was then formed with indium-111, a nuclide that has appropriate half-life and energy characteristics for gamma imaging. Antibodies treated in this way retain their antigen-binding activity and are useful in locating myocardial infarcts in vivo. PMID:7384803

  16. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    PubMed Central

    Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

    2014-01-01

    Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

  17. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    PubMed

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  18. Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids

    PubMed Central

    Thurber, Greg M.; Wittrup, K. Dane

    2010-01-01

    Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue. PMID:18451160

  19. Association of selenocysteine transfer RNA fragments with serum antibody response to Mycoplasma spp. in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to identify transfer RNA fragments (tRFs) associated with a serum antibody response to Mycoplasma spp. in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected...

  20. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  1. The role of monogamous bivalency and Fc interactions in the binding of anti-DNA antibodies to DNA antigen.

    PubMed

    Stearns, Nancy A; Pisetsky, David S

    2016-05-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. These antibodies can bind DNA avidly by monogamous bivalency, a mechanism which requires the interaction of both Fab combining regions with antigenic determinants on the same polynucleotide. To explore further this mechanism, we tested Fab and F(ab')2 fragments prepared from IgG from patient plasmas in an ELISA with native DNA antigen, detecting antibody with a peroxidase conjugated anti-Fab reagent. These studies showed that Fab fragments, which can only bind monovalently, had negligible activity. Although bivalent F(ab')2 fragments would be predicted to bind DNA, these fragments also showed poor anti-DNA activity. Control studies showed that the fragments retained antibody activity to tetanus toxoid and an EBV antigen preparation. Together, these findings suggest that anti-DNA avidity depends on monogamous bivalency, with the antibody Fc portion also influencing DNA binding, in a mechanism which can be termed Fc-dependent monogamous bivalency.

  2. The role of monogamous bivalency and Fc interactions in the binding of anti-DNA antibodies to DNA antigen.

    PubMed

    Stearns, Nancy A; Pisetsky, David S

    2016-05-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. These antibodies can bind DNA avidly by monogamous bivalency, a mechanism which requires the interaction of both Fab combining regions with antigenic determinants on the same polynucleotide. To explore further this mechanism, we tested Fab and F(ab')2 fragments prepared from IgG from patient plasmas in an ELISA with native DNA antigen, detecting antibody with a peroxidase conjugated anti-Fab reagent. These studies showed that Fab fragments, which can only bind monovalently, had negligible activity. Although bivalent F(ab')2 fragments would be predicted to bind DNA, these fragments also showed poor anti-DNA activity. Control studies showed that the fragments retained antibody activity to tetanus toxoid and an EBV antigen preparation. Together, these findings suggest that anti-DNA avidity depends on monogamous bivalency, with the antibody Fc portion also influencing DNA binding, in a mechanism which can be termed Fc-dependent monogamous bivalency. PMID:27083935

  3. Yeast display of antibody fragments: a discovery and characterization platform

    SciTech Connect

    Feldhaus, Michael; Siegel, Robert W.

    2004-07-01

    This review will focus on some of the novel attributes of the yeast surface display platform for the discovery and characterization of novel affinity reagents, optimization of those reagents, and novel uses of the platform. This is not intended to serve as an exhaustive review on the broader topic of general scFv technologies (see Winter et al., 1994; Smith and Petrenko, 1997; Bradbury et al., 2003) Furthermore, the scFv format of antibodies are easily manipulated through molecular cloning into a number of other formats such IgG, Fab, diabodies and such, for use in down steam applications and the reader is encouraged to read ?IgG?, ?Fab?, or your favorite format whenever scFv is seen in this review. This review is presented in 5 parts; (1) description of yeast display and its components, (2) library types and construction methods, (3) screening approaches for non-immune libraries and benefits, (4) screening approaches for directed evolution, kinetic on and off rates and (5) epitope complementation binning of clones.

  4. Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

    PubMed

    Hu, Xuejun; Hortigüela, María J; Robin, Sylvain; Lin, Heng; Li, Yajie; Moran, Anthony P; Wang, Wenxin; Wall, J Gerard

    2013-01-14

    Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

  5. Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: application to the four serotypes.

    PubMed

    Cabezas, Sheila; Rojas, Gertrudis; Pavon, Alequis; Alvarez, Mayling; Pupo, Maritza; Guillen, Gerardo; Guzman, Maria G

    2008-02-01

    Antibody fragments to the four Dengue virus serotypes were isolated from a human universal naïve library using phage display technology. Phage-displayed antibody fragments were selected on Dengue virus particles directly captured from infected Vero cells supernatant by an anti-dengue monoclonal antibody, in order to avoid laborious virus concentration/purification procedures. A total of nine phage-displayed antibody fragments were obtained. Seven of them were highly specific for three of the selector serotypes (two for Dengue 1, four for Dengue 3 and one for Dengue 4). One clone (Dengue 3-selected) cross-reacted with Dengue 1, whereas another (selected with Dengue 2) cross-reacted with the three remaining serotypes. The soluble variants of six antibody fragments recognized their target viruses when used at nanomolar and even subnanomolar concentrations. All phage-displayed antibody fragments were cross-reactive against several strains of distinct genotypes within the corresponding serotype(s). These antibody fragments are potentially useful for the future development of tools for viral diagnosis and serotype identification. The simple phage selection method on captured virus could be applied in a high throughput way to obtain larger panels of antibody fragments to Dengue virus for multiple applications.

  6. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

    PubMed

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

    2008-08-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  7. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    SciTech Connect

    Page, R.L.; Garg, P.K.; Gard, S. ||

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  8. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  9. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  10. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  11. Diagnosis of and therapy for solid tumors with radiolabeled antibodies and immune fragments

    SciTech Connect

    Carrasquillo, J.A.; Krohn, K.A.; Beaumier, P.; McGuffin, R.W.; Brown, J.P.; Hellstroem, K.E.; Hellstroem, I.; Larson, S.M.

    1984-01-01

    Antibodies which are directed against human tumor-associated antigens can potentially be used as carriers of radioactivity for in vivo diagnosis (radioimmunodetection) or treatment (radioimmunotherapy) of solid tumors, including colon, hepatoma, cholangiocarcinoma, and melanoma. Murine monoclonal antibodies (MOAB), produced by the hybridoma technique of Kohler and Milstein, are replacing conventional heterosera as sources of antibodies, because MOAB can be produced in large quantities as reproducible reagents with homogeneous binding properties. We have studied human melanoma using MOAB IgG and Fab fragments that recognize the human melanoma-associated antigens p97 and ''high-molecular-weight antigen''. Both antigens are found in the membrane of melanomas at much larger concentrations than in normal adult tissues. We have performed radioimmunodetection studies with whole immunoglobulin and have detected 88% of lesions greater than 1.5 cm. We have used Fab fragments for radioimmunotherapy and have found that large doses of radiolabeled antibodies (up to 342 mCi) can be repetitively given to patients without excessive end-organ toxicity. Two of three patients treated with high-dose radiolabeled antimelanoma Fab showed an effect from the treatment. Although both technical and biologic problems remain, the use of radiolabeled antibodies that are directed against tumor-associated antigens holds future promise as a new therapeutic approach to solid tumors that are resistant to conventional therapy.

  12. Probing the soybean Bowman-Birk inhibitor using recombinant antibody fragments.

    PubMed

    Muzard, Julien; Fields, Conor; O'Mahony, James John; Lee, Gil U

    2012-06-20

    The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing. PMID:22642722

  13. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Hopper, John E.; MacDonald, A. Bruce; Nisonoff, Alfred

    1970-01-01

    Idiotypic antibodies were investigated quantitatively by a method of indirect precipitation, which utilizes labeled F(ab')2 fragments of specifically purified antibenzoate antibody from the donor, anti-antibody, and an antiglobulin reagent. The contribution of allotypic and hidden determinants to these reactions was excluded. Greater fractions of an idiotypic antibody population are precipitated by this method, as compared to direct precipitation, and in two instances large proportions of idiotypic antibodies were detected in populations which failed to form precipitates by double diffusion in agar gel. The greater sensitivity of the indirect method was attributed to its capacity to detect molecules bearing a small number of antigenic determinants. Extensive studies of cross-reactions, carried out by an inhibition technique, failed to reveal any strong reactions of anti-idiotypic antibodies with heterologous antibenzoate antibody preparations, heterologous sera, or IgG, although a few weak cross-reactions were noted. One definite cross-reaction was observed by a direct binding measurement with heterologous antiserum. Antisera prepared in more than one recipient against a single donor preparation reacted with identical or overlapping subpopulations of the donor molecules. Instances in which two recipient antisera reacted with different proportions of the molecules of a single donor provided evidence for the existence of more than one idiotypic antibody population in the antibenzoate antibody of an individual rabbit. PMID:5308065

  14. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2015-01-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. PMID:26132144

  15. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  16. Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein.

    PubMed

    Skrlj, Nives; Serbec, Vladka Curin; Dolinar, Marko

    2010-03-01

    Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics. PMID:19597999

  17. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    PubMed Central

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H.-J.; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  18. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

    PubMed

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H-J; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  19. A Novel Single-Chain Antibody Fragment for Detection of Mannose 6-Phosphate-Containing Proteins

    PubMed Central

    Müller-Loennies, Sven; Galliciotti, Giovanna; Kollmann, Katrin; Glatzel, Markus; Braulke, Thomas

    2010-01-01

    Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III. PMID:20472886

  20. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    PubMed

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer. PMID:26961312

  1. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    PubMed

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

  2. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

  3. Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

    PubMed Central

    Dolk, Edward; van der Vaart, Marcel; Lutje Hulsik, David; Vriend, Gert; de Haard, Hans; Spinelli, Silvia; Cambillau, Christian; Frenken, Leon; Verrips, Theo

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications. PMID:15640220

  4. Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

    PubMed Central

    Song, Hyung-Nam; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2014-01-01

    Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH–VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. PMID:25530617

  5. Proinflammatory genes expression in granulocytes activated by native proteinase-binding fragments of anti-proteinase 3 IgG.

    PubMed

    Surmiak, M; Kaczor, M; Sanak, M

    2015-08-01

    The classical pathway of neutrophils activation due to cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) involves specific antigen binding to proteinase-3 and activation of the immunoglobulin G receptors by the constant fragment of the antibody. A requirement for this double signaling was suggested also because proteinase-3 is presented within a complex of NB-1 glycoprotein lacking transmembrane domain. An integrin Mac-1 receptor was postulated to cooperate in neutrophil stimulation by anti-proteinase 3 (anti-PR3). A characteristic profile of transcriptional activation of neutrophils by c-ANCA was described by us previously. We ascertained mRNA expression of neutrophils following stimulation with antigen-binding fragments of native anti-PR3 IgG. Expression of targeted transcripts was compared with our previous results, in which intact anti-PR3 IgG was used. Human neutrophils were isolated from healthy volunteers negative for ANCAs. Antigen-binding fragments of human anti-PR3 were prepared from sera of patients with granulomatosis with polyangiitis. We analyzed reactive oxygen species production and abundance of mRNA of 151 genes by quantitative real time-PCR in neutrophils stimulated with anti-PR3 IgG F(ab)(2). We observed a consistent upregulation of 17 genes (CYSLTR1, HPGD, IL1R1, IL1RL1, MAPK1, MAPK8, NR3C1, PLA2G7, PTGDR, CD302, DNAJB1, F2R, F2RL1, IER3, RAC1, RPL41, PTGER3), whereas other 9 genes were up-regulated only in some donors. No reactive oxygen species production was observed in neutrophils stimulated with anti-PR3 F(ab)(2). Stimulation of neutrophils with F(ab)(2) of anti-PR3 autoantibodies activated cells to a lesser extent than intact IgG. However, several cellular pathways were up-regulated, involving calcium and phosphatidylinositol 3-kinase AKT, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) signaling. Interestingly, binding of F(ab)(2) to the PR-3 present on the surface of neutrophil is sufficient for lipid

  6. Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

    PubMed

    Ahn, Jooyeon; Miura, Yutaka; Yamada, Naoki; Chida, Tsukasa; Liu, Xueying; Kim, Ahram; Sato, Ryuta; Tsumura, Ryo; Koga, Yoshikatsu; Yasunaga, Masahiro; Nishiyama, Nobuhiro; Matsumura, Yasuhiro; Cabral, Horacio; Kataoka, Kazunori

    2015-01-01

    Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors.

  7. Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

    PubMed

    Ahn, Jooyeon; Miura, Yutaka; Yamada, Naoki; Chida, Tsukasa; Liu, Xueying; Kim, Ahram; Sato, Ryuta; Tsumura, Ryo; Koga, Yoshikatsu; Yasunaga, Masahiro; Nishiyama, Nobuhiro; Matsumura, Yasuhiro; Cabral, Horacio; Kataoka, Kazunori

    2015-01-01

    Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors. PMID:25477168

  8. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model.

    PubMed

    Su, Ji-Guo; Zhang, Xiao; Han, Xiao-Ming; Zhao, Shu-Xin; Li, Chun-Hua

    2015-01-01

    Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM). Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF) of the residues, as well as the MSF in the internal distance (MSFID) between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs) in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

  9. Isolation and characterization of antibody fragments selective for toxic oligomeric tau

    PubMed Central

    Tian, Huilai; Davidowitz, Eliot; Lopez, Patricia; He, Ping; Schulz, Philip; Moe, James; Sierks, Michael R.

    2014-01-01

    Oligomeric tau species are important in the onset and progression of Alzheimer’s Disease (AD) as they are neurotoxic and can propagate tau tangle pathology. Therefore reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy (AFM) to isolate single chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated three different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human post-mortem AD brain tissue from cognitively normal post-mortem human brain tissue demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD. PMID:25616912

  10. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins. PMID:27212628

  11. SIGNIFICANCE OF BIVALENCE OF ANTIBODY IN VIRAL NEUTRALIZATION

    PubMed Central

    Rosenstein, Robert W.; Nisonoff, Alfred; Uhr, Jonathan W.

    1971-01-01

    The role of bivalence of antibody in its capacity to neutralize virus was studied with rabbit antibodies to the bacteriophage, φX174. Univalent Fab or Fab' fragments of IgG isolated from antiviral antisera obtained early in the immunization schedule had virtually no activity compared to that of the intact IgG. When the antibodies were isolated from antisera of the same rabbits several months later, the univalent fragments and IgG were essentially equal in activity. The results are interpreted on the basis that an IgG molecule, because of its bivalence, has a higher effective combining affinity (avidity) than a univalent fragment. After prolonged immunization, however, the affinity of univalent antibody becomes sufficiently high that it exceeds a threshold value, above which further increase in affinity, through bivalence, is no longer significant. The results could explain the variability in relative effectiveness of univalent antibodies observed in previous studies. These data, and the fact that F(ab')2 fragments from either "early" or "late" antisera were as effective as IgG, indicate that fragment Fc is not a significant factor in neutralization. No differences in dissociation from the virus of univalent antibody from early and late antisera could be demonstrated by dilution at temperatures up to 47°C. The attachment at sites of neutralization on the virus appears to be functionally almost irreversible in this system. PMID:5166611

  12. A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents

    PubMed Central

    Polonelli, Luciano; Ciociola, Tecla; Elviri, Lisa; Zanello, Pier Paolo; Giovati, Laura; Arruda, Denise C.; Muñoz, Julián E.; Mortara, Renato A.; Morace, Giulia; Borghi, Elisa; Galati, Serena; Marin, Oriano; Casoli, Claudio; Pilotti, Elisabetta; Ronzi, Paola; Travassos, Luiz R.; Magliani, Walter; Conti, Stefania

    2016-01-01

    A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity. PMID:27725769

  13. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging.

    PubMed

    Olafsen, Tove; Kenanova, Vania E; Sundaresan, Gobalakrishnan; Anderson, Anne-Line; Crow, Desiree; Yazaki, Paul J; Li, Lin; Press, Michael F; Gambhir, Sanjiv S; Williams, Lawrence E; Wong, Jeffrey Y C; Raubitschek, Andrew A; Shively, John E; Wu, Anna M

    2005-07-01

    We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

  14. Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo

    PubMed Central

    Tavaré, Richard; McCracken, Melissa N.; Zettlitz, Kirstin A.; Knowles, Scott M.; Salazar, Felix B.; Olafsen, Tove; Witte, Owen N.; Wu, Anna M.

    2014-01-01

    The noninvasive detection and quantification of CD8+ T cells in vivo are important for both the detection and staging of CD8+ lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8+ T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8+ T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for 64Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both 64Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice. PMID:24390540

  15. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    PubMed

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  16. Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

    PubMed Central

    Ruha, Anne-Michelle; Seifert, Steven A.; Morgan, David L.; Lewis, Brandon J.; Arnold, Thomas C.; Clark, Richard F.; Meggs, William J.; Toschlog, Eric A.; Borron, Stephen W.; Figge, Gary R.; Sollee, Dawn R.; Shirazi, Farshad M.; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D.; Boyer, Leslie V.

    2015-01-01

    Background. Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm3, fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation. PMID:25361165

  17. Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

    PubMed

    Robert, Remy; Streltsov, Victor A; Newman, Janet; Pearce, Lesley A; Wark, Kim L; Dolezal, Olan

    2010-02-01

    Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Abeta peptide (39-43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope ((3)EFRH(6)) in the Abeta peptide at the N-terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer-mediated toxicity. In this study, we describe the humanization of WO-2 using complementary determining region loop grafting onto the human germline gene and the determination of the three-dimensional structure by X-ray crystallography. This humanized version retains a high affinity for the Abeta peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease.

  18. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    PubMed Central

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

  19. Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection.

    PubMed

    Zheng, Xuexing; Wong, Gary; Zhao, Yongkun; Wang, Hualei; He, Shihua; Bi, Yuhai; Chen, Weijin; Jin, Hongli; Gai, Weiwei; Chu, Di; Cao, Zengguo; Wang, Chong; Fan, Quanshui; Chi, Hang; Gao, Yuwei; Wang, Tiecheng; Feng, Na; Yan, Feihu; Huang, Geng; Zheng, Ying; Li, Nan; Li, Yuetao; Qian, Jun; Zou, Yong; Kobinger, Gary; Gao, George Fu; Qiu, Xiangguo; Yang, Songtao; Xia, Xianzhu

    2016-01-01

    Recent successes with monoclonal antibody cocktails ZMapp(TM) and MIL77 against Ebola virus (EBOV) infections have reignited interest in antibody-based therapeutics. Since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. We produced purified equine antisera from horses hyperimmunized with EBOV virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. BALB/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. To decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate F(ab')2 fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. Full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other Fc-dependent antibody mechanisms may also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab')2 at or starting at 1 or 2 dpi were also fully protected from EBOV infection. These results justify future efficacy studies for purified equine products in NHPs.

  20. Binding of monoclonal antibody to CD16 causes calcium mobilization in large granular lymphocytes but inhibits NK killing.

    PubMed Central

    Macintyre, E A; Wallace, D W; O'Flynn, K; Abdul-Gaffar, R; Tetteroo, P A; Morgan, G; Linch, D C

    1989-01-01

    A monoclonal antibody (mAb), CLB/FcR gran I, reactive with the CD16 Fc receptor (FcRlo/FcRIII) of human cells, leads to calcium mobilization in large granular lymphocytes (LGL) but not in granulocytes. Identical responses are obtained with F(ab')2 fragments of this antibody, indicating that the response is independent of Fc-FcR binding, and that bivalent cross-linking of this receptor is adequate for optimal calcium mobilization. The calcium response was greater in CD3- LGL compared to CD3+ LGL, although the response was augmented in the latter cells by prior rosetting with sheep red blood cells (SRBC). Calcium mobilization in CD3- LGL induced by CLB/FcR gran I is associated with inhibition of natural killer cell (NK) killing, and inhibition of the enhanced NK killing induced by the anti-CD2 low-density monoclonal antibody, 9.1. This supports the view that the NK-enhancing activity of 9.1 is due to simultaneous binding to CD2 and CD16, and may in fact be transduced through the CD16 molecule. The variable reported effects of anti-CD16 antibodies on NK killing are likely to reflect the epitope bound rather than the isotype of antibody used, since F(ab')2 fragments of CLB/FcR gran I also inhibit NK killing. PMID:2564843

  1. Proteomic differences in recombinant CHO cells producing two similar antibody fragments

    PubMed Central

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin

    2016-01-01

    ABSTRACT Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  2. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. PMID:26654254

  3. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  4. Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

    PubMed Central

    Papalian, M; Lafer, E; Wong, R; Stollar, B D

    1980-01-01

    Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity. Images PMID:6153184

  5. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    PubMed

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS.

  6. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  7. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    PubMed

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera. PMID:24001307

  8. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    PubMed

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

  9. Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

    NASA Astrophysics Data System (ADS)

    Kubaštová, H.; Kleinova, V.; Seifert, D.; Fišer, M.; Kranda, K.

    2006-01-01

    The ability of the monoclonal antibody TU-20 and its scFv fragment to specifically bind to the C-end of the class III beta-tubulin makes these preparations useful as potential diagnostics for in vivo determination of neurodegenerative diseases that entail degradation of neuronal cytoskeleton. To examine this hypothesis, TU-20 and its scFv were labelled with 125I and their properties were extensively investigated. TU-20 and its scFv were labelled via chloramine-T with the yield 90 95% and 64 78%, respectively. Their quality control, performed by an ELISA and gel electrophoresis, determined adequate properties for further studies. The in vitro experiment, involving autoradiography and immunohistochemistry of mice’ brain slices, enabled confirmation of preserved immunospecificity of the radiolabelled substances. Finally, the in vivo biodistribution proved differences in elimination of either TU-20, scFv TU-20, or iodide from the mice.

