Sample records for bispecific monoclonal antibody

  1. Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.

    PubMed Central

    Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma. Images Figure 7 PMID:8795576

  2. Bispecific antibody conjugates in therapeutics

    Microsoft Academic Search

    Ying Cao; Laura Lam

    2003-01-01

    Bispecific monoclonal antibodies have drawn considerable attention from the research community due to their unique structure against two different antigens. The two-arm structure of bsMAb allows researchers to place a therapeutic agent on one arm while allowing the other to specifically target the disease site. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc. Furthermore, bsMAb may

  3. Bispecific antibodies for cancer therapy: the light at the end of the Patrick Chames1

    E-print Network

    Paris-Sud XI, Université de

    1 Bispecific antibodies for cancer therapy: the light at the end of the tunnel? Patrick Chames1, bispecific, cancer, therapy, clinical trials Abbreviation mAb: monoclonal antibodies, bsAbs: bispecific antibodies, ADCC: antibody dependent cell mediated cytotoxicity, scFv: single chain Fv fragment, Bi

  4. Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

    PubMed

    Ouyang, Hui; Wang, Limin; Yang, Shijia; Wang, Wenwen; Wang, Lin; Liu, Fengquan; Fu, Zhifeng

    2015-03-01

    The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody. PMID:25622025

  5. Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

    PubMed Central

    Nisticò, P; Mortarini, R; De Monte, L B; Mazzocchi, A; Mariani, M; Malavasi, F; Parmiani, G; Natali, P G; Anichini, A

    1992-01-01

    Intratumor heterogeneity for susceptibility to cytotoxic T lymphocytes (CTL)-mediated lysis represents a major obstacle to cancer adoptive immunotherapy. To overcome the heterogeneity observed in terms of susceptibility of target cells to cell-mediated lysis, in this study we used two purified bispecific monoclonal antibodies (bsmAbs) that recognize molecules expressed by cytotoxic effector cells (CD3 and IgG Fc receptorial molecules), as well as one high molecular weight melanoma-associated antigen (HMW-MAA). The ability of these reagents to enhance or induce a relevant in vitro cytotoxic activity by a CTL clone (CTL 49) isolated from PBL of a melanoma patient was tested on a large panel of autologous and allogeneic melanoma cell lines and clones. Functional studies revealed that the CTL 49 clone lysed all the HMW-MAA+ tumor lines in the presence of bsmAbs and that these reagents affected the target lysis in a cooperative fashion. The effectiveness of bsmAbs in overcoming the heterogeneous susceptibility of human melanoma cells to cell-mediated lysis may find practical implications in cancer adoptive immunotherapy. Images PMID:1387883

  6. Bispecific monoclonal antibodies against a viral and an enzyme: utilities in ultrasensitive virus ELISA and phage display technology

    Microsoft Academic Search

    Fei Liu; S Guttikonda; M. R Suresh

    2003-01-01

    A quadroma (hybrid-hybridoma) secreting bispecific antibodies with one paratope specific for M13 bacteriophage coat protein and another paratope specific for alkaline phosphatase (AP) was developed by electro-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter (FACS). The anti-phage M13\\/anti-AP bsMAbs were purified from anti-phage M13 monospecific MAb by a novel affinity method using Mimetic Blue

  7. 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)

  8. Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay.

    PubMed

    Kenigsberg, R L; Elliott, P J; Cuello, A C

    1991-02-15

    Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed. PMID:1999653

  9. Recombinant bispecific antibodies for cellular cancer immunotherapy.

    PubMed

    Müller, Dafne; Kontermann, Roland E

    2007-08-01

    Bispecific antibodies recognizing two different antigens present on different cells have been developed for cellular cancer therapy in which cytotoxic effector cells are recruited to tumor cells. Initial studies with bispecific antibodies have not reached satisfactory clinical endpoints, mainly due to low efficacy, Fc-mediated side effects and immunogenicity. This has resulted in a declining interest in bispecific antibodies for cancer therapy. However, growing knowledge in effector cell biology and the implementation of antibody engineering technologies has led to a revival and the development of novel or improved strategies. Various recombinant bispecific antibodies have demonstrated efficacy in vitro andin vivo, with the first recombinant antibody molecule currently in clinical trials for the treatment of B-cell malignancies. PMID:17694444

  10. In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

    PubMed Central

    Negri, D. R.; Tosi, E.; Valota, O.; Ferrini, S.; Cambiaggi, A.; Sforzini, S.; Silvani, A.; Ruffini, P. A.; Colnaghi, M. I.; Canevari, S.

    1995-01-01

    The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR). PMID:7547242

  11. Targeting of saporin to CD25-positive normal and neoplastic lymphocytes by an anti-saporin/anti-CD25 bispecific monoclonal antibody: in vitro evaluation.

    PubMed Central

    Tazzari, P. L.; Zhang, S.; Chen, Q.; Sforzini, S.; Bolognesi, A.; Stirpe, F.; Xie, H.; Moretta, A.; Ferrini, S.