  10. An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.

    PubMed

    Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2011-09-01

    An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

  11. Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

    PubMed

    Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

    2009-01-01

    Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

  12. Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies?

    PubMed Central

    Kuus-Reichel, K; Grauer, L S; Karavodin, L M; Knott, C; Krusemeier, M; Kay, N E

    1994-01-01

    While monoclonal antibodies show promise for use in the treatment of a variety of disease states, including cancer, autoimmune disease, and allograft rejection, generation of anti-antibody responses still remains a problem. For example, 50% of the patients who receive OKT3 produce blocking antibodies that interfere with its binding to T cells, thus decreasing the therapeutic effect (51). HAMA responses have also interfered with tumor imaging (39,40) and radioimmunotherapy (56). The generation of an anti-antibody response is dependent on many factors. These include the dose of antibody, the number of injections of antibody, the immunogenicity of the antibody, the form of the antibody, and the immunocompetence of the recipient. Predictably, both the number of injections of antibody and the dosage are influential in the generation of an anti-antibody response. It is apparent that human antibodies, chimeric antibodies, and mouse Fab fragments are much less likely to induce anti-antibody responses than intact mouse monoclonal antibodies or mouse F(ab')2 fragments when one injection is administered. Injections of human or chimeric antibodies appears to reduce immunogenicity, but the probability that anti-antibody responses can still be induced on multiple injections must be considered and appropriately evaluated. Several areas demand extensive investigation to enhance the clinical utility of monoclonal antibodies. First, results of thorough clinical trials with human or chimeric antibodies need to be evaluated for the induction of anti-antibodies after multiple injections of antibodies. Second, less immunogenic forms of antibodies (Fab, Fv) need to be studied for their clinical efficacies and for their abilities to induce anti-antibody responses. PMID:8556470

  13. Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

    PubMed

    Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A; Yang, Yunan; England, Christopher G; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2015-10-13

    Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions. PMID:26417085

  14. Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry

    PubMed Central

    Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A.; Yang, Yunan; England, Christopher G.; Theuer, Charles P.; Nickles, Robert J.; Cai, Weibo

    2015-01-01

    Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal “click” ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105+/+) tumors with 64Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of 64Cu-NOTA-Bs-F(ab)2. 64Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1–labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions. PMID:26417085

  15. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    PubMed

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated. PMID:26945728

  16. Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

    PubMed Central

    Tokuhara, Daisuke; ρlvarez, Beatriz; Mejima, Mio; Hiroiwa, Tomoko; Takahashi, Yuko; Kurokawa, Shiho; Kuroda, Masaharu; Oyama, Masaaki; Kozuka-Hata, Hiroko; Nochi, Tomonori; Sagara, Hiroshi; Aladin, Farah; Marcotte, Harold; Frenken, Leon G.J.; Iturriza-Gómara, Miren; Kiyono, Hiroshi; Hammarström, Lennart; Yuki, Yoshikazu

    2013-01-01

    Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections. PMID:23925294

  17. Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.

    PubMed

    Zhao, Yongqian; Moreland, Nicole J; Tay, Moon Y F; Lee, Chin Chin; Swaminathan, Kunchithapadam; Vasudevan, Subhash G

    2014-01-22

    The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex.

  18. Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

    PubMed

    Ahuja, Shivani; Rougé, Lionel; Swem, Danielle L; Sudhamsu, Jawahar; Wu, Ping; Russell, Stephen J; Alexander, Mary Kate; Tam, Christine; Nishiyama, Mireille; Starovasnik, Melissa A; Koth, Christopher M

    2015-04-01

    Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.

  19. A method to confer Protein L binding ability to any antibody fragment.

    PubMed

    Lakhrif, Zineb; Pugnière, Martine; Henriquet, Corinne; di Tommaso, Anne; Dimier-Poisson, Isabelle; Billiald, Philippe; Juste, Matthieu O; Aubrey, Nicolas

    2016-01-01

    Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection. PMID:26683650

  20. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    PubMed

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  1. A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

    PubMed

    Liang, Hui; Li, Xiaoran; Chen, Bing; Wang, Bin; Zhao, Yannan; Zhuang, Yan; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2015-07-10

    Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

  2. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

    SciTech Connect

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A. ); Hay, B.N.; Shopes, B. )

    1990-10-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

  3. Human heterophilic antibodies against equine immunoglobulins: assessment of their role in the early adverse reactions to antivenom administration.

    PubMed

    León, Guillermo; Segura, Alvaro; Herrera, María; Otero, Rafael; França, Francisco Oscar de Siqueira; Barbaro, Katia Cristina; Cardoso, João Luiz Costa; Wen, Fan Hui; de Medeiros, Carlos Roberto; Prado, José Carlos Lopes; Malaque, Ceila María Sant'Ana; Lomonte, Bruno; Gutiérrez, José María

    2008-11-01

    The presence of human heterophilic antibodies against horse immunoglobulins (HHA-HI) was determined by ELISA in sera from healthy volunteers and from patients who received equine antivenom for therapy of snake bite envenoming. These patients were selected from two independent clinical studies: one in Colombia in which patients received antivenom constituted by whole IgG (n=25); and the other in Brazil where an antivenom constituted by F(ab')(2) fragments was administered (n=31). Results show that healthy volunteers have antibodies, mainly of the IgG class, able to react with whole equine IgG. Additionally, patients have IgG antibodies that react both with whole equine IgG and F(ab')(2) fragments. In both clinical studies, no significant differences were observed in the HHA-HI titres between the patients who presented early adverse (anaphylactoid) reactions and those who did not develop them. In addition, no variation in titre was observed in samples collected before and after antivenom administration. These results do not support the hypothesis that the incidence of early adverse reactions to antivenom administration correlates with the titre of HHA-HI in the serum of patients. Nevertheless, participation of these antibodies as part of a multifactorial pathogenic mechanism associated with these reactions cannot be ruled out. PMID:18561967

  4. Production of an anti-idiotypic antibody single chain variable fragment vaccine against Edwardsiella tarda.

    PubMed

    Qin, Hong; Jin, Xiaohang; Huang, Weiquan; Liu, Yulin

    2010-02-01

    Edwardsiella tarda is the pathogen responsible for edwardsiellosis, a serious infectious disease of freshwater and marine fish species, and currently recognized to be the species pathogenic for human. An anti-idiotypic monoclonal antibody (mAb), 1E11, has been developed. It mimics the protective epitope of E. tarda and can prevent fish from infection of E. tarda. In this study, the correct variable heavy (VH) and variable light (VL) genes were obtained from 1E11 by using bioinformatics methods, and a 15 amino acid (Gly4Ser)3 linker was used to hold the two V domains together for the construction of VL-linker-VH form of single chain variable fragment (scFv) gene. Then, the scFv was subcloned into the vector pET-28a, expressed in the Escherichia coli BL21 cells, and identified by SDS-PAGE and western blotting. Red drum (Sciaenops ocellatus L.) weighing about 50 g was subjected to challenge with different E. tarda strains after 4 weeks followed by vaccination, the mortality rates and relative percentage survival were recorded and calculated, and the survival rate of fish in the scFv subgroups was obviously higher than that of control subgroups (P<0.01). Enzyme-linked immunosorbent assay results show that after 4 weeks of post-vaccination, the level of specific antibody in fish sera of scFv groups was significantly higher than control groups. This study indicates that the recombinant antibody scFv was successfully developed, and it may serve as an effective vaccine candidate against E. tarda. PMID:20119624

  5. Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

    PubMed Central

    Gokulan, Kuppan; Varughese, Kottayil I.

    2013-01-01

    Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy. PMID:24349338

  6. Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

    PubMed Central

    Jiang, W; Venugopal, K; Gould, E A

    1995-01-01

    A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus. PMID:7815482

  7. Quantitation of imaging with I-131-F(ab')/sub 2/ fragments of monoclonal antibody in patients

    SciTech Connect

    Moldofsky, P.J.; Hammond, N.D.; Mulhern, C.B. Jr.

    1984-01-01

    Iodine-131 labeled F(ab')/sub 2/ fragments of monoclonal antibody (IgG/sub 2a/ immunoglobulin with specificity for a cell surface antigen of colon carcinoma) have been used for quantitative imaging of tumor in 27 patients. Activity of I-131 F(ab')/sub 2/ fragments localized in tumor and in liver was quantitated using a modification of the method of Thomas SR, employing computer-acquired conjugate views (i.e. 180 opposed) to eliminate need for tumor or organ depth and tissue attenuation. The method was validated with an abdominal imaging phantom showing accuracy of +/- 10%. Quantitation indicates that activity reaches a peak in tumor at 48-72 hours and the ratio of activity in hepatic metastases to activity in liver peaks at approximately 72 hours. Mean activity in tumor was less than 0.01% of the administered dose per gram of tumor at any imaging time from 24 to 168 hours, while mean activity in surrounding liver was less than .002% of administered dose per gram of liver at any imaging time. Liver activity decreased monotonically with time, showing no peak activity. This non-invasive method of quantitating the distribution of F(ab')/sub 2/ fragments of monoclonal antibody in patients has proven accurate by comparison with phantom simulation. This type of quantitation is necessary for evaluating optimal imaging time, comparing relative utility of various antibodies and has use for therapeutic applications of monoclonal antibody fragments.

  8. Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

  9. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

  10. Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules.

    PubMed

    Strachan, G; Williams, S; Moyle, S P; Harris, W J; Porter, A J

    1999-09-01

    Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.

  11. Use of 18F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer

    PubMed Central

    2015-01-01

    We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon–fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18F-FDG imaging. PMID:26955657

  12. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    PubMed Central

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  13. Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.

    PubMed

    Juhl, H; Petrella, E C; Cheung, N K; Bredehorst, R; Vogel, C W

    1997-11-01

    Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy.

  14. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  15. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  16. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    PubMed

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important. PMID:26608419

  17. Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments

    PubMed Central

    Stech, Marlitt; Hust, Michael; Schulze, Corina; Dübel, Stefan; Kubick, Stefan

    2014-01-01

    Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling. PMID:25821419

  18. Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice.

    PubMed

    Bäck, Tom; Haraldsson, Börje; Hultborn, Ragnar; Jensen, Holger; Johansson, Martin E; Lindegren, Sture; Jacobsson, Lars

    2009-12-01

    Besides bone marrow, the kidneys are often dose-limiting organs in internal radiotherapy. The effects of high-linear energy transfer (LET) radiation on the kidneys after alpha-radioimmunotherapy (alpha-RIT) with the alpha-particle emitter, (211)At, were studied in nude mice by serial measurements of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals and animals bearing subcutaneous xenografts of the human ovarian cancer cell line, OVCAR-3, were used. The animals received approximately 0.4, 0.8, or 1.2 MBq in one, two, or three fractions. The mean absorbed doses to the kidneys ranged from 1.5 to 15 Gy. The renal function was studied by serial GFR measurements, using plasma clearance of (51)Cr-EDTA, up to 67 weeks after the first astatine injection. A dose-dependent effect on GFR was found and at the time interval 8-30 weeks after the first administration of astatine, the absorbed doses causing a 50% decrease in GFR were 16.4 +/- 3.3 and 14.0 +/- 4.1 Gy (mean +/- SEM), tumor- and non-tumor-bearing animals, respectively. The reduction in GFR progressed with time, and at the later time interval, (31-67 weeks) the corresponding absorbed doses were 7.5 +/- 2.4 and 11.3 +/- 2.3 Gy, respectively, suggesting that the effects of radiation on the kidneys were manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary

  19. Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice.

    PubMed

    Bäck, Tom; Haraldsson, Börje; Hultborn, Ragnar; Jensen, Holger; Johansson, Martin E; Lindegren, Sture; Jacobsson, Lars

    2009-12-01

    Besides bone marrow, the kidneys are often dose-limiting organs in internal radiotherapy. The effects of high-linear energy transfer (LET) radiation on the kidneys after alpha-radioimmunotherapy (alpha-RIT) with the alpha-particle emitter, (211)At, were studied in nude mice by serial measurements of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals and animals bearing subcutaneous xenografts of the human ovarian cancer cell line, OVCAR-3, were used. The animals received approximately 0.4, 0.8, or 1.2 MBq in one, two, or three fractions. The mean absorbed doses to the kidneys ranged from 1.5 to 15 Gy. The renal function was studied by serial GFR measurements, using plasma clearance of (51)Cr-EDTA, up to 67 weeks after the first astatine injection. A dose-dependent effect on GFR was found and at the time interval 8-30 weeks after the first administration of astatine, the absorbed doses causing a 50% decrease in GFR were 16.4 +/- 3.3 and 14.0 +/- 4.1 Gy (mean +/- SEM), tumor- and non-tumor-bearing animals, respectively. The reduction in GFR progressed with time, and at the later time interval, (31-67 weeks) the corresponding absorbed doses were 7.5 +/- 2.4 and 11.3 +/- 2.3 Gy, respectively, suggesting that the effects of radiation on the kidneys were manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary

  20. Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

    PubMed Central

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function. PMID:18783590

  1. Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

    PubMed Central

    Wu, Sau-Ching; Yeung, Jonathan C.; Duan, Yanjun; Ye, Ruiqiong; Szarka, Steven J.; Habibi, Hamid R.; Wong, Sui-Lam

    2002-01-01

    To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems. PMID:12089002

  2. Structural basis for enhanced neutralization of HIV-1 by a dimeric IgG form of the glycan-recognizing antibody 2G12

    PubMed Central

    Wu, Yunji; West, Anthony P.; Kim, Helen J.; Thornton, Matthew E.; Ward, Andrew B.; Bjorkman, Pamela J.

    2013-01-01

    SUMMARY The human IgG 2G12 recognizes high-mannose carbohydrates on the HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally-distinct dimers, demonstrating flexibility of the (Fab)2–Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12. PMID:24316082

  3. Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody.

    PubMed

    Wang, Zhiqing; Li, Long; Pennington, Janice G; Sheng, Ju; Yap, Moh Lan; Plevka, Pavel; Meng, Geng; Sun, Lei; Jiang, Wen; Rossmann, Michael G

    2013-08-01

    The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

  4. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2014-01-01

    Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  5. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2014-01-01

    Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect. PMID:24671209

  6. Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Châtelier’s Principle

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2014-01-01

    Le Châtelier’s principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier’s principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect. PMID:24671209

  7. Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

    PubMed

    Foerster, A; Hołowacz, I; Sunil Kumar, G B; Anandakumar, S; Wall, J G; Wawrzyńska, M; Paprocka, M; Kantor, A; Kraskiewicz, H; Olsztyńska-Janus, S; Hinder, S J; Bialy, D; Podbielska, H; Kopaczyńska, M

    2016-04-01

    Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

  8. High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.

    PubMed

    Leonard, Paul; Säfsten, Pär; Hearty, Stephen; McDonnell, Barry; Finlay, William; O'Kennedy, Richard

    2007-06-30

    Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day. PMID:17532001

  9. Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.

    PubMed Central

    Berton, G; Rosen, H; Ezekowitz, R A; Bellavite, P; Serra, M C; Rossi, F; Gordon, S

    1986-01-01

    Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells. Images PMID:3012541

  10. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

  11. Neonatal Immunization with Respiratory Syncytial Virus Glycoprotein Fragment Induces Protective Immunity in the Presence of Maternal Antibodies in Mice

    PubMed Central

    Noh, Youran; Shim, Byoung-Shik; Cheon, In Su; Rho, Semi; Kim, Hee Joo; Choi, Youngjoo; Kang, Chang-Yuil; Chang, Jun

    2013-01-01

    Abstract Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFNγ secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period. PMID:23869549

  12. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

    PubMed Central

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  13. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  14. Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation.

    PubMed

    Pfeffer, Martin; Maurer, Michael; Stadlmann, Johannes; Grass, Josephine; Delic, Marizela; Altmann, Friedrich; Mattanovich, Diethard

    2012-03-01

    Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway. PMID:22350260

  15. In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

    PubMed Central

    Aladin, Farah; Einerhand, Alexandra W. C.; Bouma, Janneke; Bezemer, Sandra; Hermans, Pim; Wolvers, Danielle; Bellamy, Kate; Frenken, Leon G. J.; Gray, Jim; Iturriza-Gómara, Miren

    2012-01-01

    Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains. PMID:22403728

  16. Nephritogenic Lupus Antibodies Recognize Glomerular Basement Membrane-Associated Chromatin Fragments Released from Apoptotic Intraglomerular Cells

    PubMed Central

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-01-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus. PMID:16723695

  17. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    PubMed

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  18. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John

    2016-03-01

    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications. PMID:26744234

  19. Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris.

    PubMed

    Chang, Hyun-Joo; Choi, Sung-Wook; Chun, Hyang Sook

    2008-10-01

    A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff. PMID:18575809

  20. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

    PubMed

    Muller, B H; Chevrier, D; Boulain, J C; Guesdon, J L

    1999-07-30

    Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

  1. Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Methods: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Results: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. Conclusion: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α. PMID:26793613

  2. Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants.

    PubMed Central

    Routsias, John G.; Touloupi, Evgenia; Dotsika, Eleni; Moulia, Avrilia; Tsikaris, Vassilios; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Moutsopoulos, Haralampos M.; Tzioufas, Athanasios G.

    2002-01-01

    BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with p

  3. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  4. Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D

    2009-10-01

    Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.

  5. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  6. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    PubMed

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  7. Kinetics of intralymphatically delivered monoclonal antibodies

    SciTech Connect

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.

    1985-05-01

    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations.

  8. Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

    PubMed

    Kalli, K R; Hsu, P H; Bartow, T J; Ahearn, J M; Matsumoto, A K; Klickstein, L B; Fearon, D T

    1991-12-01

    CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

  9. Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

    PubMed

    Kalli, K R; Hsu, P H; Bartow, T J; Ahearn, J M; Matsumoto, A K; Klickstein, L B; Fearon, D T

    1991-12-01

    CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities. PMID:1836011

  10. Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality. PMID:26793614

  11. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1

    PubMed Central

    Ghadge, Ghanashyam D.; Pavlovic, John; Koduvayur, Sujatha P.; Kay, Brian K.; Roos, Raymond P.

    2013-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. PMID:23607939

  12. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    PubMed

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  13. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    PubMed

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  14. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    PubMed

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods. PMID:26963935

  15. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    PubMed

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers. PMID:26256421

  16. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

  17. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    PubMed

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives. PMID:27391930

  18. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. PMID:26232710

  19. Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism.

    PubMed

    Karim-Silva, Sabrina; Moura, Juliana de; Noiray, Magali; Minozzo, Joao Carlos; Aubrey, Nicolas; Alvarenga, Larissa M; Billiald, Philippe

    2016-08-01

    Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms. PMID:27288291

  20. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  1. Phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals.

    PubMed

    Cabezas, Sheila; Rojas, Gertrudis; Pavon, Alequis; Bernardo, Lidice; Castellanos, Yinet; Alvarez, Mayling; Pupo, Maritza; Guillen, Gerardo; Guzman, Maria G

    2009-01-01

    The current study shows the usefulness of dengue-3- and dengue-4-specific phage-displayed antibody fragments as tools for viral detection and serotyping in sera from infected individuals. C6/36 HT cells were inoculated with acute-phase sera from patients, and supernatants were collected daily and analyzed by ELISA using phage-displayed antibody fragments as serotype-specific detector reagents. Serotyping of most samples was possible as early as two to three days postinoculation. Results were comparable with those obtained by indirect immunofluorescence assay but were obtained in a shorter period of time (<1 week). Phage-displayed antibody fragments were better tools for diagnosis and serotyping than their soluble counterparts. Our approach combines the advantages of viral isolation and ELISA techniques. These results could be the basis for the development of a high-throughput method for identifying dengue virus serotypes, which is crucial for the management and control of the disease.

  2. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    PubMed

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

  3. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    PubMed Central

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  4. Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds.

    PubMed

    Wang, Dezhong; Ma, Jisheng; Sun, Difei; Li, Haiyan; Jiang, Chao; Li, Xiaokun

    2015-08-01

    Seed-based expression system is an attractive platform for the production of recombinant proteins in molecular farming. Despite the many advantages of molecular farming, little is known about the effect of the different subcellular accumulation of recombinant proteins on the endoplasmic reticulum (ER) quality control system in host plants. In this study, we analyzed the expression of anti-CD20 antibody fragments in seeds of Arabidopsis thaliana (ecotype Columbia) and corresponding glycosylation mutants, and evaluated the influence of three different signal sequences on the expression levels of scFv-Fc of C2B8. The highest protein accumulation level, with a maximum of 6.12 % total soluble proteins, was observed upon fusing proteins to the signal peptide of Arabidopsis seed storage albumin 2. The ER stress responses in developing seeds at 13 days post-anthesis were also compared across different transgenic lines under normal and heat shock conditions. Based on the gene expression profiles of ER stress transducers, our results suggest that accumulation of antibody fragments in the ER exerts more stress on ER homeostasis. In addition, quantitative PCR results also implicate enhanced activation of ER-associated degradation in transgenic lines. Last but not the least, we also demonstrate the anti-tumor potency of plant-derived proteins by showing the anti-tumor activity of purified scFv-Fc proteins against Daudi cells. Together, our data implies that better understanding of the interaction between exogenous protein production and the cellular quality control system of the host plant is necessary for the development of an optimal expression strategy that will be especially beneficial to commercial protein manufacturing. PMID:25957150

  5. Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus.

    PubMed

    Cai, Kun; Wang, Hui; Bao, Shizhong; Shi, Jing; Hou, Xiaojun; Gao, Xiang; Liu, Hao; Yin, Jun

    2008-04-01

    Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus.

  6. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    PubMed

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  7. Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.

    PubMed

    Safarnejad, M R; Fischer, R; Commandeur, U

    2008-01-01

    Tomato yellow leaf curl virus (TYLCV) is a complex of geminivirus species prevalent in the tropics and sub-tropics, which causes severe diseases in economically important crops such as tomato. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. We generated two single-chain variable fragment antibodies (scFv-ScRep1 and scFv-ScRep2) that bound strongly to continuous epitopes within the TYLCV replication-associated protein (Rep). The TYLCV-Ir C1 gene (encoding Rep) was expressed as glutathione-S-transferase (GST) and maltose-binding protein (MBP) fusions. Purified MBP-Rep was used to immunize mice allowing the construction of naïve and pre-immunized scFv phage display libraries. Immunoassays showed that scFv-ScRep1 recognized an N-terminal epitope of Rep, whereas scFv-ScRep2 recognized a more central epitope. This is the first successful production of scFv antibodies against a geminivirus Rep, the initial step in the production of transgenic plants with resistance to TYLCV. PMID:19226769

  8. Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain.

    PubMed

    Harley, Carol A; Starek, Greg; Jones, David K; Fernandes, Andreia S; Robertson, Gail A; Morais-Cabral, João H

    2016-08-30

    The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained. PMID:27516548

  9. Intratracheal exposure to Fab fragments of an allergen-specific monoclonal antibody regulates asthmatic responses in mice

    PubMed Central

    Yoshino, Shin; Mizutani, Nobuaki; Matsuoka, Daiko; Sae-Wong, Chutha

    2014-01-01

    Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses. PMID:24303921

  10. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

    PubMed

    Demartis, S; Tarli, L; Borsi, L; Zardi, L; Neri, D

    2001-04-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. PMID:11357506

  11. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    NASA Astrophysics Data System (ADS)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  12. High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

    PubMed

    Papalia, Giuseppe A; Baer, Mark; Luehrsen, Kenneth; Nordin, Helena; Flynn, Peter; Myszka, David G

    2006-12-01

    A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

  13. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy.

    PubMed

    Hébert, J; Bernier, D; Mourad, W

    1990-06-01

    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  14. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  15. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    PubMed Central

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers. PMID:25993332

  16. Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

    PubMed

    Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

    2014-06-01

    Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs. PMID:24524420

  17. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    PubMed

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects. PMID:26654700

  18. Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD).

    PubMed

    Boackle, Robert J; Nguyen, Quang L; Leite, Renata S; Yang, Xiaofeng; Vesely, Jana

    2006-02-01

    in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.

  19. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  20. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases. PMID:22397408

  1. A new tool for monoclonal antibody analysis

    PubMed Central

    An, Yan; Zhang, Ying; Mueller, Hans-Martin; Shameem, Mohammed; Chen, Xiaoyu

    2014-01-01

    Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications. PMID:24927271

  2. Interaction analysis of HIV-1 antibody 2G12 and Man9GlcNAc2 ligand: Theoretical calculations by fragment molecular orbital and MD methods

    NASA Astrophysics Data System (ADS)

    Koyama, Yuka; Ueno-Noto, Kaori; Takano, Keiko

    2013-07-01

    In HIV-1 infection, human antibody 2G12 is capable of recognizing the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. To investigate the ligand binding mechanisms of antibody 2G12 with glycans aiming for the contribution to the medications, we carried out classical molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the antibody 2G12 complex with its high-mannose ligand. We found that Mannose D1 of the ligand had the largest binding affinity with the antibody, which was well consistent with experimental reports. Furthermore, significant roles of Mannose 4 and 4‧ in the ligand binding were theoretically indicated.

  3. Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D; van Keulen, L; Rosalia, R A; Weerdmeester, K; Cornelissen, A H M; De Bruin, M G M; Eblé, P L; Dekker, A

    2007-03-10

    Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.

  4. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

  5. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    PubMed

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  6. Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Taqueda, Maria E S; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2015-07-01

    In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

  7. Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D; van Keulen, L; Rosalia, R A; Weerdmeester, K; Cornelissen, A H M; De Bruin, M G M; Eblé, P L; Dekker, A

    2007-03-10

    Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo. PMID:17127019

  8. Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

    PubMed Central

    Orlova, A; Yu, X; Egelman, E H

    1994-01-01

    We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities. Images FIGURE 1 FIGURE 5 FIGURE 7 FIGURE 10 PMID:8161679

  9. Co-Expression of Anti-Rotavirus Proteins (Llama VHH Antibody Fragments) in Lactobacillus: Development and Functionality of Vectors Containing Two Expression Cassettes in Tandem

    PubMed Central

    Günaydın, Gökçe; Álvarez, Beatriz; Lin, Yin; Hammarström, Lennart; Marcotte, Harold

    2014-01-01

    Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3) as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11), by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules. PMID:24781086

  10. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells.

    PubMed

    Röhm, Martina; Handl, Alina; König, Maria; Mavoungou, Chrystelle; Handrick, René; Schindowski, Katharina

    2016-09-01

    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab')2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I) and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand. PMID:27366780

  11. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells.

    PubMed

    Röhm, Martina; Handl, Alina; König, Maria; Mavoungou, Chrystelle; Handrick, René; Schindowski, Katharina

    2016-09-01

    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab')2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I) and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand.

  12. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    PubMed

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  13. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  14. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    PubMed

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.

  15. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

    PubMed

    Encinas, P; Gomez-Casado, E; Fregeneda-Grandes; Olesen, N J; Lorenzen, N; Estepa, A; Coll, J M

    2011-03-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

  16. Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E.

    PubMed

    Schlager, S I; Dray, S

    1976-01-01

    Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use

  17. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    PubMed

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. PMID:26493138

  18. Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

    PubMed

    Hosking, Christopher G; Driguez, Patrick; McWilliam, Hamish E G; Ilag, Leodevico L; Gladman, Simon; Li, Yuesheng; Piedrafita, David; McManus, Donald P; Meeusen, Els N T; de Veer, Michael J

    2015-09-01

    Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages.

  19. Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously.

    PubMed

    Kunanopparat, Areerat; Chaivisuthangkura, Parin; Senapin, Saengchan; Longyant, Siwaporn; Rukpratanporn, Sombat; Flegel, Timothy W; Sithigorngul, Paisarn

    2011-01-01

    The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248-3045), CP-I (nucleotides 3046-3954) and CP-C (nucleotides 3955-4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

  20. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

    PubMed

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J

    2006-01-01

    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  1. Anti-endothelial cell antibodies in patients with rheumatoid arthritis complicated by vasculitis.

    PubMed Central

    Heurkens, A H; Hiemstra, P S; Lafeber, G J; Daha, M R; Breedveld, F C

    1989-01-01

    IgG antibodies reactive with human umbilical vein endothelial cells were found in 19 out of 28 patients with rheumatoid vasculitis (RV), in four out of 24 patients with rheumatoid arthritis (RA), in seven out of 10 patients with systemic lupus erythematosus (SLE), but not in healthy donors. In four patients with RV who were followed longitudinally, regression of vasculitic episodes coincided with decreasing titres of anti-endothelial antibodies (AEA). Binding activity to endothelial cells was observed in intact IgG and F(ab')2 fragments of IgG. AEA activity was unrelated to antibodies against nuclear, blood group or major histocompatibility complex antigens and did not involve immune complexes. AEA activity was not specific for endothelial cells since the AEA-positive sera and the IgG fractions prepared from these sera also reacted with fibroblasts. Adsorption of positive sera and corresponding IgG fractions with endothelial cells decreased the IgG binding reactivity on both fibroblasts and endothelial cells. These findings show that RV patients have IgG-AEA, and suggest that these antibodies may play a role in the pathogenesis of the disease. PMID:2805426

  2. Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity.

    PubMed

    Hellmert, Marco; Müller-Schiffmann, Andreas; Peters, Max Sena; Korth, Carsten; Schrader, Thomas

    2015-03-14

    Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-terminal epitope of Aβ and the variable regions of the heavy and light chains were cloned as a recombinant protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a small organic β-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform; and experimental evidence was obtained for efficient Ni(2+)-mediated complex formation with the histidine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong increase in affinity towards Aβ. Thus, the specific binding power of recombinant antibody fragments to their β-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic β-sheet binders.

  3. Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

    PubMed

    Zhao, Fengchun; Tian, Yuan; Wang, Huimin; Liu, Jiye; Han, Xiao; Yang, Zhengyou

    2016-09-01

    Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides. PMID:27411546

  4. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    PubMed

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules. PMID:24492300

  5. Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

    PubMed Central

    Bhattacharyya, Sanchari; Singh, Pranveer; Rathore, Ujjwal; Purwar, Mansi; Wagner, Denise; Arendt, Heather; DeStefano, Joanne; LaBranche, Celia C.; Montefiori, David C.; Phogat, Sanjay; Varadarajan, Raghavan

    2013-01-01

    b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals. PMID:23430741

  6. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A. N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  7. An integrated top-down and bottom-up proteomic approach to characterize the antigen binding fragment of antibodies

    SciTech Connect

    Dekker, Leendert J.; Wu, Si; vanDuijn, Martijn M.; Tolic, Nikola; Stingl, Christoph; Zhao, Rui; Luider, Theo N.; Pasa-Tolic, Ljiljana

    2014-05-31

    We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens towards which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step towards the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

  8. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  9. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  10. Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

    PubMed

    Rülker, Torsten; Voß, Luzie; Thullier, Philippe; O' Brien, Lyn M; Pelat, Thibaut; Perkins, Stuart D; Langermann, Claudia; Schirrmann, Thomas; Dübel, Stefan; Marschall, Hans-Jürgen; Hust, Michael; Hülseweh, Birgit

    2012-01-01

    Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE. PMID:22666347

  11. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

    PubMed

    Akbari, Vajihe; Sadeghi, Hamid Mir Mohammad; Jafarian-Dehkordi, Abbas; Abedi, Daryoush; Chou, C Perry

    2015-12-01

    A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications.

  12. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354.

    PubMed

    Kaufmann, Bärbel; Vogt, Matthew R; Goudsmit, Jaap; Holdaway, Heather A; Aksyuk, Anastasia A; Chipman, Paul R; Kuhn, Richard J; Diamond, Michael S; Rossmann, Michael G

    2010-11-01

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  13. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    SciTech Connect

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  14. Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa.

    PubMed Central

    Christopoulos, C

    1994-01-01

    Platelet surface immunoglobulin G (PSIgG) was measured ex vivo in nine patients with stable angina pectoris receiving continuous (48-96 h) infusions of Fab fragments of a chimaeric MoAb (human IgG with murine variable regions) to platelet glycoprotein IIb-IIIa. PSIgG was measured using flow cytometry (FC) and an Fc-specific anti-IgG polyclonal antibody, which did not cross-react with the chimaeric Fab fragment (c7E3-Fab). A variable but statistically significant (P < 0.05) elevation of PSIgG was present within 24 h after the onset of the infusion, and was more marked (P < 0.01) several days after the end of the infusion despite an exponential fall in platelet surface c7E3-Fab post-infusion. PSIgG returned to normal within 2 weeks after the end of the infusion. The timing of IgG recruitment to the platelet surface suggested the pre-existence in the patients' plasma of IgG binding to c7E3-Fab-bearing platelets. None of the patients developed thrombocytopenia. In order to assess the incidence of IgG bindable to c7E3-Fab-bearing platelets in controls clinically comparable to the c7E3-Fab infusion patients, normal platelets coated with either chimaeric (c) or murine (m) 7E3-Fab were incubated with plasmas from 21 patients with ischaemic heart disease, and recruitment of IgG to the platelet surface was measured by FC. Fourteen of the 21 plasmas contained IgG bindable to c7E3-Fab-coated platelets, whereas only one of the 21 plasmas contained IgG bindable to m7E3-Fab-coated platelets (a highly significant difference, P < 0.001). These findings indicate that infusions of Fab fragments of the chimaeric anti-platelet antibody 7E3 are often associated with elevations in PSIgG, which are probably due to pre-existing 'naturally occurring' antibodies to the Fab fragments of chimaeric (but not murine) 7E3, and most probably other chimaeric MoAbs. The possible clinical significance of such ex vivo measured activities is at present a matter for speculation, and requires further

  15. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    PubMed

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  16. Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

    PubMed Central

    Hur, Byung-ung; Choi, Hyo-jung; Yoon, Jae-bong

    2010-01-01

    Background Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize VH and VL fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of VH or VL domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli. PMID:20532123

  17. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    PubMed

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

  18. Investigation of Fragment Antibody Stability and Its Release Mechanism from Poly(Lactide-co-Glycolide)-Triacetin Depots for Sustained-Release Applications.

    PubMed

    Chang, Debby P; Garripelli, Vivek Kumar; Rea, Jennifer; Kelley, Robert; Rajagopal, Karthikan

    2015-10-01

    Achieving long-term drug release from polymer-based delivery systems continues to be a challenge particularly for the delivery of large hydrophilic molecules such as therapeutic antibodies and proteins. Here, we report on the utility of an in situ-forming and injectable polymer-solvent system for the long-term release of a model antibody fragment (Fab1). The delivery system was prepared by dispersing a spray-dried powder of Fab1 within poly(lactide-co-glycolide) (PLGA)-triacetin solution. The formulation viscosity was within the range 1.0 ± 0.3 Pa s but it was injectable through a 27G needle. The release profile of Fab1, measured in phosphate-buffered saline (PBS), showed a lag phase followed by sustained-release phase for close to 80 days. Antibody degradation during its residence within the depot was comparable to its degradation upon long-term incubation in PBS. On the basis of temporal changes in surface morphology, stiffness, and depot mass, a mechanism to account for the drug release profile has been proposed. The unprecedented release profile and retention of greater than 80% of antigen-binding capacity even after several weeks demonstrates that PLGA-triacetin solution could be a promising system for the long-term delivery of biologics. PMID:26099467

  19. Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-alpha toxin antibody fragment and alpha toxin

    PubMed Central

    Oganesyan, Vaheh; Barnes, Arnita; Tkaczyk, Christine; Ferguson, Andrew; Wu, Herren; Dall’Acqua, William F.

    2013-01-01

    Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P21, with unit-cell parameters a = 85.52, b = 148.50, c = 93.82 Å, β = 99.82°. The diffraction of the crystals extended to 2.56 Å resolution. The asymmetric unit contained two MEDI4893 Fab–AT complexes. This corresponds to a crystal volume per protein weight (V M) of 2.3 Å3 Da−1 and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time. PMID:23519809

  20. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    PubMed

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  1. Investigation of Fragment Antibody Stability and Its Release Mechanism from Poly(Lactide-co-Glycolide)-Triacetin Depots for Sustained-Release Applications.

    PubMed

    Chang, Debby P; Garripelli, Vivek Kumar; Rea, Jennifer; Kelley, Robert; Rajagopal, Karthikan

    2015-10-01

    Achieving long-term drug release from polymer-based delivery systems continues to be a challenge particularly for the delivery of large hydrophilic molecules such as therapeutic antibodies and proteins. Here, we report on the utility of an in situ-forming and injectable polymer-solvent system for the long-term release of a model antibody fragment (Fab1). The delivery system was prepared by dispersing a spray-dried powder of Fab1 within poly(lactide-co-glycolide) (PLGA)-triacetin solution. The formulation viscosity was within the range 1.0 ± 0.3 Pa s but it was injectable through a 27G needle. The release profile of Fab1, measured in phosphate-buffered saline (PBS), showed a lag phase followed by sustained-release phase for close to 80 days. Antibody degradation during its residence within the depot was comparable to its degradation upon long-term incubation in PBS. On the basis of temporal changes in surface morphology, stiffness, and depot mass, a mechanism to account for the drug release profile has been proposed. The unprecedented release profile and retention of greater than 80% of antigen-binding capacity even after several weeks demonstrates that PLGA-triacetin solution could be a promising system for the long-term delivery of biologics.

  2. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    SciTech Connect

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  3. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

  4. A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

    PubMed Central

    Gilbertson, Debra G; Frank, Amanda; Vu, Tuyen; Ardourel, Dan; Ostrander, Craig; Stevens, Brenda; Julien, Susan; Franke, Secil; Meengs, Brent; Brody, Jennifer; Presnell, Scott; Hamacher, Nels B; Lantry, Megan; Wolf, Anitra; Bukowski, Tom; Rosler, Robert; Yen, Cindy; Anderson-Haley, Monica; Brasel, Kenneth; Pan, Qi; Franklin, Hank; Thompson, Penny; Dodds, Mike; Underwood, Sara; Peterson, Scott; Sivakumar, Pallavur V; Snavely, Mark

    2010-01-01

    Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules. PMID:20065654

  5. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage.

    PubMed

    Cai, Binggang; Wang, Maorong; Zhu, Xuhui; Xu, Jing; Zheng, Wenkai; Zhang, Yiqing; Zheng, Feng; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  6. A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

    PubMed Central

    Lee, Hyunwook; Brendle, Sarah A.; Bywaters, Stephanie M.; Guan, Jian; Ashley, Robert E.; Yoder, Joshua D.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.

    2014-01-01

    ABSTRACT Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used

  7. Bifunctional antibody retargeting in vivo-activated T lymphocytes: simplifying clinical application.

    PubMed

    Chapoval, A I; Nelson, H; Thibault, C; Penna, C; Dean, P

    1995-12-01

    For antitumor x anti-CD3 bifunctional antibody (BFA) therapy to be clinically relevant in solid tumors, activated lymphocytes must be present within tumors. Toward that end, three uniquely different in vivo activation approaches were investigated in a p97 human antigen expressing syngeneic murine melanoma model. beta-Glucan (200 micrograms), staphylococcal enterotoxin B (SEB) (50 micrograms), and F(ab')2 BFA (10 micrograms) were tested for their ability to activate lymphocytes, neutralize pulmonary metastases, and treat established tumors. Systemic activation, measured as the ability of splenocytes to lyse tumor cells in vitro in the presence of BFA, was enhanced by the in vivo administration of SEB but not by beta-glucan or F(ab')2 BFA. Despite lacking a systemic effect, F(ab')2 BFA increased both direct and BFA-mediated cytotoxicity in fresh tumor infiltrating lymphocytes. beta-Glucan did not increase systemic or intratumor T cell activation. However, it significantly enhanced the ability of splenocytes to lyse NK-sensitive YAC-1 cells. When tested in a pulmonary metastases model, all three forms of immune modulation combined with F(ab')2 BFA significantly reduced the number of metastases. BFA were more effective at tumor neutralization when combined with SEB compared with adoptively transferred, in vitro-activated splenocytes. These studies demonstrate that immune modulators when combined with F(ab')2 BFA can provide effective antitumor therapy. Several clinical obstacles may be overcome by the application of these reagents. PMID:8846018

  8. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  9. Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

    PubMed

    Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

    2016-09-01

    The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine

  10. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    PubMed

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.

  11. Differential response of human thymus cells to CD2 antibodies: fragmentation of DNA of CD45RO+ and proliferation of CD45RO- subsets.

    PubMed Central

    Li, J; Campbell, D; Hayward, A R

    1992-01-01

    Human thymocytes bearing the CD45RO 'memory' cell phenotype do not proliferate in concanavalin A (Con A)-stimulated cultures and may be destined for intrathymic death. To determine whether this subset would exhibit characteristics of programmed cell death (apoptosis), we examined the integrity of the nuclear DNA by gel electrophoresis. DNA fragmentation was restricted to the CD45RO+ subset of human thymocytes following exposure to stimulating concentrations of anti-CD2 antibodies. Both CD45RO- and CD45RO+ subsets mobilized cytoplasmic Ca2+ following cell-surface CD2 ligation, but entry into the cell cycle and vigorous thymidine uptake were restricted to the CD45RO- subset. Our results provide a mechanism which may account for the failure of thymic CD45RO+ cells to respond to stimuli which elicit proliferation by the reciprocal CD45RA+ subset. Images Figure 1 Figure 2 PMID:1348052

  12. A case of Mobitz type II atrioventricular block due to Nerium oleander poisoning successfully managed with digoxin-specific Fab antibody fragments.

    PubMed

    Tatlısu, Mustafa Adem; Çekirdekçi, Elif İjlal; Akyüz, Şükrü; Nurkalem, Zekeriya

    2015-10-01

    Nerium oleander is a popular ornamental plant grown in many tropical and subtropical countries and in the Mediterranean region. It is dangerous because it has been shown to contain several types of cardiac glycosides, and hence can cause cardiac arrhythmias resembling digoxin in their toxicologic manifestations. We report a patient presenting to our hospital with Mobitz type II atrioventricular block after drinking herbal tea prepared from oleander leaves. Three hours after admission, a 200-mg empiric dose of digoxin-specific Fab antibody fragments was administered intravenously over 30 minutes. A 12-lead electrocardiogram (ECG) revealed sinus rhythm at the end of infusion. After 72 hours, the patient was discharged without any symptoms. PMID:26536992

  13. Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study.

    PubMed

    Grinstead, Jeffrey S; Schuman, Jason T; Campbell, A Patricia

    2003-12-01

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast

  14. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    PubMed Central

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  15. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    PubMed

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.

  16. High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

    PubMed

    Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

    2014-12-01

    Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies. PMID:25239038

  17. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    PubMed Central

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  18. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    PubMed

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  19. [Focus on digitalis intoxication in the elderly. Report of a case treated with digoxin-specific Fab antibody fragments].