    1993-01-01

    This study has been designed to verify the specific toxicity of saporin, a type 1 ribosome-inactivating protein (RIP), with the same activity as ricin A chain, targeted by a bispecific monoclonal antibody (bimAb) recognising both the CD25 antigen and the RIP. The CD25 antigen is expressed by lymphoid populations upon activation and by leukaemias and lymphomas with an activated membrane phenotype (Hodgkin's lymphoma, anaplastic large cell lymphoma, adult T cell leukaemia). The bimAb-saporin mixture was tested on CD25+ targets at different bimAb and saporin concentrations. Saporin, in the presence of a bimAb concentration of 10(-9) M, inhibited protein synthesis by CD25+ neoplastic lymphocytes (L540 and MT2 cell lines) with IC50S (concentrations giving 50% of inhibition) ranging from 8 x 10(-12) M to 3 x 10(-11) M. The saporin-bimAb mixture was also effective in blocking the phytohaemagglutinin-driven proliferation of normal lymphocytes, whereas it displayed the same level of toxicity exerted by saporin alone on an irrelevant CD25-negative cell line (EBV-infected B lymphoblastoid cell line). From these results it is possible to envisage a clinical use of this bimAb as a cytotoxic agent for CD25+ leukaemias and lymphomas, as well as an immunosuppressive agent for severe immune disorders such as graft-vs-host disease (GVHD) and transplanted organ rejection. PMID:8512810

  12. Enlarging the repertoire of therapeutic monoclonal antibodies platforms: domesticating half molecule exchange to produce stable IgG4 and IgG1 bispecific antibodies.

    PubMed

    Yang, Xiaoyu; Ambrogelly, Alexandre

    2014-12-01

    Half molecule exchange is the process whereby two IgG4 molecules exchange a heavy chain-light chain unit to form a new IgG4 entity with specificity towards two different antigens. While this unique property of IgG4 molecules confers anti-inflammatory properties in nature, it is not a desirable feature for a therapeutic mAb. Engineering of the IgG4 hinge region making it resemble that of an IgG1 is sufficient to dramatically reduce half molecule exchange in vitro and in vivo. The S228P modification of the hinge confers pharmaceutical properties to IgG4 equivalent to those of standard IgG1, while retaining the inability to trigger ADCC and CDC. Application of the molecular precepts underlying half molecule exchange between IgG4 molecules to IgG1 scaffolds offers the possibility to produce bispecific antibodies in vitro. PMID:25254943

  13. A multifunctional bispecific antibody protects against Pseudomonas aeruginosa.

    PubMed

    DiGiandomenico, Antonio; Keller, Ashley E; Gao, Cuihua; Rainey, Godfrey J; Warrener, Paul; Camara, Mareia M; Bonnell, Jessica; Fleming, Ryan; Bezabeh, Binyam; Dimasi, Nazzareno; Sellman, Bret R; Hilliard, Jamese; Guenther, Caitlin M; Datta, Vivekananda; Zhao, Wei; Gao, Changshou; Yu, Xiang-Qing; Suzich, JoAnn A; Stover, C Kendall

    2014-11-12

    Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4?Pa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens. PMID:25391481

  14. Clinical pharmacology of bispecific antibody constructs.

    PubMed

    Rathi, Chetan; Meibohm, Bernd

    2015-03-01

    The confluence of rapid scientific advancements especially in protein engineering and recombinant technology, unmet medical needs, and commercial incentives have led to the development of the next generation of therapeutic proteins. Bispecific antibody constructs are one of the novel strategies that is being pursued, combining the ability to bind simultaneously to two distinct targets and the advantages of purpose-designed and optimized antibody-based scaffolds. Their pharmacokinetic and pharmacodynamic properties, including their immunogenic potential, are closely related to their structural features and ability to interact with disposition mechanisms of immunoglobulin molecules. Catumaxomab and blinatumomab are bispecific constructs that are approved for clinical use and have provided clinical pharmacology data for this novel class of therapeutics. This knowledgebase on the clinical behavior of bispecific therapeutic proteins is poised to rapidly evolve over the next few years with many development programs having entered the clinical development stage. PMID:25707960

  15. [Monoclonal antibodies].

    PubMed

    Koyama, K

    1988-08-01

    Since the first successful application of somatic cell fusion by Kohler and Milstein to generate hybridomas secreting monoclonal antibodies (Mabs), this hybridoma technique has been widely applied for production of Mabs against a wide variety of antigens. In the lecture, general procedures for establishment of hybridomas and characteristic properties of Mabs were briefly explained and followed by presentation of our clinical studies using Mabs produced in our laboratory to hCG, sperm and tumor cells. 1. Monoclonal antibodies to hCG. Three hybridomas (5D4, 6E4, 2F8) were established by fusing mouse myeloma cells (P3U1) with spleen cells from BALB/c mice immunized with partially purified hCG. Mabs were obtained either from hybridoma culture media or from ascites of mice inoculated the hybridoma cells intraperitoneally. Three Mabs obtained were all IgG1 and each had a unique binding specificity to hCG, hCG-beta, hCG-alpha and LH. They were applied for development of specific, sensitive and easy to perform hCG assays. A reverse passive hemagglutination assay of urinary hCG with a sensitivity of 12.5 IU/l and a sandwich-enzyme immunoassay of serum hCG with a sensitivity of 0.1 mIU/ml were presented. 2. Monoclonal antibodies to human spermatozoa. For elucidation of mechanisms for induction of auto- or iso-sperm immunization and infertility by antisperm antibodies, antigen analysis of seminal components was essential. For this purpose, several Mabs with strong sperm immobilizing (SI) and agglutinating (SA) activities were generated in mice, rats and humans and the corresponding antigens were analysed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3075226

  16. Bispecific antibody platforms for cancer immunotherapy.

    PubMed

    Lameris, Roeland; de Bruin, Renée C G; Schneiders, Famke L; van Bergen en Henegouwen, Paul M P; Verheul, Henk M W; de Gruijl, Tanja D; van der Vliet, Hans J

    2014-12-01

    Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward. PMID:25195094

  17. Universal Bispecific Antibody for Targeting Tumor Cells for Destruction by Cytotoxic T Cells

    NASA Astrophysics Data System (ADS)

    Gilliland, Lisa K.; Clark, Michael R.; Waldmann, Herman

    1988-10-01

    Previous studies have demonstrated that bispecific hybrid antibodies produced by cell-cell fusion or chemically conjugated heteroaggregates can direct cytotoxic T lymphocytes to kill target cells for which they have no intrinsic specificity, a phenomenon we call effector cell retargeting (ECR). These studies used bispecific reagents with one specificity directed to CD3 or Ti on the effector cell and the other directed to a target cell antigen. To avoid the need to create different hybrid hybridomas for each target antigen we have developed a universal means to elicit ECR through the use of an antiglobulin step. We have constructed a bispecific hybrid antibody with dual specificity for CD3 and a rat immunoglobulin light chain allotype. This bispecific antibody could mediate ECR to a range of target cells, each coated with distinct surface-binding rat monoclonal antibodies. A particular advantage of targeting to surface-bound monoclonal antibodies is that all other available effector systems may also attack the same antibody-coated target cell.

  18. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.

    PubMed

    Wagner, Koen; Kwakkenbos, Mark J; Claassen, Yvonne B; Maijoor, Kelly; Böhne, Martino; van der Sluijs, Koenraad F; Witte, Martin D; van Zoelen, Diana J; Cornelissen, Lisette A; Beaumont, Tim; Bakker, Arjen Q; Ploegh, Hidde L; Spits, Hergen

    2014-11-25

    Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners. PMID:25385586

  19. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  20. Targeting of Indium Ill-labeled Bivalent Hapten to Human Melanoma Mediated by Bispecific Monoclonal Antibody Conjugates: Imaging of Tumors Hosted in Nude Mice

    Microsoft Academic Search

    Jean-Marc Le Doussal; Anne Gruaz-Guyon; Marie Martin; Emmanuel Gautherot; Michel Delaage; Jacques Barbet

    1990-01-01

    Antibody conjugates were prepared by coupling F(ab')2 or Fab' frag ments of an antibody specific for the human high molecular weight- melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the anti body conjugate mediated binding of the '''In-labeled

  1. Immunotherapy of Human Tumors with T-Cell-activating Bispecific Antibodies: Stimulation of Cytotoxic Pathways in Vivo1

    Microsoft Academic Search

    Stefan Bauer; Christoph Renner; Jan-Peter Juwana; Gerhard Held; Sascha Ohnesorge; Klaus Gerlach; Michael Pfreundschuh

    1999-01-01

    Bispecific monoclonal antibodies (Bi-mAbs) specific for a tumor-asso- ciated antigen and the CD3 or CD28 antigen on T lymphocytes represent one of the most successful experimental strategies for the immunotherapy of cancer. We report that the in vivo administration of both a-CD3\\/CD30 and a-CD28\\/CD30 Bi-mAbs results in the specific activation of xenotrans- planted, resting human T cells infiltrating the CD30-positive

  2. A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity

    PubMed Central

    Tan, Wenlong; Meng, Yanchun; Li, Hui; Chen, Yang; Han, Siqi; Zeng, Jing; Huang, Ang; Li, Bohua; Zhang, Yanyun; Guo, Yajun

    2013-01-01

    Treatment of chronic hepatitis B virus (HBV) infection with interferon and viral reverse transcriptase inhibitor regimens results in poor viral clearance, loss of response, and emergence of drug-resistant mutant virus strains. These problems continue to drive the development of new therapeutic approaches to combat HBV. Here, we engineered a bispecific antibody using two monoclonal antibodies cloned from hepatitis B surface antigen (HBsAg)-specific memory B cells from recombinant HBsAg-vaccinated healthy volunteers. Next, we evaluated its efficacy in neutralizing HBV in HepaRG cells. This bispecific antibody, denoted as C4D2-BsAb, had superior HBV-neutralizing activity compared with the combination of both parental monoclonal antibodies, possibly through steric hindrance or induction of HBsAg conformational changes. Moreover, C4D2-BsAb has superior endocytotic characteristics into hepatocytes, which inhibits the secretion of HBsAg. These results suggest that the anti-HBsAg bispecific antibody may be an effective treatment method against HBV infection. PMID:24492346

  3. Bispecific small molecule–antibody conjugate targeting prostate cancer

    PubMed Central

    Kim, Chan Hyuk; Axup, Jun Y.; Lawson, Brian R.; Yun, Hwayoung; Tardif, Virginie; Choi, Sei Hyun; Zhou, Quan; Dubrovska, Anna; Biroc, Sandra L.; Marsden, Robin; Pinstaff, Jason; Smider, Vaughn V.; Schultz, Peter G.

    2013-01-01

    Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant ?CD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ?100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors. PMID:24127589

  4. Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy

    SciTech Connect

    Sharkey, Robert M.