    PubMed

    Pautas, Éric; Lopez, Clara; Gouronnec, Adeline; Gravelaine, Sophie; Peyron, Isabelle; Lapostolle, Frédéric

    2012-12-01

    The indications for digoxin are currently limited to rare cases of heart failure and/or atrial fibrillation. Its use should be even more rare in geriatrics its pharmacological characteristics, associated with age-related changes and comorbidities, particularly increase the risk of digoxin poisoning in the elderly. However, at least a third of aged patients suffering from heart failure and/or atrial fibrillation is treated by digitalis. Digoxin intoxication can provoke gastrointestinal troubles, neurological disturbances and, above all, cardiac conduction impairment and dysrythmias, which explain its severity and high mortality rate. Presently, first-line therapy is the administration of digoxin specific antibodies. Poor prognosis factors, frequently found in digoxin intoxications in the elderly, have been established for guiding the prescription of antibodies and their dosage. It is important for geriatricians to be able to recognize poisoning signs and the conditions in which an antidote treatment is necessary. This will permit a more effective management of the case, with the support of a poison control center and possible referral of the patient to an intensive care unit.

  20. Astatine-211 labeling of an anti-melanoma antibody and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate : comparisons In Vivo with the para-[{sup 125}1]iodobenzoyl conjugate.

    SciTech Connect

    Hadley, S. W.; Wilbur, D. S.; Gray, M. A.; Atcher, R. W.; Chemistry; NeoRx Corp.; Univ. of Washington Medical Center

    1991-01-01

    Astatine-211 labeling of an anti-melanoma antibody, NR-ML-05, and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [{sup 211}At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[{sup 125}I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

  1. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.

    PubMed

    Goncalvez, Ana P; Men, Ruhe; Wernly, Claire; Purcell, Robert H; Lai, Ching-Juh

    2004-12-01

    Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of

  2. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  3. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    SciTech Connect

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  4. Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment.

    PubMed

    Zhen, Le; Ford, Nicole; Gale, Debra K; Roesijadi, Guritno; Rorrer, Gregory L

    2016-05-15

    A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene).

  5. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    SciTech Connect

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji ); Kawaminami, Shunro )

    1991-07-02

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C{sub H}1 domain. A variety of Fv analogues labeled with {sup 2}H in the aromatic rings and with {sup 13}C and/or {sup 15}N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with {sup 1}H and {sup 13}C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the {sup 1}H-{sup 15}N shift correlation spectra. Use of the {beta}-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.

  6. Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei.

    PubMed

    Colombi, Débora; Oliveira, Maria L S; Campos, Ivana B; Monedero, Vicente; Pérez-Martinez, Gaspar; Ho, Paulo L

    2006-12-01

    Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough. PMID:17106803

  7. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  8. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

  9. Soluble, but not immobilized, anti-IgM antibody inhibits post-activation events leading to T-cell-dependent B-cell differentiation.

    PubMed Central

    Zamorano, J; Rivas, D; Gayo, A; Mozo, L; Gutiérrez, C

    1995-01-01

    The potential for surface immunoglobulin-binding ligands to modify B-cell differentiation responses induced by activated T cells has been investigated. Activated T cells in human splenic mononuclear cells cultured on anti-CD3-coated plates induced B cells to produce large amounts of IgM and IgG. In this experimental system, cross-linking of B-cell antigen receptors by soluble, bivalent monoclonal or polyclonal anti-IgM antibodies completely inhibited IgM production, and greatly diminished IgG production, in a dose-dependent manner. Similar results were obtained using a F(ab')2 fragment of a goat anti-IgM antibody. Inhibition of B-cell differentiation by bivalent cross-linking reagents did not require the presence of antigen-presenting cells (APC), as comparable results were obtained in co-cultures of purified T and B cells. In contrast, enhanced immunoglobulin secretion was seen when surface IgM was cross-linked using anti-IgM antibody immobilized on the culture plate. Interestingly, activated T cells induced similar levels of expression on B cells of the activation antigens CD23, CD25 and CD71, and of class II molecules, irrespective of any treatment with soluble or immobilized anti-IgM antibody. This indicates that soluble anti-IgM specifically inhibits B-cell differentiation without altering initial events of T-cell-dependent B-cell activation. Images Figure 3 Figure 5 PMID:7642212

  10. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  11. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-01

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties. PMID:27007881

  12. Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

    PubMed

    Maruta, Yasuhiro; Kuwata, Takeo; Tanaka, Kazuki; Alam, Muntasir; Valdez, Kristel Paola Ramirez; Egami, Yoshika; Suwa, Yoshiaki; Morioka, Hiroshi; Matsushita, Shuzo

    2016-09-21

    The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

  13. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  14. Synthesis and pre-clinical evaluation of an (18)F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer.

    PubMed

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an (18)F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of (18)F-labeled scFv-B43.13 ([(18)F]FBz-scFv-B43.13) was studied with PET. [(18)F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  15. Synthesis and pre-clinical evaluation of an 18F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer

    PubMed Central

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an 18F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of 18F-labeled scFv-B43.13 ([18F]FBz-scFv-B43.13) was studied with PET. [18F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  16. A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments.

    PubMed

    Nanaware-Kharade, Nisha; Thakkar, Shraddha; Gonzalez, Guillermo A; Peterson, Eric C

    2015-07-10

    To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (± 0.3) to 26 (± 2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles.

  17. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    PubMed

    Pereira, Soraya S; Moreira-Dill, Leandro S; Morais, Michelle S S; Prado, Nidiane D R; Barros, Marcos L; Koishi, Andrea C; Mazarrotto, Giovanny A C A; Gonçalves, Giselle M; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Pereira da Silva, Luiz H; Duarte dos Santos, Claudia N; Fernandes, Carla F C; Stabeli, Rodrigo G

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus

  18. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

    PubMed

    Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

    2008-07-22

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

  19. Suppression of natural killer cell activity by rabbit antibody to human beta 2-microglobulin (beta 2m) is an Fc-mediated phenomenon and is not beta 2m specific.

    PubMed

    Jones, R A; Richards, S J; Patel, D; Scott, C S

    1991-07-01

    Natural killer (NK) cell cytotoxicity constitutes an important component of the host immune defence system. The NK effector cell has been relatively well defined in terms of immunophenotypic characteristics, but in contrast to the functional T-cell receptor molecule associated with major histocompatibility complex (MHC)-restricted cytotoxic activity, the NK cell receptor has not to date been defined. However, several studies have suggested that the beta 2-microglobulin (beta 2m) molecule is functionally associated with NK cell activity. Using various heterospecific and monoclonal antibodies, this study has shown that intact rabbit IgG antibody bound either directly or indirectly to peripheral mononuclear cell (PMNC) effector populations significantly reduced their lytic activity against K562 targets. Substitution of F(ab)2 fragments for rabbit IgG antibodies, or the use of monoclonal antibodies alone, failed to reduce peripheral blood mononuclear cell (PMNC) lytic activity. Addition of non-NK cell components labelled with rabbit anti-beta 2m to purified NK-enriched effector cell populations also suppressed K562 lysis. In contrast, pre-treatment of a NK-enriched PMNC fraction with rabbit anti-beta 2m enhanced target lysis. These results strongly suggest that antibody-induced suppression of PMNC NK activity is mediated via rabbit Fc attached to co-existing non-NK cells in the mononuclear fraction, and are inconsistent with the previously suggested functional association between NK activity and membrane beta 2m.

  20. Improved pharmacokinetics of Yttrium-90 delivery with multivalent Fab{prime} fragments

    SciTech Connect

    Turner, A.; Boyce, B.A.; Antoniw, P.

    1994-05-01

    Humanised monoclonal antibodies have circulating half-lives in man of many days and one of the problems in using Yttrium-90 labelled versions for tumor therapy is the level of consequent whole body radiation. F(ab`){sub 2} fragments are rapidly cleared from the circulation but have relatively poor tumor uptake in biodistribution experiments. We initially developed a chemically cross-linked F(ab`)2 species (divalent Fab` maleimide or DFM) capable of carrying Yttrium-90 complexed to a 12N4 macrocycle. Although the DFM derived from the Fab` of cB72.3 had the desired pharmacokinetics of blood clearance with good tumor uptake in athymic nude mice carrying human tumor xenografts, significant retention of radioactivity by the kidneys was observed. We then progressed to higher oligomers and found that the trivalent Fab` maleimide (TFM), obtained via the structure below (n=3), when labelled with Y-90 possessed acceptable pharmacokinetics and good tumor uptake without kidney accumulation of radioactivity. The clinical evaluation of this technology is in progress.

  1. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

  2. Antibody fragment-armed mesoporous silica nanoparticles for the targeted delivery of bevacizumab in ovarian cancer cells.

    PubMed

    Zhang, Ying; Guo, Jing; Zhang, Xiao-Ling; Li, Da-Peng; Zhang, Ting-Ting; Gao, Fu-Feng; Liu, Nai-Fu; Sheng, Xiu-Gui

    2015-12-30

    In order to enhance the therapeutic efficacy and intracellular concentration of bevacizumab (BVC), we have designed a novel tumor endothelial marker 1 (TEM1)/endosialin (Ab-/scFv)-conjugated mesoporous silica nanoparticles (MSN) to target ovarian cancer cell. The Ab-/scFv-conjugated MSN were prepared by the conjugation of amine functional group of antibody of the carboxyl group of MSN. The resultant MSN was nanosized, spherical shaped, and exhibited a controlled release phenomenon in pH 7.4 conditions. Furthermore, BMSN/Ab was found to increase the cellular uptake and intracellular distribution of BVC in OVCAR-5 cancer cells. The Ab- conjugated MSN exhibited a superior anticancer effect with profound apoptosis in cancer cells in a time- and concentration dependent manner. Consistently, BMSN/Ab effectively inhibited the colony formation in transwell plate. Finally, BMSN/Ab showed a notable increase in the proportion of cells in G2/M phase of cell cycle indicating promising anticancer efficacy profile. Overall, Ab-/scFv-conjugated MSN might provide an effective strategy for the therapeutic management of ovarian cancers.

  3. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

    PubMed

    Schmidt, Florian I; Lu, Alvin; Chen, Jeff W; Ruan, Jianbin; Tang, Catherine; Wu, Hao; Ploegh, Hidde L

    2016-05-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci. PMID:27069117

  4. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    PubMed

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases. PMID:25447207

  5. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future. PMID:27329045

  6. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.

  7. Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors.

    PubMed

    Cheng, Ming; Santich, Brian H; Xu, Hong; Ahmed, Mahiuddin; Huse, Morgan; Cheung, Nai-Kong V

    2016-06-01

    Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.

  8. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    PubMed

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  9. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    PubMed

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  10. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    PubMed

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents. PMID:25994015

  11. Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors

    PubMed Central

    Cheng, Ming; Santich, Brian H.; Xu, Hong; Ahmed, Mahiuddin; Huse, Morgan; Cheung, Nai-Kong V.

    2016-01-01

    ABSTRACT Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca2+ flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity. PMID:27471647

  12. Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

    PubMed

    Kramski, Marit; Lichtfuss, Gregor F; Navis, Marjon; Isitman, Gamze; Wren, Leia; Rawlin, Grant; Center, Rob J; Jaworowski, Anthony; Kent, Stephen J; Purcell, Damian F J

    2012-10-01

    Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission. PMID:22730083

  13. Lung injury mediated by antibodies to endothelium. I. In the rabbit a repeated interaction of heterologous anti-angiotensin-converting enzyme antibodies with alveolar endothelium results in resistance to immune injury through antigenic modulation

    PubMed Central

    1983-01-01

    To study the effects of relatively long-term interaction of antibodies with surface antigens of lung endothelium, rabbits were intravenously injected for a maximum of 4 d with goat anti-rabbit lung angiotensin- converting enzyme (Gt anti-RbACE) antibodies. On day 1 69%, on day 2 13%, and on days 3 and 4 of injection none of the rabbits developed lethal pulmonary edema. By immunofluorescence microscopy, deposits of GtIgG, frequently in association with RbC3, were found along the endothelium of alveolar capillary walls in all rabbits studied on day 1, in 57% on day 2, in 33% on day 3, and in none of them on day 4. While in vitro anti-ACE antibodies bound in a linear pattern to the lung endothelium, the binding pattern in vivo was distinctly granular. The in vivo interaction of antibodies with ACE also redistributed ACE in a granular pattern along capillary walls. In contrast to the granular deposition of injected anti-ACE IgG and F(ab')2 fragments of anti-ACE IgG, Fab fragments of anti-ACE IgG localized, without fixing C3, in a linear pattern along the endothelium of lung capillaries and did not modify the normal distribution of ACE. However, when the injection of Fab fragments of Gt anti-RbACE IgG was followed by an injection of Rb anti-GtIgG serum, granular deposits of Gt Fab fragments, RbIgG and RbC3 were seen along alveolar capillary walls. Biochemical measurement of ACE activity in lung homogenates provided data in agreement with those obtained by immunofluorescence microscopy, showing diminished activity to none on day 4, with some return of ACE activity on day 5, 24 h after the last injection of antibody, and normal values on day 21. The results obtained indicate that divalent antibodies to an antigen expressed on the plasma membrane of rabbit lung endothelial cells promotes a rapid redistribution of antigenic receptors, fixation of complement and, in surviving rabbits, disappearance of the antigen from the endothelial cells that are no longer susceptible to

  14. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  15. Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments.

    PubMed

    Opperman, Pamela A; Maree, Francois F; Van Wyngaardt, Wouter; Vosloo, Wilna; Theron, Jacques

    2012-08-01

    Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.

  16. An ultra scale-down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli.

    PubMed

    Li, Qiang; Mannall, Gareth J; Ali, Shaukat; Hoare, Mike

    2013-08-01

    Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab') and the impact of fermentation harvest time. This approach is followed by microwell-based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high-pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab' release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale.

  17. Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.

    PubMed

    Isidro, Inês A; Portela, Rui M; Clemente, João J; Cunha, António E; Oliveira, Rui

    2016-09-01

    Despite the growing importance of the Pichia pastoris expression system as industrial workhorse, the literature is almost absent in systematic studies on how culture medium composition affects central carbon fluxes and heterologous protein expression. In this study we investigate how 26 variations of the BSM+PTM1 medium impact central carbon fluxes and protein expression in a P. pastoris X-33 strain expressing a single-chain antibody fragment. To achieve this goal, we adopted a hybrid metabolic flux analysis (MFA) methodology, which is a modification of standard MFA to predict the rate of synthesis of recombinant proteins. Hybrid MFA combines the traditional parametric estimation of central carbon fluxes with non-parametric statistical modeling of product-related quantitative or qualitative measurements as a function of central carbon fluxes. It was observed that protein yield variability was 53.6 % (relative standard deviation) among the different experiments. Protein yield is much more sensitive to medium composition than biomass growth, which is mainly determined by the carbon source availability and main salts. Hybrid MFA was able to describe accurately the protein yield with normalized RMSE of 6.3 % over 5 independent experiments. The metabolic state that promotes high protein yields is characterized by high overall metabolic rates through main central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy generating pathways. PMID:27129458

  18. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  19. The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6.

    PubMed

    Phillips, Damilola D; Garboczi, David N; Singh, Kavita; Hu, Zonglin; Leppla, Stephen H; Leysath, Clinton E

    2013-08-01

    The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  20. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  1. Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

    PubMed

    Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

    2010-01-01

    Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb. PMID:19597742

  2. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    PubMed

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  3. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv).

    PubMed

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G

    1997-09-15

    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  4. Astatine-211 labeling of an antimelanoma antibody and its Fab fragment using N-succinimidyl p-astatobenzoate: comparisons in vivo with the p-[125I]iodobenzoyl conjugate.

    PubMed

    Hadley, S W; Wilbur, D S; Gray, M A; Atcher, R W

    1991-01-01

    Astatine-211 labeling of an antimelanoma antibody, NR-ML-05, and its Fab fragment with N-succinimidyl p-[211At]astatobenzoate (2a) has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 in the reaction mixture. The dry distillation technique employed gave recoveries of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [211At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[125I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

  5. Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2).

    PubMed

    Perri, R T; Wilson, B S; Kay, N E

    1986-04-01

    Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states. PMID:2938967

  6. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  7. High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.

    PubMed

    Gurkan, Cemal; Symeonides, Stefan N; Ellar, David J

    2004-02-01

    The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors. PMID:12962542

  8. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    PubMed Central

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  9. In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

    PubMed

    Nabuurs, Rob J A; Rutgers, Kim S; Welling, Mick M; Metaxas, Athanasios; de Backer, Maaike E; Rotman, Maarten; Bacskai, Brian J; van Buchem, Mark A; van der Maarel, Silvère M; van der Weerd, Louise

    2012-01-01

    This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits. PMID:22675537

  10. In Vivo Detection of Amyloid-β Deposits Using Heavy Chain Antibody Fragments in a Transgenic Mouse Model for Alzheimer's Disease

    PubMed Central

    Welling, Mick M.; Metaxas, Athanasios; de Backer, Maaike E.; Rotman, Maarten; Bacskai, Brian J.; van Buchem, Mark A.; van der Maarel, Silvère M.; van der Weerd, Louise

    2012-01-01

    This study investigated the in vivo properties of two heavy chain antibody fragments (VHH), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled VHH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled VHH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All VHH showed rapid renal clearance (10–20 min). Twenty-four hours post-injection 99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for 99mTc-ni3A or DTPA(111In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled VHH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both VHH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that VHH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits. PMID:22675537

  11. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    PubMed

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  12. Effects of digoxin-specific antibody Fab fragment (Digibind) on blood pressure and renal water-sodium metabolism in 5/6 reduced renal mass hypertensive rats.

    PubMed

    Kaide, J; Ura, N; Torii, T; Nakagawa, M; Takada, T; Shimamoto, K

    1999-06-01

    The importance of increased endogenous digitalis-like factor (EDLF) in volume-expanded hypertension has been generally agreed. To further clarify the role of EDLF on the development of hypertension and renal water-sodium handling in 5/6 reduced renal mass hypertensive rats (RRM), we studied the effects of acute administration of digoxin-specific antibody Fab fragment (Digibind) in the early phase and the chronic phase of hypertension in RRM. RRM and sham-operated rats were given 1% saline for 1 or 4 weeks. RRM were injected Digibind (60 mg/kg) or vehicle (0.9% saline) intravenously in the first or fourth week under thiobutabarbital anesthesia. All sham-operated rats were administered Digibind under the same condition. Digibind altered neither blood pressure, heart rate, urine volume, nor urinary sodium excretion in sham-operated rats. However, Digibind produced a gradual but significant decline in mean arterial pressure to the level slightly above that in sham-operated rats from 153 +/- 5 to 131 +/- 5 mm Hg in the first week and from 181 +/- 6 to 129 +/- 4 mm Hg in the fourth week without any significant change in heart rate. The decrease in mean arterial pressure at 160 min after Digibind administration in the fourth week (-48 +/- 5 mm Hg) was greater than that in the first week (-22 +/- 4 mm Hg). No differences were observed in urine volume, urinary sodium excretion, or plasma norepinephrine concentration between Digibind and vehicle-treated RRM in either week. These data suggest that EDLF would contribute to both the early and chronic phase in the development of hypertension in RRM.

  13. Anti-CD3 induced thymocyte apoptosis in vivo require the antibody Fc domain.

    PubMed

    Xue, Y; Castanos-Velez, E; Biberfeld, P; Jondal, M

    2000-05-01

    We have earlier found that explanted thymic epithelial cells (TEC) can produce glucocorticoid (GC) activity in vitro and that the GC receptor (GR) antagonist RU486 partially inhibit thymic apoptosis induced by the anti-CD3 monoclonal antibodies (MoAb) 2C11, both in vivo and in new-born thymic organ cultures. To explain the inhibitory effect of RU486 in this system we have now investigated the importance of the 2C11 Fc as this MoAb bind with high affinity to cellular FcR. We have found both that whole 2C11 MoAb can bind to explanted TEC in vitro and that F(ab)'2 fragments from this MoAb loose this ability, in addition with the capacity to induce thymic apoptosis in vivo. We interpret our results to indicate that the injected 2C11 MoAb may establish a close contact between GC producing, FcR positive TEC cells and CD3 positive thymocytes and thereby subject the later to high paracrine GC concentrations and subsequent induction of apoptosis.

  14. Immunoscintigraphy with indium-111 labeled monoclonal antibodies: The importance of a good display method

    SciTech Connect

    Liehn, J.C.; Hannequin, P.; Nasca, S.; Lebrun, D.; Fernandez-Valoni, A.; Valeyre, J. )

    1989-03-01

    A major drawback of In-111-labeled monoclonal antibodies (MoAb) is the presence of intense liver, renal, and bone marrow nonspecific activity. This makes the display of the images hardly optimal and their visual interpretation difficult. In this study, the intrinsic color scale (which consists of selecting the limits of the color scale as the highest and the lowest pixel value of the image) was compared to a new, simple algorithm for the determination of the limits of the color scale. This algorithm was based on the count density in the iliac crest areas. OC-125 or anti-CEA In-111 MoAb F(ab')2 fragments were used in 32 patients with suspected recurrence of ovarian (19 patients) or colorectal cancer (13 patients). Final diagnosis was assessed by surgery (21 patients), biopsy (five patients), or followup (six patients). A 10-minute abdomino-pelvic anterior view was recorded two days after injection. These views are displayed using the two methods and interpreted by two observers. Using their responses in each quadrant of the pelvis, the authors calculated two ROC curves. The comparison of the ROC curves showed better performances for the new method. For example, for the same specificity (73%), the sensitivity of the new method was significantly better (78% versus 68%). This result confirmed the importance of a good methodology for displaying immunoscintigraphic images.

  15. Evaluation of the Naturally Acquired Antibody Immune Response to the Pv200L N-terminal Fragment of Plasmodium vivax Merozoite Surface Protein-1 in Four Areas of the Amazon Region of Brazil

    PubMed Central

    Storti-Melo, Luciane M.; Souza-Neiras, Wanessa C.; Cassiano, Gustavo C.; Taveira, Leonardo C.; Cordeiro, Antônio J.; Couto, Vanja S. C. A.; Póvoa, Marinete M.; Cunha, Maristela G.; Echeverry, Diana M.; Rossit, Andréa R. B.; Arévalo-Herrera, Myriam; Herrera, Sócrates; Machado, Ricardo L. D.

    2011-01-01

    Frequency and levels of IgG antibodies to an N-terminal fragment of the Plasmodium vivax MSP-1 (Pv200L) protein, in individuals naturally exposed to malaria in four endemic areas of Brazil, were evaluated by enzyme-linked immunosorbent assay. Plasma samples of 261 P. vivax-infected individuals from communities of Macapá, Novo Repartimento, Porto Velho, and Plácido de Castro in the Amazonian region with different malaria transmission intensities. A high mean number of studied individuals (89.3%) presented with antibodies to the Pv200L that correlated with the number of previous malaria infections; there were significant differences in the frequency of the responders (71.9–98.7) and in the antibody levels (1:200–1:51,200) among the four study areas. Results of this study provide evidence that Pv200L is a naturally immunogenic fragment of the PvMSP-1 and is associated with the degree of exposure to parasites. The fine specificity of antibodies to Pv200L is currently being assessed. PMID:21292879

  16. Studies in the rat of antibody-coated and N-ethylmaleimide-treated erythrocyte clearance by the spleen. II. Effects of immune complex infusion.

    PubMed Central

    Yousaf, N; Howard, J C; Williams, B D

    1986-01-01

    The effects of immune complex infusion on the clearance of antibody (R3/13)-coated and NEM-treated rat erythrocytes by the splenic component of the rat mononuclear phagocyte system (MPS) were investigated. Equivalence complexes of BSA-anti-BSA produced a significant delay in the clearance of the NEM cells (pre-infusion T1/2 19.7 +/- 3.9 min, post-infusion T1/2 26.5 +/- 3.8 min, mean +/- SE, n = 6, P less than 0.01), but this effect could be abolished by agents that prevented the changes in the splenic blood flow that followed complement activation. The immune complexes formed in 10-fold antigen excess (mean size 15S) did not delay the clearance of the NEM cells. Clearance of R3/13-coated cells was impaired by the infusion of immune complexes prepared at equivalence. 10-fold antigen excess or complexes prepared with F(ab')2 fragments of rabbit anti-BSA antibody. The inhibition of red cell clearance was independent of changes in blood flow, but the degree of inhibition produced did not correlate well with the dose of immune complex injected. PMID:3759130

  17. Neutralization of the neuromuscular inhibition of venom and taipoxin from the taipan (Oxyuranus scutellatus) by F(ab')2 and whole IgG antivenoms.

    PubMed

    Herrera, María; de Cássia de O Collaço, Rita; Villalta, Mauren; Segura, Álvaro; Vargas, Mariángela; Wright, Christine E; Paiva, Owen K; Matainaho, Teatulohi; Jensen, Simon D; León, Guillermo; Williams, David J; Rodrigues-Simioni, Léa; Gutiérrez, José María

    2016-01-22

    The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization.

  18. Neutralization of the neuromuscular inhibition of venom and taipoxin from the taipan (Oxyuranus scutellatus) by F(ab')2 and whole IgG antivenoms.

    PubMed

    Herrera, María; de Cássia de O Collaço, Rita; Villalta, Mauren; Segura, Álvaro; Vargas, Mariángela; Wright, Christine E; Paiva, Owen K; Matainaho, Teatulohi; Jensen, Simon D; León, Guillermo; Williams, David J; Rodrigues-Simioni, Léa; Gutiérrez, José María

    2016-01-22

    The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization. PMID:26621539

  19. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab{prime}){sub 2}

    SciTech Connect

    Anderson, C.J.; Schwarz, S.W.; Connett, J.M. ||

    1995-05-01

    Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab{prime}){sub 2} was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2} was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N{prime},N{double_prime},N{prime}{double_prime}-tetraacetic acid (BAT) was conjugated to 1A3-F(ab{prime}){sub 2} using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab{prime}. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab{prime}){sub 2} from TETA-1A3-Fab{prime} after conjugation prior to labeling with {sup 64}Cu. The biodistribution of {sup 64}Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab{prime}){sub 2}, the immunoreactivity of {sup 64}Cu-labeled 1A3-F(ab{prime}){sub 2} was significantly improved over that of non-FPLC-purified {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2}, and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2}.The improved dosimetry of {sub 64}Cu-labeled FPLC purified BAT-2IT-1A3-F(ab{prime}){sub 2} should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs.

  20. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

    PubMed

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2008-08-01

    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  1. Specific Single Chain Variable Fragment (ScFv) Antibodies to Angiotensin II AT2 Receptor: Evaluation of the Angiotensin II Receptor Expression in Normal and Tumor-bearing Mouse Lung

    PubMed Central

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2010-01-01

    Summary To gain insight into the mechanism by which angiotensin II type 2 receptor (AT2) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT2 single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT2 receptor protein. The specificity of the antibodies was verified using AT2 over-expressing COS-7 cells and AT2 naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT2 and AT1 immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis. PMID:18438736

  2. Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

    NASA Astrophysics Data System (ADS)

    Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

    2001-09-01

    To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

  3. Antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.

    1988-06-28

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.

  4. A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation.

    PubMed

    Boukerche, H; Ruchaud-Sparagano, M H; Rouen, C; Brochier, J; Kaplan, C; McGregor, J L

    1996-02-01

    P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M(r)) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIb alpha). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825 +/- 2886, mean +/- SD) Kd = 1.5 +/- 0.5 nM) compared to resting platelets (2801 +/- 1278, mean +/- SD) (Kd = 1.5 +/- 0.6 nM), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab')2 or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell-cell contact, may play an active role in platelet-platelet interactions. PMID:8603015

  5. Pathogenic relevance of IgG and IgM antibodies against desmoglein 3 in blister formation in pemphigus vulgaris.

    PubMed

    Tsunoda, Kazuyuki; Ota, Takayuki; Saito, Masataka; Hata, Tsuyoshi; Shimizu, Atsushi; Ishiko, Akira; Yamada, Taketo; Nakagawa, Taneaki; Kowalczyk, Andrew P; Amagai, Masayuki

    2011-08-01

    Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas anti-Dsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab')(2) fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus.

  6. Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional sup 1 H NMR analyses fo the antigen-antibody interactions

    SciTech Connect

    Takahashi, Hideo; Igarashi, Takako; Shimada, Ichio; Arata, Yoji )

    1991-03-19

    The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire C{sub H}1 domain is deleted. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides {sup 1}H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2{prime}-H and Tyr C3{prime},5{prime}-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansly group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, the authors have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed.

  7. Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli.

    PubMed

    Liu, Aiping; Ye, Yang; Chen, Weifeng; Wang, Xiaohong; Chen, Fusheng

    2015-02-01

    Aflatoxin B1 (AFB1) is a toxic secondary metabolic product, which threatens human and animal health. Antibody is a key factor for immunoassay against toxic stuff like AFB1, and single-chain Fv antibody fragment (scFv) has become a popular format of genetically engineered antibody. In this study, four hybridoma cell lines against AFB1 were obtained, and then scFvs 2E6 derived from hybridoma cell line 2E6 were constructed in different V(H)/V(L) orientations. Subsequently, scFvs 2E6 were expressed in E. coli BL21(DE3) mainly in the form of inclusion body. SDS-PAGE, Western blot and ELISA were employed to characterize scFvs 2E6. The results revealed that the yield of inclusion body of scFvs 2E6 in either V(H)/V(L) orientation was similar; however, only the scFv in V(H)-linker-V(L) orientation showed anti-AFB1 bioactivity after refolding. The present study underscores the importance of choosing optimal V(H)/V(L) orientation for scFv construction, and scFv may be favorable for immunoassays in food industry. PMID:25540048

  8. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

    PubMed

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold

    2002-04-01

    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  9. Tumor immunoscintigraphy by means of radiolabeled monoclonal antibodies: Multicenter studies of the Italian National Research Council--Special Project Biomedical Engineering

    SciTech Connect

    Siccardi, A.G. )

    1990-02-01

    Four radioimmunopharmaceuticals ({sup 99m}Tc- and 111In-labeled anti-melanoma and {sup 111}In- and {sup 131}I-labeled anti-carcinoembryonic antigen F(ab')2 fragments derived from monoclonal antibodies 225.28S and F023C5) were developed by means of a collaborative effort coordinated by the Italian National Research Council, Special Project Biomedical Engineering. After appropriate pilot studies, the radioimmunopharmaceuticals, prepared by Sorin Biomedica (Saluggia, Italy), were distributed to 31 Nuclear Medicine departments in Italy and in 10 other European countries within the framework of three immunoscintigraphy multicenter studies. A total of 1245 patients were studied, 898 of whom carried 1725 documented tumor lesions; 1596 of 2193 tumor lesions (468 of which were previously unknown) were imaged by immunoscintigraphy in 785 of 990 lesion-bearing patients. Among the occult lesions, 173 were imaged in 92 patients admitted to the study as lesion-free patients. The results have been analyzed in terms of the reliability, reproducibility, and diagnostic usefulness of the method and of each immunoradiopharmaceutical.

  10. Novel Human Three-Domain Antibody Fragments Against sTNFα as Well as tmTNFα with High Affinity Generated by the Combination of Ribosome Display and E. coli Expression System.

    PubMed

    Zhao, X-L; Tian, L-F; Zhang, S-J; Li, J-M; Feng, H; Wang, L-M; Wang, S; Wang, J; Wang, T; Chen, W-Q

    2016-04-01

    Human tumour necrosis factor α (hTNFα) has been proved to be a validated therapeutic target in a number of immune-mediated inflammatory diseases (IMIDs). Fully human monoclonal antibodies (mAbs) that can neutralize soluble hTNFα (sTNFα) as well as transmembrane hTNFα (tmTNFα) are more desirable hTNFα antagonists. Here, we report that novel anti-hTNFα human low-molecular-weight MAbs have been selected and identified using both sTNFα and tmTNFα as target antigens by the combination of ribosome display and E. coli expression system for the first time. As a newly born engineering small molecular antibody, three-domain antibody fragment (VH /κ) provides an alternative promising molecular principle to generate biological agents for TNFα-dependent IMIDs. In this study, a panel of novel human VH /κs (F09, F21, F49 and F409) with high affinity (10(-10) -10(-9) mol/l) to neutralize sTNFα as well as tmTNFα was generated by the combination of ribosome display and E. coli expression system. Among the four clones, F21 and F409 could reduce cytotoxicity on L929 cells induced by sTNFα as well as tmTNFα effectively, and both of them had great potential to inhibit hTNFα-mediated NF-κB activation. Soluble F21 and F409 were also able to inhibit the binding of hTNFα to TNFR1 and TNFR2. The new human antibodies described here have desirable capability to neutralize sTNFα as well as tmTNFα effectively with high affinity and reasonable stability; this may provide an alternative approach for patients who are not responding adequately to currently available anti-TNFα agents. PMID:26860639

  11. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

    PubMed

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute

    2011-07-01

    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806. PMID:21592580

  12. Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis - Tandem mass spectrometry as nanoESI infusion platform and separation method.

    PubMed

    Said, Nassur; Gahoual, Rabah; Kuhn, Lauriane; Beck, Alain; François, Yannis-Nicolas; Leize-Wagner, Emmanuelle

    2016-04-28

    Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules. PMID:27046210

  13. Clinical safety of a polyvalent F(ab')2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. VAO (Venin Afrique de l'Ouest) Investigators.

    PubMed

    Chippaux, J P; Lang, J; Eddine, S A; Fagot, P; Rage, V; Peyrieux, J C; Le Mener, V

    1998-01-01

    A large-scale clinical trials was conducted, according to World Health Organization Good Clinical Practice guidelines, in 7 centres in north Cameroon to determine the safety and efficacy of a polyvalent antivenom composed of purified F(ab')2. This study included 223 patients presenting clinically with obvious snake bite, predominantly due to Echis ocellatus (viper), the most abundant species in this savannah region. Clinical surveillance was maintained for 5 d in all patients and until the twenty-sixth day in 74% of cases. Two 10 mL ampoules of polyvalent F(ab')2 equine antivenom (Ipser Africa) were administered to each patient by intravenous infusion. If necessary, treatment was repeated 1 h after the end of the first infusion, and then with a frequency determined by the patient's clinical condition. Before initiation of antivenom treatment, the main clinical disorders observed on admission were oedema (93.7%) and haemorrhage (48.9%), with a clotting time longer than 30 min in 65.4% of patients. Clinical cure was obtained in 213 patients (96.8%). No amputation was necessary, and the case fatality rate was only 1.3%. On average, 4.6 (+/- 3.7) ampoules were administered per patient; 43% of subjects recovered after only a single infusion of 2 ampoules. Early adverse reactions, of varying degrees of severity, were observed in 6.3% of patients. A severe early reaction, anaphylactic shock, was observed in only one patient (0.4%). Serum sickness was observed in another patient. Polyvalent F(ab')2 equine antivenom given by repeated 20 mL intravenous infusions is a safe and effective treatment for envenomation caused by African vipers. PMID:10326114

  14. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

    PubMed Central

    Yan, Jie; Wang, Yuan; Shao, Shi-He; Mao, Ya-Fei; Li, Hua-Wen; Luo, Yi-Hui

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases. METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients’ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed. RESULTS: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients’ serum samples (96/109) were CagA antibody-positive. The

  15. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    PubMed Central

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  16. Effect of tumor mass and antigenic nature on the biodistribution of labeled monoclonal antibodies in mice

    SciTech Connect

    Watanabe, Y.; Endo, K.; Koizumi, M.; Kawamura, Y.; Saga, T.; Sakahara, H.; Kuroki, M.; Matsuoka, Y.; Konishi, J.

    1989-06-01

    The effect of tumor mass and antigenic nature on the biodistribution of 111In- and 125I-labeled monoclonal antibodies (MoAbs) was studied using F(ab')2 fragments of three representative anti-tumor MoAbs and SW1116 human colorectal carcinoma grown in nude mice. The 19-9, F33-104 anti-CEA, and 17-1A MoAbs showed specific binding to SW1116 cells. The former two MoAbs recognize circulating CA 19-9 with molecular weights of more than 5,000,000 and CEA of Mr 170,000-180,000, respectively, whereas 17-1A reacts with a nonshedding antigen. Both percentage injected dose per gram tumor and tumor-to-blood ratios were inversely proportional to the tumor mass in nude mice administered 111In- and 125I-labeled 19-9, but liver uptake increased as tumor size increased. Analysis of serum samples and tumor homogenates demonstrated the presence of a high-molecular-weight species, probably due to the antibody binding to CA 19-9. In the case of 111In-labeled anti-CEA MoAb, tumor uptake also decreased and liver uptake increased with tumor size, but this effect was less obvious than that of 19-9. In contrast, tumor and liver uptake of 125I-labeled anti-CEA MoAb, 111In- and 125I-labeled 17-1A and control antibodies were independent of tumor mass. The absolute tumor uptake and tumor-to-blood ratios of all 125I-labeled antibodies were lower than those of the 111In-labeled ones. And the effect of tumor mass was also weaker with 125I-labeled antibodies, probably due to in vivo dehalogenation. These results indicate that the effect of tumor size on the incorporation of labeled MoAb into tumors is dependent on the antigenic nature to be targeted and/or radionuclides used for labeling and that high concentrations of circulating high molecular weight antigens may limit in vivo use of MoAb conjugates.

  17. Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2.

    PubMed

    Zeytin, H E; Tripathi, P K; Bhattacharya-Chatterjee, M; Foon, K A; Chatterjee, S K

    2000-11-01

    Anti-idiotype antibody, 1A7, functionally mimics the tumor-associated antigen disialoganglioside GD2, which is overexpressed on the surface of a number of neuroectodermal tumors such as melanoma, neuroblastoma, soft tissue sarcoma, and small cell carcinoma of the lung. Immunization of mice with 1A7 generated the production of anti-GD2 antibodies. In a phase I clinical trial, immunization of patients with 1A7, mixed with the adjuvant QS21, demonstrated that 1A7 could act as a surrogate antigen for GD2 and induce strong humoral immune responses in advanced stage melanoma patients. DNA vaccines have recently been shown to invoke humoral as well as cellular responses in injected hosts against the transgene product. To evaluate the efficiency of DNA vaccines encoding anti-idiotype antibodies, we constructed expression plasmids encoding the variable heavy (VH) and variable light (VL) chains of 1A7. The plasmids were made in two configurations, expressing either the VH (pc1A7VHLnVL) or the VL (pc1A7VLLnVH) chain of 1A7 at the amino terminus, linked together by a 15-amino acid linker (Ln). In vitro transcription/translation assays and transfection of CHO-K1 cells with the plasmids demonstrated that a approximately 30-kDa protein was expressed by both configurations of the single-chain variable fragment. This protein can be specifically precipitated by monoclonal anti-GD2 antibody, 14G2a. Following intramuscular injection in mice, the plasmids were detectable in the injected tissues for at least 3 months and the injected plasmids actively transcribed the single-chain variable fragment 1A7 gene at the injected site. A single, intramuscular immunization of a group of C57BL/6 mice with pc1A7VLLnVH in phosphate-buffered saline induced humoral immune responses against 1A7 as well as GD2, the nominal antigen. Multiple immunizations, however, were required to elicit stronger immune responses. PMID:11129285

  18. Prevention and Treatment of Influenza with Hyperimmune Bovine Colostrum Antibody

    PubMed Central

    Ng, Wy Ching; Wong, Victor; Muller, Brian; Rawlin, Grant; Brown, Lorena E.

    2010-01-01

    Background Despite the availability of specific vaccines and antiviral drugs, influenza continues to impose a heavy toll on human health worldwide. Passive transfer of specific antibody (Ab) may provide a useful means of preventing or treating disease in unvaccinated individuals or those failing to adequately seroconvert, especially now that resistance to antiviral drugs is on the rise. However, preparation of appropriate Ab in large scale, quickly and on a yearly basis is viewed as a significant logistical hurdle for this approach to control seasonal influenza. Methodology/Principal Findings In this study, bovine colostrum, which contains approximately 500 g of IgG per milking per animal, has been investigated as a source of polyclonal antibody for delivery to the respiratory tract. IgG and F(ab')2 were purified from the hyperimmune colostrum of cows vaccinated with influenza A/Puerto Rico/8/34 (PR8) vaccine and were shown to have high hemagglutination-inhibitory and virus-neutralizing titers. In BALB/c mice, a single administration of either IgG or F(ab')2 could prevent the establishment of infection with a sublethal dose of PR8 virus when given as early as 7 days prior to exposure to virus. Pre-treated mice also survived an otherwise lethal dose of virus, the IgG- but not the F(ab')2-treated mice showing no weight loss. Successful reduction of established infection with this highly virulent virus was also observed with a single treatment 24 hr after virus exposure. Conclusions/Significance These data suggest that a novel and commercially-scalable technique for preparing Ab from hyperimmune bovine colostrum could allow production of a valuable substitute for antiviral drugs to control influenza with the advantage of eliminating the need for daily administration. PMID:21049034

  19. Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution.

    PubMed Central

    Jacobo-Molina, A; Clark, A D; Williams, R L; Nanni, R G; Clark, P; Ferris, A L; Hughes, S H; Arnold, E

    1991-01-01

    Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process. Images PMID:1720554

  20. Human Parathyroid Hormone IMMUNOLOGICAL CHARACTERIZATION OF ANTIBODIES AGAINST A GLANDULAR EXTRACT AND THE SYNTHETIC AMINO-TERMINAL FRAGMENTS 1-12 AND 1-34 AND THEIR USE IN THE DETERMINATION OF IMMUNOREACTIVE HORMONE IN HUMAN SERA

    PubMed Central

    Fischer, Jan A.; Binswanger, Ulrich; Dietrich, Felix M.