    2005-02-04

    The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

  5. Improved pretargeted delivery of radiolabelled hapten to human tumour xenograft in mice by avidin chase of circulating bispecific antibody

    Microsoft Academic Search

    Eric Mirallié; Catherine Saï-Maurel; Alain Faivre-Chauvet; Nicolas Regenet; Chien-Hsing Chang; David M. Goldenberg; Jean-François Chatal; Jacques Barbet; Philippe Thedrez

    2005-01-01

    Purpose: Pretargeted therapy with radiolabelled bivalent haptens and bispecific antibodies has shown prom- ising results, but blood clearance of the activity-carrying haptens under conditions designed for radioimmunothera- py is relatively slow. Thus, the chase of excess circulating bispecific antibody by biotinylation of the bispecific anti- body and injection of avidin before hapten administration was tested with a view to increasing

  6. Ipilimumab augments antitumor activity of bispecific antibody-armed T cells

    PubMed Central

    2014-01-01

    Background Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC). Methods PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt’s lymphoma cell line (Daudi). Results In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p?=?0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p?bispecific antibodies. PMID:25008236

  7. Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines*

    PubMed Central

    Spiess, Christoph; Bevers, Jack; Jackman, Janet; Chiang, Nancy; Nakamura, Gerald; Dillon, Michael; Liu, Hongbin; Molina, Patricia; Elliott, J. Michael; Shatz, Whitney; Scheer, Justin M.; Giese, Glen; Persson, Josefine; Zhang, Yin; Dennis, Mark S.; Giulianotti, James; Gupta, Prateek; Reilly, Dorothea; Palma, Enzo; Wang, Jianyong; Stefanich, Eric; Scheerens, Heleen; Fuh, Germaine; Wu, Lawren C.

    2013-01-01

    Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes. PMID:23880771

  8. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. (University Hospital of Cleveland, OH (USA))

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.48 references.

  9. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    NASA Astrophysics Data System (ADS)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  10. Rational design and generation of recombinant control reagents for bispecific antibodies through CDR mutagenesis.

    PubMed

    Choi, Bryan D; Gedeon, Patrick C; Kuan, Chien-Tsun; Sanchez-Perez, Luis; Archer, Gary E; Bigner, Darell D; Sampson, John H

    2013-09-30

    Developments in the field of bispecific antibodies have progressed rapidly in recent years, particularly in their potential role for the treatment of malignant disease. However, manufacturing stable molecules has proven to be costly and time-consuming, which in turn has hampered certain aspects of preclinical evaluation including the unavailability of appropriate "negative" controls. Bispecific molecules (e.g., bispecific tandem scFv) exhibit two specificities, often against a tumor antigen as well as an immune-activation ligand such as CD3. While for IgG antibodies, isotype-matched controls are well accepted, when considering smaller antibody fragments it is not possible to adequately control for their biological activity through the use of archetypal isotypes, which differ dramatically in affinity, size, structure, and design. Here, we demonstrate a method for the rapid production of negative control tandem scFvs through complementarity determining region (CDR) mutagenesis, using a recently described bispecific T-cell engager (BiTE) targeting a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) as an example. Four independent control constructs were developed by this method through alteration of residues spanning individual CDR domains. Importantly, while target antigen affinity was completely impaired, CD3 binding affinity was conserved in each molecule. These results have a potential to enhance the sophistication by which bispecific antibodies can be evaluated in the preclinical setting and may have broader applications for an array of alternative antibody-derived therapeutic platforms. PMID:23806556

  11. Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules

    SciTech Connect

    Jordan, Jacob L.; Arndt, Joseph W.; Hanf, Karl; Li, Guohui; Hall, Janine; Demarest, Stephen; Huang, Flora; Wu, Xiufeng; Miller, Brian; Glaser, Scott; Fernandez, Erik J.; Wang, Deping; Lugovskoy, Alexey; (UV); (Biogen)

    2010-01-12

    Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LT{beta}R) binding Fv domain of an anti-LT{beta}R/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability.

  12. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

    PubMed

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

    2015-03-20

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. PMID:25583986

  13. Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

    PubMed Central

    Laventie, Benoît-Joseph; Rademaker, Hendrik Jan; Saleh, Maher; de Boer, Ernie; Janssens, Rick; Bourcier, Tristan; Subilia, Audrey; Marcellin, Luc; van Haperen, Rien; Lebbink, Joyce H. G.; Chen, Tao; Prévost, Gilles; Grosveld, Frank; Drabek, Dubravka

    2011-01-01

    Panton–Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti–LukS-PV HCAb, three anti–LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti–LukS-PV HCAb also binds to ?-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. PMID:21930905

  14. Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins.

    PubMed

    Laventie, Benoît-Joseph; Rademaker, Hendrik Jan; Saleh, Maher; de Boer, Ernie; Janssens, Rick; Bourcier, Tristan; Subilia, Audrey; Marcellin, Luc; van Haperen, Rien; Lebbink, Joyce H G; Chen, Tao; Prévost, Gilles; Grosveld, Frank; Drabek, Dubravka

    2011-09-27

    Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to ?-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. PMID:21930905

  15. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  16. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  17. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.