    1974-01-01

    Antibodies to a urea-trichloroacetic acid extract [hPTH-(TCA)] of human parathyroid tumors and to the synthetic NH2-terminal fragments of human parathyroid hormone hPTH-(1-12) and -(1-34) were developed in goats to characterize immunochemically various PTH preparations and to estimate immunoreactive PTH (iPTH) in human sera. They were quantitated on the basis of their capacity to bind [131I]-hPTH-(1-12), [131I]hPTH-(1-34) or [131I]bovine PTH (bPTH-(1-84)). The quality of the antibodies was assessed by reference to inhibition of their interaction with labeled peptides by synthetic hPTH comprising 34 NH2-terminal amino acid residues or fragments thereof [hPTH-(1-12), -(13-34), -(18-34), -(25-34), -(18-24)] or by the Sephadex G-100-purified full-length peptide hPTH-(1-84) [hPTH-(1-84)G-100]. The synthetic peptides used in this work correspond in their structure to the NH2-terminal amino acid sequence 1-34, as elucidated by Brewer and collaborators (1972. Proc. Natl. Acad. Sci. U. S. A.69: 3583-3588). Inhibition studies were also carried out with bPTH-(1-34) and bPTH-(1-84). Anti-hPTH-(TCA) exhibited specificities directed to determinants in the COOH-terminal and NH2-terminal part of hPTH-(1-84) and exhibited cross-reactivity with bPTH-(1-84). Anti-hPTH-(1-34), on the other hand, showed immunological specificities mainly directed to antigenic determinants located in the COOH-terminal half of hPTH-(1-34). In addition, some reactivity with the NH2-terminal hPTH-(1-12) and with the extractive full-length peptides of human and bovine origin was observed. Antibodies to hPTH-(1-12) cross-reacted with hPTH-(1-34) and -(1-84)G-100. iPTH was radioimmunologically determined in human sera by the following systems: (a) [131I]bPTH-(1-84), anti-hPTH-(TCA) and hPTH-(1-84)G-100 as standard; (b) [131I]hPTH-(1-34), anti-hPTH-(1-34) and hPTH-(1-34) as standard. With system (a), COOH-terminal fragments of hPTH-(1-84) having a molecular weight of approximately 7,000 were detected, and

  1. Complement fragments C3b and iC3b coupled to latex induce a respiratory burst in human neutrophils.

    PubMed

    Hoogerwerf, M; Weening, R S; Hack, C E; Roos, D

    1990-02-01

    The complement fragments C3b and iC3b were purified from human serum by affinity chromatography with Sepharose-coupled monoclonal antibody against the C3d region of C3. The resulting preparations were more than 95% pure and contained less than 0.1% native IgG. Purified C3b and iC3b were coupled to latex beads (0.8 micron diameter) by means of F(ab')2 fragments of monoclonal antibodies against the beta chain or the C3d region of C3, thus orienting the C3b and the iC3b on the latex with the C3b- and iC3b-specific regions outwards. These particles were found to activate the respiratory burst of freshly isolated human neutrophils to 20-30% of the maximal capacity. Latex particles randomly coated with C3b or iC3b were about 3 times less stimulatory. C3b, iC3b and IgG coupled to latex in an oriented fashion were about equally effective in stimulating the respiratory burst. Neutrophils from a patient with a total deficiency of CR3 responded normally to C3b-coated latex but did not respond to iC3b-coated latex. A monoclonal antibody against the alpha chain of CR3 inhibited the activation by iC3b-coated latex and a polyclonal antibody against CR1 partially inhibited the activation by C3b-coated latex. We found an additive effect between IgG-coated latex and C3b-coated latex, regardless of the presence of IgG and C3b on the same particle or on different particles. Thus, binding of ligands to either CR1 or CR3 per se is sufficient to induce an activating signal to the NADPH oxidase in human neutrophils.

  2. Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect.

    PubMed

    Sugaya, Akinori; Hyodo, Ichinosuke; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Takashima, Hiroki; Sato, Ryuta; Tsumura, Ryo; Furuya, Fumiaki; Yasunaga, Masahiro; Harada, Mitsunori; Tanaka, Ryosuke; Matsumura, Yasuhiro

    2016-03-01

    Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti-TF antibody (clone1849)-conjugated epirubicin-incorporating micelles (NC-6300), and reported that this anti-TF1849-NC-6300 showed enhanced antitumor activity against TF-high expressed human pancreatic cancer cells, when compared with NC-6300 alone. However, clone 1849 antibody inhibited TF-associated blood coagulation activity. We studied another anti-TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti-TF1859-NC-6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC-6300, three forms of the 1859 antibody (whole IgG, F[ab']2 , and Fab') were conjugated to NC-6300. The antitumor effect of each anti-TF1859-NC-6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high-expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti-TF1859-NC-6300 showed higher cytocidal effects than NC-6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti-TF1859-NC-6300 significantly suppressed tumor growth when compared to NC-6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti-TF1859-IgG-NC-6300 had a higher antitumor tendency in TF-high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti-TF1859-NC-6300 in a TF-high expressing tumor; anti-TF1859-IgG-NC-6300 could be used to simplify the manufacturing process of the antibody-micelle conjugation for future clinical studies.

  3. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  4. Inhibition of human immunodeficiency virus type 1 integrase by the Fab fragment of a specific monoclonal antibody suggests that different multimerization states are required for different enzymatic functions.

    PubMed

    Barsov, E V; Huber, W E; Marcotrigiano, J; Clark, P K; Clark, A D; Arnold, E; Hughes, S H

    1996-07-01

    We have characterized a murine monoclonal antibody (MAb 35), which was raised against human immunodeficiency virus type 1 (HIV-1) integration protein (IN), and the corresponding Fab 35. Although MAb 35 does not inhibit HIV-1 IN, Fab 35 does. MAb 35 (and Fab 35) binds to an epitope in the C-terminal region of HIV-1 IN. Fab 35 inhibits 3'-end processing, strand transfer, and disintegration; however, DNA binding is not affected. The available data suggest that Fab 35 inhibits enzymatic activities of IN by interfering with the ability of IN to form multimers that are enzymatically active. This implies that the C-terminal region of HIV-1 IN participates in interactions that are essential for the multimerization of IN. Titration of the various IN-mediated enzymatic activities suggests that different degrees of multimerization are required for different activities of HIV-1 IN.

  5. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  6. Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of Alzheimer disease.

    PubMed

    Giménez-Llort, Lydia; Rivera-Hernández, Geovanny; Marin-Argany, Marta; Sánchez-Quesada, José L; Villegas, Sandra

    2013-01-01

    The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid β (Aβ) peptide in neuroblastoma cell cultures by withdrawing Aβ oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia -like behaviors. Cognitive deficits included long- and short-term learning and memory deficits and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aβ oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.

  7. Advances in Antibody Design.

    PubMed

    Tiller, Kathryn E; Tessier, Peter M

    2015-01-01

    The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

  8. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    PubMed

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  9. Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

    PubMed Central

    Vlasova, Anastasia N.; Chattha, Kuldeep S.; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M.; Garaicoechea, Lorena L.; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J.; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea. PMID:23658521

  10. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    PubMed Central

    Hosking, Christopher G.; McWilliam, Hamish E. G.; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P.; Ilag, Leodevico L.; Meeusen, Els N. T.; de Veer, Michael J.

    2015-01-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  11. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    PubMed

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

  12. Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

    PubMed

    Makvandi-Nejad, Shokouh; Sheedy, Claudia; Veldhuis, Linda; Richard, Gabrielle; Hall, J Christopher

    2010-08-31

    Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

  13. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement of immunoglobulin G Fd fragments aids in the diagnosis of plasma antibody-forming cell abnormalities....

  14. Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

    PubMed Central

    Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2015-01-01

    Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

  15. Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

    PubMed

    Li, Qiang; Aucamp, Jean P; Tang, Alison; Chatel, Alex; Hoare, Mike

    2012-08-01

    An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.

  16. Anti-neutrophil cytoplasmic antibody-enriched IgG induces adhesion of human T lymphocytes to extracellular matrix proteins.

    PubMed

    Tomer, Y; Lider, O; Gilburd, B; Hershkoviz, R; Meroni, P L; Wiik, A; Shoenfeld, Y

    1997-06-01

    Recent studies have shown that anti-neutrophil cytoplasmic antibodies (ANCA) can activate neutrophils to adhere to endothelium, degranulate, and cause endothelial cell injury. These data have lead to the hypothesis that the T cell inflammatory response causing the vasculitis in Wegener's granulomatosis (WG) is secondary to stimulation of neutrophils by ANCA. So far there is no evidence for a direct effect of ANCA on lymphocytes. The present study was designed to examine whether lymphocytes can be directly stimulated by ANCA to adhere to endothelial extracellular matrix (ECM) proteins. Human and mouse ANCA-enriched IgG were tested for their ability to increase adhesion of human T lymphocytes to fibronectin, laminin, and intact ECM. Incubation of human T lymphocytes with human ANCA-enriched IgG increased adhesion of the lymphocytes in a dose-dependent manner to fibronectin, laminin, and intact ECM (the percentage adhesion to intact ECM was 55.7 +/- 3.1 and 45.0 +/- 1.0% for lymphocytes incubated with human IgG containing ANCA or control human IgG, respectively; P = 0.0045). The same induction of adhesion to fibronectin, laminin, and intact ECM was observed when the cells were incubated with the F(ab)2 fragment of ANCA-enriched IgG. Similarly, ANCA-enriched IgG produced in mice increased the adhesion of lymphocytes to fibronectin (the percentage adhesion to fibronectin was 29.7 +/- 4.3 and 16.6 +/- 1.9% for lymphocytes incubated with mouse IgG-ANCA or control mouse IgG, respectively; P = 0.0008). These results may suggest that ANCA can directly stimulate lymphocytes to adhere to endothelial ECM and to induce the vasculitic lesions of WG. It remains to be shown by which mechanisms ANCA stimulate lymphocytes to adhere to ECM. PMID:9175913

  17. Magma Fragmentation

    NASA Astrophysics Data System (ADS)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmatic volatiles, decompression of low-viscosity magma leads to rapid expansion. The magma is torn into fragments, as it is stretched into hydrodynamically unstable sheets and filaments. If the magma is highly viscous, resistance to bubble growth will instead lead to excess gas pressure and the magma will deform viscoelastically by fracturing like a glassy solid, resulting in the formation of a violently expanding gas-pyroclast mixture. In either case, fragmentation represents the conversion of potential energy into the surface energy of the newly created fragments and the kinetic energy of the expanding gas-pyroclast mixture. If magma comes into contact with external water, the conversion of thermal energy will vaporize water and quench magma at the melt-water interface, thus creating dynamic stresses that cause fragmentation and the release of kinetic energy. Lastly, shear deformation of highly viscous magma may cause brittle fractures and release seismic energy.

  18. Topical skin treatment with Fab fragments of an allergen-specific IgG1 monoclonal antibody suppresses allergen-induced atopic dermatitis-like skin lesions in mice.

    PubMed

    Sae-Wong, Chutha; Mizutani, Nobuaki; Kangsanant, Sureeporn; Yoshino, Shin

    2016-05-15

    Fab fragments (Fabs), which lack effector functions due to the absence of the Fc portion, maintain the ability to bind to specific allergens. In the present study, we examined whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) were able to regulate allergen-induced atopic dermatitis-like skin lesions in mice. BALB/c mice passively sensitized with ovalbumin (OVA)-specific IgE mAb were repeatedly challenged with OVA applied to the skin after sodium dodecyl sulfate treatment. Fabs prepared by the digestion of anti-OVA IgG1 mAb (O1-10) with papain were applied to the skin 30min before the OVA challenges followed by measurement of clinical symptoms including erythema/hemorrhage, edema, scarring/dryness, and excoriation/erosion of the skin. Treatment with O1-10 Fabs, but not intact O1-10, showed inhibition of clinical symptoms (P<0.01) induced by the repeated OVA challenges in the sensitized mice; O1-10 Fabs suppressed histological changes such as epidermal hyperplasia (P<0.01) and the accumulation of mast cells (P<0.01) and neutrophils (P<0.01). Furthermore, treatment with O1-10 Fabs inhibited the increase in levels of IL-13 (P<0.01) and IL-17A production (P<0.05) in the lymph nodes of the sensitized mice. Additionally, the increased level of OVA in serum following the repeated OVA challenges in the sensitized mice was reduced by the treatment (P<0.05). These results suggest that topical application of pathogenic allergen-specific IgG1 mAb Fabs to the skin of mice is effective in suppressing allergen-induced atopic dermatitis-like skin lesions, suggesting that allergen-specific mAb Fabs could be used as a tool to regulate allergen-induced atopic dermatitis. PMID:26970183

  19. Dose Range Evaluation of Mycograb C28Y Variant, a Human Recombinant Antibody Fragment to Heat Shock Protein 90, in Combination with Amphotericin B-Desoxycholate for Treatment of Murine Systemic Candidiasis ▿

    PubMed Central

    Louie, Arnold; Stein, Daniel S.; Zack, Julia Z.; Liu, Weiguo; Conde, Haley; Fregeau, Christine; VanScoy, Brian D.; Drusano, George L.

    2011-01-01

    Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t1/2α of ∼0.198 h and a t1/2β of ∼1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis. PMID:21502626

  20. Targeting antibodies to the cytoplasm.

    PubMed

    Marschall, Andrea L J; Frenzel, André; Schirrmann, Thomas; Schüngel, Manuela; Dübel, Stefan

    2011-01-01

    A growing number of research consortia are now focused on generating antibodies and recombinant antibody fragments that target the human proteome. A particularly valuable application for these binding molecules would be their use inside a living cell, e.g., for imaging or functional intervention. Animal-derived antibodies must be brought into the cell through the membrane, whereas the availability of the antibody genes from phage display systems allows intracellular expression. Here, the various technologies to target intracellular proteins with antibodies are reviewed.

  1. Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.

    PubMed Central

    Garrigues, J.; Garrigues, U.; Hellström, I.; Hellström, K. E.

    1993-01-01

    BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that

  2. Long-lasting protective antiviral immunity induced by passive immunotherapies requires both neutralizing and effector functions of the administered monoclonal antibody.

    PubMed

    Nasser, Roudaina; Pelegrin, Mireia; Michaud, Henri-Alexandre; Plays, Marc; Piechaczyk, Marc; Gros, Laurent

    2010-10-01

    Using FrCas(E) retrovirus-infected newborn mice as a model system, we have shown recently that a long-lasting antiviral immune response essential for healthy survival emerges after a short treatment with a neutralizing (667) IgG2a isotype monoclonal antibody (MAb). This suggested that the mobilization of adaptive immunity by administered MAbs is key for the success in the long term for the MAb-based passive immunotherapy of chronic viral infections. We have addressed here whether the anti-FrCas(E) protective endogenous immunity is the mere consequence of viral propagation blunting, which would simply give time to the immune system to react, and/or to actual immunomodulation by the MAb during the treatment. To this aim, we have compared viral replication, disease progression, and antiviral immune responses between different groups of infected mice: (i) mice treated with either the 667 MAb, its F(ab')(2) fragment, or an IgM (672) with epitopic specificity similar to that of 667 but displaying different effector functions, and (ii) mice receiving no treatment but infected with a low viral inoculum reproducing the initial viral expansion observed in their infected/667 MAb-treated counterparts. Our data show that the reduction of FrCas(E) propagation is insufficient on its own to induce protective immunity and support a direct immunomodulatory action of the 667 MAb. Interestingly, they also point to sequential actions of the administered MAb. In a first step, viral propagation is exclusively controlled by 667 neutralizing activity, and in a second one, this action is complemented by FcgammaR-binding-dependent mechanisms, which most likely combine infected cell cytolysis and the modulation of the antiviral endogenous immune response. Such complementary effects of administered MAbs must be taken into consideration for the improvement of future antiviral MAb-based immunotherapies.

  3. Comparison of SPECT imaging using monoclonal antibodies with computed tomography (CT) and ultrasonography (US) for detection of recurrences of colorectal carcinoma: A prospective clinical study

    SciTech Connect

    Chatal, J.F.; Saccavini, J.C.; Douillard, J.Y.; Curtet, C.; Kremer, M.; Le Mevel, B.

    1985-05-01

    A prospective clinical study compared SPECT imaging, ultrasonography (US), and computed tomography (CT) in 22 patients clinically or biologically (increased CEA and/or CA 19-9 serum concentration) suspected of recurrence of colorectal carcinoma. The recordings were performed 3 to 5 days after injection of 111 to 129.5 MBq of cocktail of I-131-labeled anti-CEA and 19-9 (F(ab')2 fragments) monoclonal antibodies. Twenty nine tumor sites were demonstrated by surgery or concordant results of conventional diagnostic methods. SPECT visualized 21 of these 29 tumor sites (72%). It was negative in 4 cases with no demonstrated recurrence (by any method and follow-up). With respect to localization of tumor sites, SPECT visualized 7/12 liver metastases, 8/8 local pelvic recurrences and 6/8 abdominal recurrences. CT and US, systematically performed blind after SPECT, respectively visualized 9/10 and 9/12 liver metastases, 7/12 and 4/13 pelvic and abdominal recurrences. Image interpretation of SPECT was difficult due to poor tumor contrast and the large number of low-intensity, nonspecific radioactive foci. A focus had to recur in at least 3 successive slices to be considered pathological. Four tumor sites were visualized with SPECT and not with US and CT (negative or uncertain results). SPECT would appear to be useful for localizing pelvic or abdominal recurrences in cases in which interpretation of US and CT images is difficult, often because their nonspecific approach does not make it possible to differentiate a tumor recurrence from post-operative anatomical changes.

  4. [Pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody in human body].

    PubMed

    Li, Yunchun; Tan, Tianzhi; Mo, Tingshu; Lu, Wusheng; Deng, Houfu; Yang, Xiaochuan; Li, Xiao

    2007-08-01

    To study pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody (hepatoma monoclonal antibody HAb18 F(ab')2) in vivo. 24 cases of primary hepatocelluar carcinoma (PHC) were equally divided into the low dose group, middle dose group and high dose group. After the relevant injection was administrated into the hepatic artery of each case, intravenous blood and urine samples were separately collected at different time for determination of the radioactive count ratio (min(-1)). The proportion of 131I-HAb18 F(ab')2 in serum of each blood sample was determined, and the radioactive count ratio (min(-1)) of druggery for each blood sample was revised according to the proportion. The pharmacokinetic parameters were calculated using DAS ver 1.0 (Drug And Statistics for Windows) program. The component of urine radiomaterial was determined and the percentages of urine radioactivity in administration dosage were calculated. The catabolism of the injection with time accorded with dynamics two-compartment model. The catabolism product was mainly free-131I and was excreted via kidney; the urine radioactivity was 47.70%-51.16% of administration dosage during 120 h after administration of drug. Therefore, the pharmacokinetics of the injection can satisfy the clinical demands. The drug dose recommended for clinical use was 27.75 MBq of the injection for each kg of human body.

  5. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  6. Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1.

    PubMed

    Forthal, Donald N; Landucci, Gary; Phan, Tran B; Becerra, Juan

    2005-02-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.

  7. Anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  8. Prospective, consecutive case series of 158 snakebite patients treated at Savannakhet provincial hospital, Lao People's Democratic Republic with high incidence of anaphylactic shock to horse derived F(ab')2 antivenom.

    PubMed

    Vongphoumy, Inthanomchanh; Chanthilat, Phankham; Vilayvong, Phongmany; Blessmann, Joerg

    2016-07-01

    Snakebites are a seriously neglected public health problem in Lao PDR. Community-based cross-sectional surveys in two districts of Savannakhet province in Southern Laos revealed an incidence of up to 1105 snakebites per 100,000 persons per year. In contrast the number of snakebite patients treated in district and provincial hospitals are low. In order to improve health care for snakebite victims, antivenom was introduced to Savannakhet provincial hospital in July 2013 and medical staff has been trained in management of venomous snakebites at the same time. After the intervention the number of snakebite patients treated at the provincial hospital increased significantly from 4 patients in 2012 to 158 snakebite patients between July 2013 and November 2015. They were included into a prospective, consecutive case series. Median age was 32 years (range 1.5-70 years) and male-to-female ratio 2.2:1. Forty patients were bitten by Malayan pit vipers, 26 by green pit vipers, 24 by cobras, including 3 cases of venom ophthalmia, 5 by kraits, 8 by non-venomous species and in 55 cases the snake could not be identified. Forty-three out of 158 patients received horse derived F(ab')2 antivenom from Queen Saovabha Memorial Institute (QSMI) in Bangkok. Twenty-three patients (53%) developed early adverse reactions (EARs) within one hour after antivenom administration, including 13 patients (30%) with severe anaphylaxis. This extremely high rate of severe EARs turns the use of antivenom into a risky intervention. In contrast a retrospective chart review from Chulalongkorn University in Bangkok found only 3.5% early reactions including 1.2% severe anaphylactic reactions using the same antivenom from QSMI between 1997 and 2006. The reason for this enormous difference remains unclear. A better understanding of the aetiology and pathophysiology behind antivenom induced anaphylaxis is crucial in order to identify patients at risk and to improve safety of antivenom administration. PMID:26995210

  9. Monoclonal antibodies.

    PubMed

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  10. REGULATION OF THE IMMUNE RESPONSE

    PubMed Central

    Sinclair, Nicholas R. StC.

    1969-01-01

    The ability of 7S and F(ab')2 antibody fragments to suppress priming with low doses of antigen was compared. The 7S preparation was approximately 100–1000 times more potent than the F(ab')2 preparation when the agglutinin titers of the two preparations were the same. The presence of any ability to suppress priming in the F(ab')2 preparation may reflect an inherent capacity of the F(ab')2 antibody or contamination with small amounts of 7S antibody. The difference between 7S and F(ab')2 antibody in ability to suppress priming is attributed to the lack of the Fc portion on the F(ab')2 antibody. The Fc portion may be needed to prevent rapid excretion of antibody from the body, to induce rapid phagocytosis of antigen-antibody complexes with consequent breakdown and elimination of antigen, or to inactivate or suppress the antigen-sensitive cells from reacting to antigenic determinants. More detailed studies will permit a better assessment of the importance of these three possible regulatory roles of the Fc portion of the immunoglobulin in the immune response. PMID:5305714

  11. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    PubMed Central

    2012-01-01

    Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). Findings Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly

  12. Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor).

    PubMed

    Nuijens, J H; Huijbregts, C C; Eerenberg-Belmer, A J; Meijers, J C; Bouma, B N; Hack, C E

    1989-08-01

    We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.

  13. Increased serum levels of IgA1-IgG immune complexes and anti-F(ab')2 antibodies in patients with primary IgA nephropathy.