    PubMed

    Yu, Y Joy; Atwal, Jasvinder K; Zhang, Yin; Tong, Raymond K; Wildsmith, Kristin R; Tan, Christine; Bien-Ly, Nga; Hersom, Maria; Maloney, Janice A; Meilandt, William J; Bumbaca, Daniela; Gadkar, Kapil; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Ernst, James A; Scearce-Levie, Kimberly; Couch, Jessica A; Dennis, Mark S; Watts, Ryan J

    2014-11-01

    Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ?-secretase (BACE1) can cross the BBB and reduce brain amyloid-? (A?) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain A? in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced A? both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain. PMID:25378646

  18. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. (Hoag Cancer Center, Newport Beach, CA (USA))

    1989-10-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references.

  19. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  20. Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification.

    PubMed

    Kim, Hyori; Park, Sunyoung; Lee, Hwa Kyoung; Chung, Junho

    2013-01-01

    We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein. PMID:24071736

  1. Monoclonal antibodies in haematopathology

    SciTech Connect

    Grignani, F.; Martelli, M.F.; Mason, D.Y.

    1985-01-01

    This book contains over 40 selections. Some of the titles are: Oncogene (c-myc, c-myb) amplification in acute myelogenous leukaemia; Ultrastructural characterization of leukaemic cells with monoloclonal antibodies; Origin of B-cell malignancies; Immunohistology of gut lymphomas; and Spurious evidence of lineage infidelity in monocytic leukaemia.

  2. Radioimmunoguided surgery using monoclonal antibody

    SciTech Connect

    Martin, E.W. Jr.; Mojzisik, C.M.; Hinkle, G.H. Jr.; Sampsel, J.; Siddiqi, M.A.; Tuttle, S.E.; Sickle-Santanello, B.; Colcher, D.; Thurston, M.O.; Bell, J.G.

    1988-11-01

    The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery.

  3. Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

    PubMed

    Xu, Yiren; Lee, John; Tran, Cuong; Heibeck, Tyler H; Wang, Willie D; Yang, Junhao; Stafford, Ryan L; Steiner, Alexander R; Sato, Aaron K; Hallam, Trevor J; Yin, Gang

    2015-01-01

    Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer. PMID:25427258

  4. CD3 directed bispecific antibodies induce increased lymphocyte–endothelial cell interactions in vitro

    Microsoft Academic Search

    G Molema; J W Cohen Tervaert; B J Kroesen; W Helfrich; D K F Meijer; L F M H de Leij

    2000-01-01

    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab?)2 to carcinoma patients in a phase I\\/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-? and interferon-? levels. Yet, no

  5. A monoclonal antibody against PMEL.

    PubMed

    Shi, Fangyuan; Xu, Zhenjie; Chen, Hongdong; Wang, Xin; Cui, Jihong; Zhang, Ping; Zhang, Ping; Xie, Xin

    2014-10-01

    PMEL, also known as Pmel17 or gp100, is a melanocyte-specific glycoprotein that is essential for the formation of stage II melanosomes. As it has a highly restricted expression pattern in normal tissues and a transient presence on the cell surface, PMEL is believed to be a potential target for antibody drug conjugate therapy in some pigmentary diseases. The production of a high specificity and high affinity monoclonal antibody against human PMEL was helpful for the antibody drug conjugate therapy study. In the present study, monoclonal antibodies (MAbs) against PMEL were obtained by immunizing BALB/c mice with the recombinant PMEL-GST fusion protein. Three mAbs (A3F, G11B, and J7E) with a titer of 1:6000, 1:10,000, and 1:3000, respectively, were obtained. Immunoglobulin subclass assay revealed that A3F was IgG2b, G11B was IgG1, and J7E was IgG2a. Specificity analysis by Western blotting demonstrated that A3F and J7E cross-reacted with GPNMB or LAMP; however, G11B reacted with PMEL only. Immunohistochemistry experiments showed that G11B could bind human PMEL antigen in normal skin. Flow cytometry assay demonstrated that G11B could bind to the surface of PMEL positive melanoma cells but not PMEL negative cells. Taken together, these results show that this G11B provides a useful tool for the antibody drug conjugate therapy study in some pigmentary diseases. PMID:25118787

  6. Monoclonal Antibody Therapy for Cancer

    Microsoft Academic Search

    Christoph Rader

    \\u000a Since the approval of rituximab (Rituxan®) for the treatment of B-cell non-Hodgkin’s lymphoma (B-NHL) in 1997, nine additional monoclonal antibodies (mAbs) have been\\u000a approved by the FDA for cancer therapy. Currently, more than 1,300 clinical studies registered at ClinicalTrials.gov investigate\\u000a mAb therapy of cancer, including more than 150 phase III clinical trials. In concert with their clinical acceptance, mAbs\\u000a in

  7. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  8. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  9. Application of 300× enhanced fluorescence on a plasmonic chip modified with a bispecific antibody to a sensitive immunosensor.