    PubMed Central

    Schena, F P; Pastore, A; Ludovico, N; Sinico, R A; Benuzzi, S; Montinaro, V

    1989-01-01

    A solid-phase ELISA was used to detect IgA1 immune complexes (IgA1 ICs) containing IgG and IgM in 38 serum samples from 30 patients with primary IgA nephropathy (IgAN) and 14 subjects with non-IgA chronic glomerulonephritis. A jackfruit lectin, jacalin, was used as the substrate for the selective binding of human IgA1 ICs in serum PEG precipitate (7%). The presence of IgG, A and M antibodies against the F(ab')2 region of IgG was also investigated by the solid-phase ELISA. Six patients were studied during remission and relapse (fever, upper respiratory tract infection and macroheamaturia). The results showed significant increases in serum levels of IgA1 ICs (P less than 0.001) in 39.4% of the IgAN patients, IgA1-IgG ICs (P less than 0.001) in 68.4%, and IgA1-IgM ICs (P less than 0.002) in 10.5% of the patients. A significant increase in IgA1-IgG ICs was observed during relapse (P less than 0.02). Significantly high values of IgG (P less than 0.003) and IgA (P less than 0.001) antibodies directed at the F(ab')2 region of IgG were found. A significant increase in anti F(ab')2 antibodies (class IgA and IgM) was seen in the acute phase of the disease. The data suggest that an increased production of IgA1 ICs occurs in IgAN patients; ICs are mainly IgA1-IgG ICs during relapse. The presence of high serum levels of IgG and IgA antibodies against the F(ab')2 region of IgG indicates that in addition to the multiple anomalies of IgA regulation described in IgAN patients there may be further aberrances. PMID:2788538

  14. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  15. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  16. Antimitochondrial antibody

    MedlinePlus

    ... antibodies (AMA) are substances ( antibodies ) that form against mitochondria. The mitochondria are an important part of cells. They are ... often, in people with other kinds of liver disease and some autoimmune diseases. Risks Risks for having ...

  17. Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives.

    PubMed

    Vaz de Andrade, Edmar; Freitas, Sonia Maria; Ventura, Manuel Mateus; Maranhão, Andréa Queiroz; Brigido, Marcelo Macedo

    2005-11-30

    Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.

  18. Controlled delivery of antibodies from injectable hydrogels.

    PubMed

    Fletcher, Nathan A; Babcock, Lyndsey R; Murray, Ellen A; Krebs, Melissa D

    2016-02-01

    Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

  19. Antibody validation

    PubMed Central

    Bordeaux, Jennifer; Welsh, Allison W.; Agarwal, Seema; Killiam, Elizabeth; Baquero, Maria T.; Hanna, Jason A.; Anagnostou, Valsamo K.; Rimm, David L.

    2013-01-01

    Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence. PMID:20359301

  20. Fractals and fragmentation

    NASA Technical Reports Server (NTRS)

    Turcotte, D. L.

    1986-01-01

    The use of renormalization group techniques on fragmentation problems is examined. The equations which represent fractals and the size-frequency distributions of fragments are presented. Method for calculating the size distributions of asteriods and meteorites are described; the frequency-mass distribution for these interplanetary objects are due to fragmentation. The application of two renormalization group models to fragmentation is analyzed. It is observed that the models yield a fractal behavior for fragmentation; however, different values for the fractal dimension are produced . It is concluded that fragmentation is a scale invariant process and that the fractal dimension is a measure of the fragility of the fragmented material.

  1. Immune response to phosphorylcholine. IX. Characterization of hybridoma anti-TEPC15 antibodies.

    PubMed

    Wittner, M K; Bach, M A; Köhler, H

    1982-02-01

    Hybridoma antibodies against the PC-binding T15 BALB/c myeloma protein were raised by cell fusion with anti-T15 A/He immune cells. The idiotype specificity of these monoclonal anti-T15 antibodies was determined with a panel of different myeloma and hybridoma immunoglobulins. Two types of anti-T15 antibodies are seen. One reacts with a number of different IgA myeloma proteins and with serum IgA of certain strains of mice; this reactivity most likely is due to allotypy. The other group consists of anti-T15 antibodies that are specific for the T15 idiotype and are therefore termed anti-idiotypic. The bindings of the anti-idiotype antibodies to T15 were specifically inhibited by T15 (F(ab')2 but not by other PC-binding myeloma proteins of different idiotypes. The relationship of the idiotype-specific anti-T15 antibodies to the PC-binding site of the T15 idiotype was analyzed by hapten inhibition of anti-idiotypic binding and by inhibition of BALB/c anti-PC splenic hemolytic plaque formation. Anti-T15 antibodies, for which the T15 binding is inhibited by PC or PC-BSA, also specifically inhibit anti-PC plaque formation. These antibodies are labeled site and near-site anti-idiotypic antibodies. Site and near-site-specific anti-idiotypic antibodies recognize different idiotopes on the T15 molecules. The possible differential biologic activities of these anti-idiotopes in idiotype network regulation is considered.

  2. A comparison of 67Cu- and 131I-labelled forms of monoclonal antibodies SEN7 and SWA20 directed against small-cell lung cancer.

    PubMed

    Smith, A; Zangemeister-Wittke, U; Waibel, R; Schenker, T; Schubiger, P A; Stahel, R A

    1994-01-01

    The intact anti-SCLC monoclonal antibody (MAb) SEN7 and its F(ab')2 were labelled with the beta-emitting isotope 67Cu. Both materials retained their biological activity in vitro as determined by the Lindmo assay. In a direct comparison of in vivo distribution in a xenograph model, 131I- and 67Cu-labelled intact SEN7 showed similar absolute tumour accumulation. Blood levels were markedly lower in the case of the 67Cu-labelled antibody, resulting in improved tumour:blood ratios which reached a maximum of 13:1 compared with only 4.5:1 for 131I-SEN7. In the case of the 67Cu-labelled F(ab')2, very high accumulation of the nuclide was observed in the kidney. Levels of radio copper in liver and spleen were also found to be significantly raised when compared with radio iodine. SWA20, a MAb which had previously failed to show any selective in vivo accumulation in tumour xenografts when labelled with radio iodine showed higher and more stable tumour accumulation when labelled with 67Cu.

  3. Selectable fragmentation warhead

    SciTech Connect

    Bryan, C.S.; Paisley, D.L.; Montoya, N.I.; Stahl, D.B.

    1992-12-31

    This report discusses a selectable fragmentation warhead which is capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

  4. Selectable fragmentation warhead

    DOEpatents

    Bryan, Courtney S.; Paisley, Dennis L.; Montoya, Nelson I.; Stahl, David B.

    1993-01-01

    A selectable fragmentation warhead capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

  5. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

    PubMed

    Spruyt, L L; Glennie, M J; Beyers, A D; Williams, A F

    1991-12-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

  6. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16)

    PubMed Central

    1991-01-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i. PMID:1683892

  7. Therapeutic antibodies in ophthalmology

    PubMed Central

    Magdelaine-Beuzelin, Charlotte; Pinault, Coralie; Paintaud, Gilles

    2010-01-01

    More than a century after the first successful use of serotherapy, antibody-based therapy has been renewed by the availability of recombinant monoclonal antibodies. As in the past, current clinical experience has prompted new pharmacological questions and induced much debate among practitioners, notably in the field of ophthalmology. An examination of the history of antibodies as treatments for ocular disorders reveals interesting parallels to the modern era. The fact that a treatment administered by a systemic route could be efficacious in a local disease was not widely accepted and the “chemical” nature of antibodies was not clearly understood in the late 19th century. Clinical studies by Henry Coppez, a Belgian ophthalmologist, established in 1894 that antidiphtheric antitoxins could be used to treat conjunctival diphtheria. Nearly 20 years later, Coppez and Danis described age-related macular degeneration, a disorder which today benefits from ranibizumab therapy. The product, a locally-administered recombinant monoclonal Fab fragment, is directed against vascular endothelial growth factor A. Interestingly, its full-size counterpart, bevacizumab, which is approved for the treatment of solid tumors, has also demonstrated efficacy in age-related macular degeneration when administered either intravenously or locally, which raises new questions about antibody pharmacology and biodistribution. In order to shed some light on this debate, we recount the early history of serotherapy applied to ophthalmology, review the exact molecular differences between ranibizumab and bevacizumab, and discuss what is known about IgG and the blood-retina barrier and the possible role of FcRn, an IgG transporter. PMID:21358858

  8. Universality of fragment shapes

    PubMed Central

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  9. Universality of fragment shapes.

    PubMed

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  10. Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library.

    PubMed

    Zhang, Xiao; Xu, Chongxin; Zhang, Cunzheng; Liu, Yuan; Xie, Yajing; Liu, Xianjin

    2014-04-01

    ScFvs are composed of the variable regions of the heavy and light chains via a short linker that maintain the specific antigen binding abilities of antibodies. In this study, we constructed a naïve mouse phage displayed library to generate scFvs against Cry1Ab toxin. After affinity panning, positive phage-scFvs were isolated, sequenced and characterized by ELISA. The best binding ability scFv-G9 was expressed and purified. SDS-PAGE indicated that the relative molecular mass of scFv was estimated at 28 kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008 μg mL(-1), a working range 0.018-6.23 μg mL(-1), and the linear curve displayed an acceptable correlation coefficient of 0.98. The cross-reactivity showed that scFv-G9 had strongly binding ability to Cry1Ac toxin, but not to Cry1B, Cry1C and Cry1F toxin. The average recoveries of Cry1Ab toxin from spiked leaf and rice samples were in the range 92.1-94.8%, and 91.6-98.6%, respectively, with a coefficient of variation (C.V) less than 5.0%. These results showed promising applications of scfv-G9 for detecting Cry1Ab toxin with new DAS-ELISA.

  11. Fragmentation properties of metals

    SciTech Connect

    Grady, D.E.; Kipp, M.E.

    1996-06-01

    In the present study we are developing an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests, both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

  12. Derivatized gold clusters and antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.; Furuya, F.R.

    1994-11-01

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be as small as 5.0 nm. Methods and reagents are disclosed in which antibodies, Fab' or F(ab')[sub 2] fragments are covalently bound to a stable cluster of gold atoms. The gold clusters may contain 6, 8, 9, 11, 13, 55 or 67 gold atoms in their inner core. The clusters may also contain radioactive gold. The antibody-cluster conjugates are useful in electron microscopy applications as well as in clinical applications that include imaging, diagnosis and therapy. 7 figs.

  13. Derivatized gold clusters and antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, James F.; Furuya, Frederic R.

    1994-11-01

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be as small as 5.0 nm. Methods and reagents are disclosed in which antibodies, Fab' or F(ab').sub.2 fragments thereof are covalently bound to a stable cluster of gold atoms. The gold clusters may contain 6, 8, 9, 11, 13, 55 or 67 gold atoms in their inner core. The clusters may also contain radioactive gold. The antibody-cluster conjugates are useful in electron microscopy applications as well as in clinical applications that include imaging, diagnosis and therapy.

  14. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    SciTech Connect

    Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

    2009-04-24

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  15. Antibody-based immunosuppressive agents for corneal transplantation.

    PubMed

    Thiel, M A; Kaufmann, C; Coster, D J; Williams, K A

    2009-10-01

    The progress in antibody engineering over the last 20 years has created the tools for the development of novel antibody-based drugs and constructs, such as small antibody fragments, suitable for topical administration. In rheumatology, oncology, transplantation medicine and ophthalmology, therapeutic antibody constructs, and antibody fragments have been responsible for the clinical progress seen over the last decade. Although antibody-based therapies have become a well-established immunosuppressive option in solid organ transplantation, there are only very few reports with regard to corneal transplantation. The following review explains some of the important aspects of engineered antibody-based therapeutic agents and summarises the current use of such immunosuppressive therapies in transplantation medicine and corneal transplantation.

  16. The binding of monoclonal antibodies to cell-surface molecules. A quantitative analysis with immunoglobulin G against two alloantigenic determinants of the human transplantation antigen HLA-A2.

    PubMed Central

    Ways, J P; Parham, P

    1983-01-01

    Monoclonal IgG1 (immunoglobulin G1) PA2.1 and MA2.1 antibodies recognize polymorphic sites of the human transplantation antigen HLA-A2. They are distinguishable because MA2.1 binds HLA-A2 and HLA-B17, whereas PA2.1 binds HLA-A2 and HLA-A28. The affinities of PA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-A28 are similar and relatively low (1.9 X 10(7) M-1). The affinities of MA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-B17 are similar and high (1.2 X 10(9) M-1). The difference in affinity is due to the rates of dissociation, which give half-times of dissociation of 290 min for MA2.1-Fab and 4 min for PA2.1-Fab. For both Fab, equilibrium measurements and kinetic determinations gave consistent estimates for affinity. When PA2.1-F(ab)2 or IgG is incubated with cells it reaches equilibrium within 3 h, with most molecules bound bivalently to the cell. Under similar conditions, MA2.1-F(ab)2 does not reach equilibrium and a significant proportion of molecules bound with one and two sites are found. For the lower-affinity antibody (PA2.1), estimates of the binding constants for one- and two-site interactions could be made. By simple Scatchard analysis the avidity of F(ab)2 or IgG is 1.3 X 10(9) M-1, giving an enhancement factor of 68 between bivalent and univalent binding. This is a measure of the equilibrium constant for the interchange between bivalent and univalent binding. Analysis of the results with more realistic models indicates that the actual value is larger (10(3)-10(4) M-1) than 68 M-1. The avidities of F(ab)2 and IgG for HLA-A2 are identical, showing the Fc does not interfere with bivalent binding to cells. PMID:6197968

  17. Novel antibodies as anticancer agents.

    PubMed

    Zafir-Lavie, I; Michaeli, Y; Reiter, Y

    2007-05-28

    In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents. PMID:17530025

  18. An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

    PubMed

    Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

    2015-08-01

    Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

  19. The second life of antibodies.

    PubMed

    Navolotskaya, E V

    2014-01-01

    Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (~75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289-292 of the C(H2)-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341-344), immunorphin (364-373), immunocortin (11-20), and peptide p24 (335-358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed. PMID:24512657

  20. Anti-CD2 antibodies induce T cell unresponsiveness in vivo

    PubMed Central

    1991-01-01

    The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo. PMID:1682413

  1. Selectable fragmentation warhead

    SciTech Connect

    Bryan, C.S.; Paisley, D.L.; Montoya, N.I.; Stahl, D.B.

    1993-07-20

    A selectable fragmentation warhead is described comprising: a case having proximal and distal ends; a fragmenting plate mounted in said distal end of said casing; first explosive means cast adjacent to said fragmenting plate for creating a predetermined number of fragments from said fragmenting plate; three or more first laser-driven slapper detonators located adjacent to said first explosive means for detonating said first explosive means in a predetermined pattern; smoother-disk means located adjacent to said first means for accelerating said fragments; second explosive means cast adjacent to said smoother-disk means for further accelerating said fragments; at least one laser-driven slapper detonators located in said second explosive means; a laser located in said proximal end of said casing; optical fibers connecting said laser to said first and second laser-driven slapper detonators; and optical switch means located in series with said optical fibers connected to said plurality of first laser-driven slapper detonators for blocking or passing light from said laser to said plurality of first laser-driven slapper detonators.

  2. Opaque rock fragments

    SciTech Connect

    Abhijit, B.; Molinaroli, E.; Olsen, J.

    1987-05-01

    The authors describe a new, rare, but petrogenetically significant variety of rock fragments from Holocene detrital sediments. Approximately 50% of the opaque heavy mineral concentrates from Holocene siliciclastic sands are polymineralic-Fe-Ti oxide particles, i.e., they are opaque rock fragments. About 40% to 70% of these rock fragments show intergrowth of hm + il, mt + il, and mt + hm +/- il. Modal analysis of 23,282 opaque particles in 117 polished thin sections of granitic and metamorphic parent rocks and their daughter sands from semi-arid and humid climates show the following relative abundances. The data show that opaque rock fragments are more common in sands from igneous source rocks and that hm + il fragments are more durable. They assume that equilibrium conditions existed in parent rocks during the growth of these paired minerals, and that the Ti/Fe ratio did not change during oxidation of mt to hm. Geothermometric determinations using electron probe microanalysis of opaque rock fragments in sand samples from Lake Erie and the Adriatic Sea suggest that these rock fragments may have equilibrated at approximately 900/sup 0/ and 525/sup 0/C, respectively.

  3. Auroral fragmentation into patches

    NASA Astrophysics Data System (ADS)

    Shiokawa, Kazuo; Hashimoto, Ayumi; Hori, Tomoaki; Sakaguchi, Kaori; Ogawa, Yasunobu; Donovan, Eric; Spanswick, Emma; Connors, Martin; Otsuka, Yuichi; Oyama, Shin-Ichiro; Nozawa, Satonori; McWilliams, Kathryn

    2014-10-01

    Auroral patches in diffuse auroras are very common features in the postmidnight local time. However, the processes that produce auroral patches are not yet well understood. In this paper we present two examples of auroral fragmentation which is the process by which uniform aurora is broken into several fragments to form auroral patches. These examples were observed at Athabasca, Canada (geomagnetic latitude: 61.7°N), and Tromsø, Norway (67.1°N). Captured in sequences of images, the auroral fragmentation occurs as finger-like structures developing latitudinally with horizontal-scale sizes of 40-100 km at ionospheric altitudes. The structures tend to develop in a north-south direction with speeds of 150-420 m/s without any shearing motion, suggesting that pressure-driven instability in the balance between the earthward magnetic-tension force and the tailward pressure gradient force in the magnetosphere is the main driving force of the auroral fragmentation. Therefore, these observations indicate that auroral fragmentation associated with pressure-driven instability is a process that creates auroral patches. The observed slow eastward drift of aurora during the auroral fragmentation suggests that fragmentation occurs in low-energy ambient plasma.

  4. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ...

  5. Fragment capture device

    DOEpatents

    Payne, Lloyd R.; Cole, David L.

    2010-03-30

    A fragment capture device for use in explosive containment. The device comprises an assembly of at least two rows of bars positioned to eliminate line-of-sight trajectories between the generation point of fragments and a surrounding containment vessel or asset. The device comprises an array of at least two rows of bars, wherein each row is staggered with respect to the adjacent row, and wherein a lateral dimension of each bar and a relative position of each bar in combination provides blockage of a straight-line passage of a solid fragment through the adjacent rows of bars, wherein a generation point of the solid fragment is located within a cavity at least partially enclosed by the array of bars.

  6. Fragmentation in Biaxial Tension

    SciTech Connect

    Campbell, G H; Archbold, G C; Hurricane, O A; Miller, P L

    2006-06-13

    We have carried out an experiment that places a ductile stainless steel in a state of biaxial tension at a high rate of strain. The loading of the ductile metal spherical cap is performed by the detonation of a high explosive layer with a conforming geometry to expand the metal radially outwards. Simulations of the loading and expansion of the metal predict strain rates that compare well with experimental observations. A high percentage of the HE loaded material was recovered through a soft capture process and characterization of the recovered fragments provided high quality data, including uniform strain prior to failure and fragment size. These data were used with a modified fragmentation model to determine a fragmentation energy.

  7. A modified ELISA technique for the titration of antibodies to polio virus as an alternative to a virus neutralization test.

    PubMed

    Hagenaars, A M; van Delft, R W; Nagel, J; van Steenis, G; van Wezel, A L

    1983-04-01

    An ELISA based on inhibition of antibody binding for the determination of antibodies to polio virus type I is described. F(ab1)2-fragments of bovine antibodies to polio virus type I are used as the capture antibody thus lowering the background staining. A good correlation was found between the poliovirus neutralizing antibody level and the antibody titers as determined by ELISA.

  8. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  9. Edelman's view on the discovery of antibodies.

    PubMed

    Ribatti, Domenico

    2015-04-01

    Gerald M. Edelman began working to the structure of antibodies when joined as graduate student the laboratory of Henry Kunkel in 1958 at the "Rockefeller University" in New York, obtaining his doctorate in 1960. Edelman's focus on the structure of antibodies led to the 1972 Nobel Prize in Physiology or Medicine along with Rodney R. Porter. Edelman and Porter decided to approach the problem of antibodies structure by splitting. In 1959, Porter published a report in which he used the enzyme papain to cleave the antibody molecule into three pieces of about 50,000 Da, corresponding to the two Fab (antigen-binding) and constant Fc (crystallizable) fragments. In the same year, Edelman showed that reduction of the disulfide bonds of antibodies in the presence of denaturizing agents led to dissociation of the molecule into smaller pieces, now known to be the light (L) and heavy (H) chains.

  10. 21 CFR 866.5530 - Immunoglobulin G (Fc fragment specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunoglobulin G (resulting from breakdown of immunoglobulin G antibodies) in urine, serum, and other body fluids. Measurement of immunoglobulin G Fc fragments aids in the diagnosis of plasma cell...

  11. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  12. The maturation of antibody technology for the HIV epidemic.

    PubMed

    Winnall, Wendy R; Beasley, Matthew D; Center, Rob J; Parsons, Matthew S; Kiefel, Ben R; Kent, Stephen J

    2014-08-01

    Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV. PMID:24797582

  13. Bacterial expression and purification of recombinant bovine Fab fragments.

    PubMed

    O'Brien, Philippa M; Maxwell, Gavin; Campo, M Saveria

    2002-02-01

    We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli. PMID:11812221

  14. Antibody Engineering and Therapeutics

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  15. Heavy fragment radioactivities

    SciTech Connect

    Price, P.B.