    PubMed

    Tawa, Keiko; Umetsu, Mitsuo; Nakazawa, Hikaru; Hattori, Takamitsu; Kumagai, Izumi

    2013-09-11

    The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300× enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface. PMID:23945148

  10. Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

    PubMed Central

    DAN, ZHAOKUI; TAN, ZUI; XIA, HONGLI; WU, GAN

    2013-01-01

    The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing. Then, the soluble recombinant antibody in bacterial lysates was purified by an NTA column and molecular sieve chromatography in turn. Finally, the binding specificity of the purified antibody was tested by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that the construction of the expression plasmid was successful and the purified recombinant protein, which had a molecular weight of ?56 kDa, was specific to GPIIb/IIIa and D-dimer. In conclusion, a plasmid expressing a bispecific antibody was constructed by a new method of blunt-end ligation. The soluble recombinant protein is a promising platform for target-oriented thrombolytic therapy. PMID:24137225

  11. Preparation of anfi-CD3 and anti-IgM&mgr; chain bispecific antibody by cell fusion.

    PubMed

    Su, Jin; Zhang, Ya-Li; Guo, Zhi-Bing; Che, Xiao-Yan; Wang, Xiao-Ning

    2001-01-01

    OBJECTIVE: To prepare anti-CD3 and anti-Igm&mgr; chain bispecific antibody (BsAb) by means of cell fusion and assess the stability and activity of the hybrid hybridoma. METHODS: Mouse hybridoma cell line secreting anti-human CD3 monoclonal antibody (mAb) was trans formed into HAT-sensitive cells by 8-azaguanine, which was subsequently transfected by plasmid pCDaA3 containing neoR marker gene via FuGENETM6. The resulted mutant phenotype, named alphaCD3 HATs G418R, was fused with the hybridoma producing anti-IgM&mgr; chain mAb, and enzyme-linked immunosorbent assay and flow cytometry were employed to identify the fusion cells producing the target BsAb. RESULTS: Six rounds of cell fusion was performed and a total of l 080 wells inoculated, yielding 5 hybrid hybridoma cell lines. Two of the 5 cell lines were subcloned and continuously cultured in vitro for 2 months without losing their capability to secrete BsAb. CONCLUSION: Cell fusion technique can be utilized to prepare BsAb-producing hybrid hybridoma that has similar stability and activity to its parent hybridoma. PMID:12426160

  12. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  13. Multimodal Cancer Therapy Involving Oncolytic Newcastle Disease Virus, Autologous Immune Cells, and Bi-Specific Antibodies

    PubMed Central

    Schirrmacher, Volker; Fournier, Philippe

    2014-01-01

    This paper focuses on oncolytic Newcastle disease virus (NDV). This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities PMID:25309868

  14. Original article Monoclonal antibodies against goldfish

    E-print Network

    Paris-Sud XI, Université de

    immunoglobulin and antibody levels by ELISA in carp (Cyprinus carpio) AK Siwicki C Vergnet J Charlemagne M Dunier decreased until day 28. monoclonal antibody / ELISA / ELISPOT / antibody-secreting cells / Cyprinus carpio suite. anticorps monoc/ona//EL/S/Cyprinus carpio

  15. Cost modeling for monoclonal antibody manufacturing

    E-print Network

    Simpson, Christina M. (Christina Margaret)

    2011-01-01

    The Novartis BioPharmOps division is responsible for manufacturing large molecule products, including monoclonal antibodies, for late stage clinical trials and commercial sales. The BioPharmOps site in Huningue, France is ...

  16. Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

    PubMed Central

    Sampei, Zenjiro; Igawa, Tomoyuki; Soeda, Tetsuhiro; Okuyama-Nishida, Yukiko; Moriyama, Chifumi; Wakabayashi, Tetsuya; Tanaka, Eriko; Muto, Atsushi; Kojima, Tetsuo; Kitazawa, Takehisa; Yoshihashi, Kazutaka; Harada, Aya; Funaki, Miho; Haraya, Kenta; Tachibana, Tatsuhiko; Suzuki, Sachiyo; Esaki, Keiko; Nabuchi, Yoshiaki; Hattori, Kunihiro

    2013-01-01

    In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients. PMID:23468998

  17. Cold denaturation of monoclonal antibodies

    PubMed Central

    Lazar, Kristi L; Patapoff, Thomas W

    2010-01-01

    The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from ?5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ?Gu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ?Gu versus temperature data from fluorescence gave a ?Cp of 8.0 kcal mol?1 K?1, a maximum apparent stability of 23.7 kcal mol?1 at 18°C, and an apparent cold denaturation temperature (TCD) of ?23°C. ?Gu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near ?20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs. PMID:20093856

  18. Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo.

    PubMed

    Wallace, P K; Kaufman, P A; Lewis, L D; Keler, T; Givan, A L; Fisher, J L; Waugh, M G; Wahner, A E; Guyre, P M; Fanger, M W; Ernstoff, M S

    2001-02-01

    Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation. PMID:11223077

  19. Monoclonal Antibodies as Diagnostics; an Appraisal

    PubMed Central

    Siddiqui, M. Z.

    2010-01-01

    Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use. PMID:20582184

  20. Therapeutic bispecific antibodies: The selection of stable single-chain fragments to overcome engineering obstacles.

    PubMed

    Mabry, Robert; Snavely, Mark

    2010-08-01

    The clinical success of mAbs continues to reinforce antibody engineering as an essential tool for the development of biologics. Research focused on discovering the next generation of therapeutics has prompted a revisiting of the concept of bispecific antibodies (bsAbs). Recently, clinical programs investigating combinations of mAb therapies have renewed interest in the applications of bsAbs. However, because of challenges with production, efforts directed toward the development of bsAbs have yet to yield a product approved by the FDA. The current status of these proteins implies that the strategies for constructing therapeutic bsAbs will likely require a highly refined design plan at the outset of the engineering process. Antibody fragments are attractive building blocks for the assembly of bsAbs. Of the recombinant antibody fragments, single-chain variable fragments (scFvs) offer the advantage of expression as a single polypeptide, thereby greatly simplifying production. However, issues with stability have plagued these proteins and limit the application of scFvs as therapeutics. Recent advances in selection processes using display platforms have been reported that facilitate the 'evolution' of scFvs to obtain stabilities comparable with those of mAbs. The timely advances in scFv engineering parallel the resurgence of bsAbs and enable the construction of dual-targeting proteins that can be manufactured as therapeutics. PMID:20721825

  1. Therapeutic potential of monovalent monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Cobbold, S. P.; Waldmann, H.

    1984-03-01

    One therapeutic use for monoclonal antibody technology1 is the elimination of categories of unwanted cells by virtue of their distinct cell surface antigens. The efficiency of cell destruction by complement lysis or opsonization depends on a number of factors such as antibody specificity and isotype as well as certain properties of the target antigen2. In some instances cells can escape destruction by redistributing and eventually losing the antigen-antibody complexes from their surface3,4. This process, known as antigenic modulation, generally depends on bivalent antibody binding. Starting from the observation that rabbit antisera can be made more effective at killing tumour cells if they are first rendered univalent by limited proteolysis, we have now prepared a number of monovalent rat monoclonal antibodies to human cell-surface antigens. We find that these antibodies are no longer able to bring about modulation of their target antigens and have an enhanced facility for lysis with human complement. These special properties should greatly increase the therapeutic potential of monoclonal antibodies.

  2. Magic Bullets and Monoclonals: An Antibody Tale

    NSDL National Science Digital Library

    Margie Patlak (Federation of American Societies for Experimental Biology Office of Public Affairs)

    2010-07-12

    FASEB Breakthroughs in Bioscience article. This most recent article describes the century of fundamental immunology research that led to todayÂ?s cutting edge monoclonal antibody therapies, used to treat millions of patients for several types of cancer, autoimmune and inflammatory disorders, and infectious disease.

  3. Subcutaneous Administration of Monoclonal Antibodies in Oncology.

    PubMed

    Jackisch, C; Müller, V; Maintz, C; Hell, S; Ataseven, B

    2014-04-01

    Treatment with monoclonal antibodies (mabs) has become an established component of oncological therapy. The monoclonal antibodies available for this purpose are mainly administered intravenously in individually adapted doses according to body weight over longer treatment times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous administration of drugs is an established therapy option. For the subcutaneous administration of larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue represents a problem. The co-formulation with recombinant human hyaluronidase makes the relatively pain-free administration of larger fluid volumes and thus the subcutaneous administration of monoclonal antibodies possible, as illustrated by the development of a subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and flexible form of administration for patients with HER2-positive breast cancer that, with its fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term therapy not only for the patients but also for the medical personnel. PMID:25076790

  4. Subcutaneous Administration of Monoclonal Antibodies in Oncology

    PubMed Central

    Jackisch, C.; Müller, V.; Maintz, C.; Hell, S.; Ataseven, B.

    2014-01-01

    Treatment with monoclonal antibodies (mabs) has become an established component of oncological therapy. The monoclonal antibodies available for this purpose are mainly administered intravenously in individually adapted doses according to body weight over longer treatment times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous administration of drugs is an established therapy option. For the subcutaneous administration of larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue represents a problem. The co-formulation with recombinant human hyaluronidase makes the relatively pain-free administration of larger fluid volumes and thus the subcutaneous administration of monoclonal antibodies possible, as illustrated by the development of a subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and flexible form of administration for patients with HER2-positive breast cancer that, with its fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term therapy not only for the patients but also for the medical personnel. PMID:25076790

  5. Monoclonal antibodies reactive with chicken interleukin-17

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In our previous study chicken interleukin -17 (chIL-17) gene was cloned from the expressed sequence tag (EST) cDNA library and initially analyzed. To further investigate biological properties of chicken IL-17, six monoclonal antibodies (mAbs) against bacterially expressed protein were produced and c...

  6. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  7. Human Monoclonal Antibody Against Mesothelin

    Cancer.gov

    Mesothelin is a cell surface protein that is naturally expressed at very low levels, but that is significantly increased in aggressive tumors such as mesotheliomas, and pancreatic and ovarian tumors. Therefore, mesothelin is an excellent candidate for tumor-targeted immunotherapeutics. However, the only antibodies against mesothelin that are currently available for clinical trials are of murine origin. The use of these antibodies may be limited by their potential to elicit adverse immune responses in patients with repeated doses.

  8. A new bispecific antibody targeting non-overlapping epitopes on IGF2: design, in vitro characterization and pharmacokinetics in macaques.