    1987-12-10

    This recently discovered mode of radioactive decay, like alpha decay and spontaneous fission, is believed to involve tunneling through the deformation-energy barrier between a very heavy nucleus and two separated fragments the sum of whose masses is less than the mass of the parent nucleus. In all known cases the heavier of the two fragments is close to doubly magic /sup 208/Pb, and the lighter fragment has even Z. Four isotopes of Ra are known to emit /sup 14/C nuclei; several isotopes of U as well as /sup 230/Th and /sup 231/Pa emit Ne nuclei; and /sup 234/U exhibits four hadronic decay modes: alpha decay, spontaneous fission, Ne decay and Mg decay.

  16. Okazaki fragment metabolism.

    PubMed

    Balakrishnan, Lata; Bambara, Robert A

    2013-02-01

    Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are ~1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

  17. Allogenous tooth fragment reattachment

    PubMed Central

    Maitin, Nitin; Maitin, Shipra; Rastogi, Khushboo; Bhushan, Rajarshi

    2013-01-01

    Coronal fractures of the anterior teeth are a common form of dental trauma and its sequelae may impair the establishment and accomplishment of an adequate treatment plan. Among the various treatment options, reattachment of a crown fragment obtained from a previously extracted tooth is a conservative treatment that should be considered for crown fractures of anterior teeth. This article reports reattachment of an allogenous tooth fragment in a fractured maxillary lateral incisor in a 38-year-old patient. It is suggested that allogenous reattachment in a fractured anterior tooth serves to be a better alternative and should be further researched. Aesthetic and functional rehabilitation of a fractured complicated anterior crown using allogenous tooth fragment is a better alternative to other more conventional treatment options. PMID:23845684

  18. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

    López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; González-González, Everardo; Trujillo-de Santiago, Grissel; Ponce-Ponce de León, César Alejandro; Zhang, Yu Shrike; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Alvarez, Mario Moisés

    2015-01-01

    Background Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV. Methods/Principal Findings We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures. Conclusion/Significance Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications. PMID:26489048

  19. IMPACT fragmentation model developments

    NASA Astrophysics Data System (ADS)

    Sorge, Marlon E.; Mains, Deanna L.

    2016-09-01

    The IMPACT fragmentation model has been used by The Aerospace Corporation for more than 25 years to analyze orbital altitude explosions and hypervelocity collisions. The model is semi-empirical, combining mass, energy and momentum conservation laws with empirically derived relationships for fragment characteristics such as number, mass, area-to-mass ratio, and spreading velocity as well as event energy distribution. Model results are used for several types of analysis including assessment of short-term risks to satellites from orbital altitude fragmentations, prediction of the long-term evolution of the orbital debris environment and forensic assessments of breakup events. A new version of IMPACT, version 6, has been completed and incorporates a number of advancements enabled by a multi-year long effort to characterize more than 11,000 debris fragments from more than three dozen historical on-orbit breakup events. These events involved a wide range of causes, energies, and fragmenting objects. Special focus was placed on the explosion model, as the majority of events examined were explosions. Revisions were made to the mass distribution used for explosion events, increasing the number of smaller fragments generated. The algorithm for modeling upper stage large fragment generation was updated. A momentum conserving asymmetric spreading velocity distribution algorithm was implemented to better represent sub-catastrophic events. An approach was developed for modeling sub-catastrophic explosions, those where the majority of the parent object remains intact, based on estimated event energy. Finally, significant modifications were made to the area-to-mass ratio distribution to incorporate the tendencies of different materials to fragment into different shapes. This ability enabled better matches between the observed area-to-mass ratios and those generated by the model. It also opened up additional possibilities for post-event analysis of breakups. The paper will discuss

  20. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fd fragment specific) immunological test system. 866.5540 Section 866.5540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement...

  1. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoglobulin G (Fd fragment specific) immunological test system. 866.5540 Section 866.5540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement...

  2. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoglobulin G (Fd fragment specific) immunological test system. 866.5540 Section 866.5540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement...

  3. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoglobulin G (Fd fragment specific) immunological test system. 866.5540 Section 866.5540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement...

  4. Blocking Ligand Occupancy of the αVβ3 Integrin Inhibits the Development of Nephropathy in Diabetic Pigs

    PubMed Central

    Maile, Laura A.; Busby, Walker H.; Gollahon, Katherine A.; Flowers, William; Garbacik, Nikol; Garbacik, Stefani; Stewart, Kara; Nichols, Timothy; Bellinger, Dwight; Patel, Amit; Dunbar, Paul; Medlin, Matt

    2014-01-01

    Hyperglycemia stimulates secretion of αVβ3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVβ3 integrin. This study determined whether blocking ligand occupancy of αVβ3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs received an F(ab)2 fragment of an antibody directed against the extracellular domain of the β3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 ± 50 μg of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 ± 57 μg/mg), whereas diabetic animals treated with the anti-β3 F(ab)2 excreted 119 ± 55 μg/mg (P < .05). Mesangial volume/glomerular volume increased to 21 ± 2.4% in control-treated diabetic animals compared with 14 ± 2.8% (P < .01) in animals treated with active antibody. Diabetic animals treated with control F(ab)2 had significantly less glomerular podocin staining compared with nondiabetic animals, and this decrease was attenuated by treatment with anti-β3 F(ab)2. Glomerular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals (212 ± 14 nm) compared with nondiabetic animals (170 ± 8.8 nm), but it was unchanged (159.9 ± 16.4 nm) in animals receiving anti-β3 F(ab)2. Podocyte foot process width was greater in control, F(ab)2-treated, animals (502 ± 34 nm) compared with animals treated with the anti-β3 F(ab)2 (357 ± 47 nm, P < .05). Renal β3 tyrosine phosphorylation decreased from 13 934 ± 6437 to 6730 ± 1524 (P < .01) scanning units in the anti-β3-treated group. We conclude that administration of an antibody that inhibits activation of the β3-subunit of αVβ3 that is induced by hyperglycemia attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it may have utility in preventing the progression of this disease complication. PMID:25171599

  5. Target fragmentation in radiobiology

    NASA Technical Reports Server (NTRS)

    Wilson, John W.; Cucinotta, Francis A.; Shinn, Judy L.; Townsend, Lawrence W.

    1993-01-01

    Nuclear reactions in biological systems produce low-energy fragments of the target nuclei seen as local high events of linear energy transfer (LET). A nuclear-reaction formalism is used to evaluate the nuclear-induced fields within biosystems and their effects within several biological models. On the basis of direct ionization interaction, one anticipates high-energy protons to have a quality factor and relative biological effectiveness (RBE) of unity. Target fragmentation contributions raise the effective quality factor of 10 GeV protons to 3.3 in reasonable agreement with RBE values for induced micronuclei in bean sprouts. Application of the Katz model indicates that the relative increase in RBE with decreasing exposure observed in cell survival experiments with 160 MeV protons is related solely to target fragmentation events. Target fragment contributions to lens opacity given an RBE of 1.4 for 2 GeV protons in agreement with the work of Lett and Cox. Predictions are made for the effective RBE for Harderian gland tumors induced by high-energy protons. An exposure model for lifetime cancer risk is derived from NCRP 98 risk tables, and protraction effects are examined for proton and helium ion exposures. The implications of dose rate enhancement effects on space radiation protection are considered.

  6. Fragment Separator ACCULINNA-2

    SciTech Connect

    Krupko, S. A.; Fomichev, A. S.; Chudoba, V.; Daniel, A. V.; Golovkov, M. S.; Gorshkov, V. A.; Oganessian, Yu. Ts.; Sidorchuk, S. I.; Slepnev, R. S.; Stepantsov, S. V.; Ter-Akopian, G. M.; Wolski, R.; Grigorenko, L. V.; Tarasov, O. B.; Ershov, S. N.; Lukyanov, V. K.; Danilin, B. V.; Korsheninnikov, A. A.; Goldberg, V. Z.; Mukha, I. G.

    2010-04-30

    Project of a new in-flight fragment separator is proposed as a part of the third generation DRIBs facilities in Dubna. As compared to the existing separator ACCULINNA, beam intensity should be increased by a factor 10-15, the beam quality improved and the RIB assortment should broaden considerably at ACCULINNA-2. Research program and structure are outlined for the new instrument.

  7. Comment on diquark fragmentation

    SciTech Connect

    Fredriksson, S.; Larsson, T.

    1983-07-01

    We discuss diquark fragmentation and suggest that a spectator uu system in deep-inelastic lepton-nucleon scattering has a larger breakup probability than a ud system. The reason for this is argued to be that half of the leftover ud systems are in bound (ud)/sub 0/ diquark configurations, while no such bound uu diquarks exist.

  8. Role of muscarinic-3 receptor antibody in systemic sclerosis: correlation with disease duration and effects of IVIG.

    PubMed

    Kumar, Sumit; Singh, Jagmohan; Kedika, Ramalinga; Mendoza, Fabian; Jimenez, Sergio A; Blomain, Erik S; DiMarino, Anthony J; Cohen, Sidney; Rattan, Satish

    2016-06-01

    Gastrointestinal dysmotility in systemic sclerosis (SSc) is associated with autoantibodies against muscarinic-3 receptor (M3-R). We investigated the temporal course of the site of action of these autoantibodies at the myenteric neurons (MN) vs. the smooth muscle (SM) M3-R in relation to disease duration, and determined the role of intravenous immunoglobulin (IVIG) in reversing these changes. Immunoglobulins purified from SSc patients (SScIgG) were used to assess their differential binding to MN and SM (from rat colon) employing immunohistochemistry (IHC). Effect of SScIgG on neural and direct muscle contraction was determined by cholinergic nerve stimulation and bethanechol-induced SM contraction. Effects of IVIG and its antigen-binding fragment F(ab')2 on SScIgG binding were studied by enzyme-linked immunosorbent assay (ELISA) of rat colonic longitudinal SM myenteric plexus (LSMMP) lysate and to second extracellular loop peptide of M3-R (M3-RL2). SScIgG from all patients demonstrated significantly higher binding to MN than to SM. With progression of SSc duration, binding at MN and SM increased in a linear fashion with a correlation coefficient of 0.696 and 0.726, respectively (P < 0.05). SScIgG-mediated attenuation of neural and direct SM contraction also increased with disease duration. ELISA analysis revealed that IVIG and F(ab')2 significantly reduced SScIgG binding to LSMMP lysate and M3-RL2. Dysmotility in SSc occurs sequentially, beginning with SScIgG-induced blockage of cholinergic neurotransmission (neuropathy), which progresses to inhibition of acetylcholine action at the SM cell (myopathy). IVIG reverses this cholinergic dysfunction at the neural and myogenic receptors by anti-idiotypic neutralization of SScIgG.

  9. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    SciTech Connect

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue

    2009-05-13

    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  10. Purification and properties of non-precipitating rabbit antibodies

    PubMed Central

    Margni, R.; Binaghi, R.

    1972-01-01

    Precipitating and non-precipitating anti-egg albumin and anti-dinitrophenyl rabbit antibodies were specifically purified from hyperimmunized sera. Both populations of antibody were similar with regard to electrophoretic mobility and molecular size. Non-precipitating antibodies brought about passive haemagglutination and PCA, although with less efficiency than precipitating antibodies. On the other hand, only precipitating antibodies fixed complement and produced a reverse Arthus reaction. The F(ab′)2 fragment obtained from non-precipitating antibody did not precipitate with antigen. These results are compatible with the hypothesis that non-precipitability is due to a particular configuration of the molecule that makes it impossible for one molecule of antibody to combine with two different molecules of antigen simultaneously. PMID:4259530

  11. Polynucleotides encoding anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  12. Cryobiology of coral fragments.

    PubMed

    Hagedorn, Mary; Farrell, Ann; Carter, Virginia L

    2013-02-01

    Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1M dimethyl sulfoxide at 1.0, 1.5 and 2.0h exposures, and 1.5M dimethyl sulfoxide at 1.0 and 1.5h exposures, P>0.05, ANOVA), whereas 2M concentrations did not (P<0.05, ANOVA). A seasonal response to chilling was observed in the coral tissue, but not in the zooxanthellae. During the winter when the fragments were chilled, the coral tissue remained relatively intact (∼25% loss) post-treatment, but the zooxanthellae numbers in the tissue declined after 5min of chilling (P<0.05, ANOVA). However, in the late spring, coral tissue (∼75% loss) and zooxanthellae numbers declined in response to chilling alone (P<0.05, ANOVA). When a cryoprotectant (1M dimethyl sulfoxide) was used in concert with chilling it protected the coral against tissue loss after 45min of cryoprotectant exposure (P>0.05, ANOVA), but it did not protect against the loss of zooxanthellae (P<0.05, ANOVA). The zooxanthellae are the most sensitive element in the coral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity.

  13. Fragmentation of cancer cells

    NASA Astrophysics Data System (ADS)

    Vanapalli, Siva; Kamyabi, Nabiollah

    Tumor cells have to travel through blood capillaries to be able to metastasize and colonize in distant organs. Among the numerous cells that are shed by the primary tumor, very few survive in circulation. In vivo studies have shown that tumor cells can undergo breakup at microcapillary junctions affecting their survival. It is currently unclear what hydrodynamic and biomechanical factors contribute to fragmentation and moreover how different are the breakup dynamics of highly and weakly metastatic cells. In this study, we use microfluidics to investigate flow-induced breakup of prostate and breast cancer cells. We observe several different modes of breakup of cancer cells, which have striking similarities with breakup of viscous drops. We quantify the breakup time and find that highly metastatic cancer cells take longer to breakup than lowly metastatic cells suggesting that tumor cells may dynamically modify their deformability to avoid fragmentation. We also identify the role that cytoskeleton and membrane plays in the breakup process. Our study highlights the important role that tumor cell fragmentation plays in cancer metastasis. Cancer Prevention and Research Institute of Texas.

  14. Fracture, failure, and fragmentation

    SciTech Connect

    Dienes, J.K.

    1984-01-01

    Though continuum descriptions of material behavior are useful for many kinds of problems, particularly those involving plastic flow, a more general approach is required when the failure is likely to involve growth and coalescence of a large number of fractures, as in fragmentation. Failures of this kind appear frequently in rapid dynamic processes such as those resulting from impacts and explosions, particularly in the formation of spall fragments. In the first part of this paper an approach to formulating constitutive relations that accounts for the opening, shear and growth of an ensemble of cracks is discussed. The approach also accounts for plastic flow accompanying fragmentation. The resulting constitutive relations have been incorporated into a Lagrangean computer program. In the second part of this paper a theoretical approach to coalescence is described. The simplest formulation makes use of a linear Liouville equation, with crack growth limited by the mean free path of cracks, assumed constant. This approach allows for an anisotropic distribution of cracks. An alternative approach is also described in which the decrease of the mean free path with increasing crack size is accounted for, but the crack distribution is assumed isotropic. A reduction of the governing Liouville equation to an ordinary differential equation of third order is possible, and the result can be used to determine how mean-free-path decreases with increasing crack size.

  15. [Antinuclear antibodies].

    PubMed

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  16. The Structure of Natural and Recombinant Antibodies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    Immunoglobulins (Ig) isotypes A, D, E, G, and M are glycoproteins which are mainly composed of a "Y"-shaped Ig monomer (~150 kDa), consisting of two light and two heavy chains. Both light and heavy chains contain variable (N-terminal) and constant regions (C-terminal). Each light chain consists of one variable domain and one constant domain, whereas each heavy chain has one variable domain and three constant domains. However, heavy-chain antibodies consisting of only heavy chains and lacking the light chains are found in camelids and cartilaginous fishes. Unlike other immunoglobulins, the heavy chain of avian antibody IgY (~180 kDa) consists of four constant domains. The single-chain variable fragment (scFv; ~25 kDa) of an antibody contains variable regions of antibody heavy and light chains. The fragment antigen-binding (Fab; ~50 kDa) region has the full antibody light chain but the heavy chain is composed of a variable region and one constant domain.

  17. Monoclonal antibody against the N-terminal end of human plasma fibronectin.

    PubMed Central

    Vartio, T; Salonen, E M; De Petro, G; Barlati, S; Miggiano, V; Stähli, C; Virgallita, G; Takács, B; Vaheri, A

    1983-01-01

    Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin. Images Fig. 1. Fig. 2. PMID:6194791

  18. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

    SciTech Connect

    Eisenberg, R.J.; Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.

    1982-02-01

    We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.

  19. Virtual fragment preparation for computational fragment-based drug design.

    PubMed

    Ludington, Jennifer L

    2015-01-01

    Fragment-based drug design (FBDD) has become an important component of the drug discovery process. The use of fragments can accelerate both the search for a hit molecule and the development of that hit into a lead molecule for clinical testing. In addition to experimental methodologies for FBDD such as NMR and X-ray Crystallography screens, computational techniques are playing an increasingly important role. The success of the computational simulations is due in large part to how the database of virtual fragments is prepared. In order to prepare the fragments appropriately it is necessary to understand how FBDD differs from other approaches and the issues inherent in building up molecules from smaller fragment pieces. The ultimate goal of these calculations is to link two or more simulated fragments into a molecule that has an experimental binding affinity consistent with the additive predicted binding affinities of the virtual fragments. Computationally predicting binding affinities is a complex process, with many opportunities for introducing error. Therefore, care should be taken with the fragment preparation procedure to avoid introducing additional inaccuracies.This chapter is focused on the preparation process used to create a virtual fragment database. Several key issues of fragment preparation which affect the accuracy of binding affinity predictions are discussed. The first issue is the selection of the two-dimensional atomic structure of the virtual fragment. Although the particular usage of the fragment can affect this choice (i.e., whether the fragment will be used for calibration, binding site characterization, hit identification, or lead optimization), general factors such as synthetic accessibility, size, and flexibility are major considerations in selecting the 2D structure. Other aspects of preparing the virtual fragments for simulation are the generation of three-dimensional conformations and the assignment of the associated atomic point charges

  20. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  1. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  2. New Scalings in Nuclear Fragmentation

    SciTech Connect

    Bonnet, E.; Bougault, R.; Galichet, E.; Gagnon-Moisan, F.; Guinet, D.; Lautesse, P.; Marini, P.; Parlog, M.

    2010-10-01

    Fragment partitions of fragmenting hot nuclei produced in central and semiperipheral collisions have been compared in the excitation energy region 4-10 MeV per nucleon where radial collective expansion takes place. It is shown that, for a given total excitation energy per nucleon, the amount of radial collective energy fixes the mean fragment multiplicity. It is also shown that, at a given total excitation energy per nucleon, the different properties of fragment partitions are completely determined by the reduced fragment multiplicity (i.e., normalized to the source size). Freeze-out volumes seem to play a role in the scalings observed.

  3. Fragmentation function measurements at Belle

    SciTech Connect

    Seidl, Ralf; Vossen, Anselm; Leitgab, Martin; Grosse-Perdekamp, Matthias; Giordano, Francesca; Ogawa, Akio

    2011-12-14

    The precision measurement of fragmentation functions is an important requirement to study the spin structure of the nucleon. Unpolarized fragmentation functions at reasonably low scale and high fractional energy are necessary to complement the measurements mostly performed at LEP in order to obtain high enough precision for measurements at semi-inclusive DIS experiments and at RHIC. Those can be obtained from the abundant data collected with the Belle detector at the e{sup +}e{sup -} collider KEKB. In addition one can cleanly measure the transversely polarized fragmentation functions such as the Collins fragmentation function and the interference fragmentation functions. Both have been obtained with great precision at Belle.

  4. Fragment oriented molecular shapes.

    PubMed

    Hain, Ethan; Camacho, Carlos J; Koes, David Ryan

    2016-05-01

    Molecular shape is an important concept in drug design and virtual screening. Shape similarity typically uses either alignment methods, which dynamically optimize molecular poses with respect to the query molecular shape, or feature vector methods, which are computationally less demanding but less accurate. The computational cost of alignment can be reduced by pre-aligning shapes, as is done with the Volumetric-Aligned Molecular Shapes (VAMS) method. Here, we introduce and evaluate fragment oriented molecular shapes (FOMS), where shapes are aligned based on molecular fragments. FOMS enables the use of shape constraints, a novel method for precisely specifying molecular shape queries that provides the ability to perform partial shape matching and supports search algorithms that function on an interactive time scale. When evaluated using the challenging Maximum Unbiased Validation dataset, shape constraints were able to extract significantly enriched subsets of compounds for the majority of targets, and FOMS matched or exceeded the performance of both VAMS and an optimizing alignment method of shape similarity search. PMID:27085751

  5. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  6. Detection of complement activation using monoclonal antibodies against C3d

    PubMed Central

    Thurman, Joshua M.; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A.; Mitchell, Lynne M.; Hourcade, Dennis E.; Hannan, Jonathan P.; Kovacs, James M.; Coughlin, Beth; Woodell, Alex S.; Pickering, Matthew C.; Rohrer, Bärbel; Holers, V. Michael

    2013-01-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation–associated tissue inflammation. PMID:23619360

  7. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  8. Dissecting antibodies with regards to linear and conformational epitopes.

    PubMed

    Forsström, Björn; Axnäs, Barbara Bisławska; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

    2015-01-01

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

  9. Anti Transglutaminase Antibodies Cause Ataxia in Mice

    PubMed Central

    Boscolo, Sabrina; Lorenzon, Andrea; Sblattero, Daniele; Florian, Fiorella; Stebel, Marco; Marzari, Roberto; Not, Tarcisio; Aeschlimann, Daniel; Ventura, Alessandro; Hadjivassiliou, Marios; Tongiorgi, Enrico

    2010-01-01

    Background Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the mo