    PubMed

    Feng, Yang; Zhao, Qi; Chen, Weizao; Wang, Yanping; Crowder, Karalyne; Dimitrov, Dimiter S

    2014-12-01

    The insulin-like growth factor 2 (IGF2) is an important target for cancer therapy. We have previously proposed an approach for fast and irreversible removal of IGF2 from the circulation by using monoclonal antibodies (mAbs) that bind to two or more non-overlapping epitopes on the same molecule. We provided initial evidence for the formation of oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (Fc?Rs) with high avidity using an antibody domain with relatively low affinity as one of the anti-IGF2 mAbs. Recently, we identified a mAb, m708.5, in a scFv format which binds to both IGF2 and IGF1 with very high (pM) affinity. Interestingly, and rather surprisingly, this mAb did not compete with our other high affinity mAb, m610.27, for binding to IGF2. Therefore, we generated a new bispecific mAb, m67, by combining m708.5 and m610.27. As expected m67 potently inhibited binding of IGF2 to cells expressing the IGF1R and its phosphorylation, and resulted in formation of multimolecular complexes when incubated with IGF2 and bound with high avidity to cells expressing Fc?RII; the complexes were internalized in a macrophage-like cell line. However, although m67 exhibited a reasonably long half-life (6.4 ± 0.6 days) in cynomolgus macaques and high stability in serum, its administration to three animals did not result in any measurable decrease in the IGF2 concentration likely due to the complexity of the IGF2 interactions in the blood and the relatively low (2mg/kg) dose of the mAb leading to a relatively low maximal blood concentration of 120nM. In spite of the lack of effect on the IGF2 concentration in this particular experimental setup, m67 exhibited good drugability properties and could be highly effective in other animal models and in humans. Studies with animal models of cancer are ongoing to evaluate the potential of m67 as a new candidate mAb-based therapeutic. PMID:25220345

  9. A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity

    PubMed Central

    Castoldi, R; Ecker, V; Wiehle, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C

    2013-01-01

    Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling. PMID:23812422

  10. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  11. Recent developments in monoclonal antibody radiolabeling techniques

    SciTech Connect

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  12. Monoclonal Antibodies to Plant Growth Regulators

    PubMed Central

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.

    1986-01-01

    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  13. Next generation and biosimilar monoclonal antibodies

    PubMed Central

    2011-01-01

    The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 3–4, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

  14. Identification of Streptococcus sobrinus with monoclonal antibodies.

    PubMed

    de Soet, J J; van Dalen, P J; Appelmelk, B J; de Graaff, J

    1987-12-01

    Identification of Streptococcus sobrinus is often difficult to perform because of the great resemblance of the organism to other oral streptococcal species. Therefore, monoclonal antibodies were prepared which were shown to be highly specific for S. sobrinus. Cross-reactivity with other oral microorganisms has not been observed in an enzyme-linked immunosorbent assay and an immunofluorescence assay. These monoclonal antibodies belonged to the subclass immunoglobulin G2b. To be certain that the strains used in cross-reactivity tests were S. sobrinus, their DNA base composition was measured as a golden standard. Additional tests like colony morphology and sugar fermentation with the API 20 Strep system (Analytab Products, Montalieu-Vercieu, France) were performed. These additional tests turned out to be necessary because 100% correct identification could not be obtained by separate tests. Immunological characterization with the clones OMVU10 and OMVU11 proved to be discriminative between S. sobrinus and other streptococcal species. PMID:3323224

  15. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  16. Monoclonal antibody targets, kills leukemia cells

    Cancer.gov

    Researchers at the University of California, San Diego Moores Cancer Center have identified a humanized monoclonal antibody that targets and directly kills chronic lymphocytic leukemia (CLL) cells. The findings, published in the online Early Edition of the Proceedings of the National Academy of Sciences on March 25, 2013 represent a potential new therapy for treating at least some patients with CLL, the most common type of blood cancer in the United States.

  17. Historical Development of Monoclonal Antibody Therapeutics

    Microsoft Academic Search

    A. Nissim; Y. Chernajovsky

    Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology,\\u000a mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents.\\u000a Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune,\\u000a cardiovascular, and infectious diseases.

  18. Mapping nucleolar proteins with monoclonal antibodies

    Microsoft Academic Search

    JOERG KISTLER; YVONNE DUNCOMBE; ULRICH K. LAEMMLI

    1984-01-01

    Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleo- protein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may

  19. Retargeting T Cells to GD2 Pentasaccharide on Human Tumors Using Bispecific Humanized Antibody.

    PubMed

    Xu, Hong; Cheng, Ming; Guo, Hongfen; Chen, Yuedan; Huse, Morgan; Cheung, Nai-Kong V

    2015-03-01

    Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >10(5)-fold selectivity over normal tissues, releasing Th1 cytokines (TNF?, IFN?, and IL2) when GD2(+) tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells. Cancer Immunol Res; 3(3); 266-77. ©2014 AACR. PMID:25542634

  20. Bispecific anti-CD3 x anti-HER2 antibody mediates T cell cytolytic activity to HER2-positive colorectal cancer in vitro and in vivo.

    PubMed

    Han, Huamin; Ma, Juan; Zhang, Keming; Li, Wei; Liu, Changzhen; Zhang, Yu; Zhang, Ganlin; Ma, Pan; Wang, Lei; Zhang, Ge; Tao, Hua; Gao, Bin

    2014-12-01

    Targeting HER2 overexpressed breast cancer cells with anti?HER2 monoclonal antibodies inhibits tumor growth. Here we investigated whether HER2 can serve as a target for T cell-mediated immunotherapy of human colorectal carcinoma. Specific cytolytic activity of activated T cells (ATCs) armed with anti?CD3 x anti?HER2 bispecific antibody (HER2Bi-Ab) against HER2+ tumor cells was evaluated by bioluminescent signal generated by luciferase reporter on tumor cells in vitro and in vivo. In contrast to unarmed ATCs, increased cytotoxic activity of HER2Bi-armed ATCs against HER2+ tumor cells was observed. Moreover, HER2Bi-armed ATCs expressed higher level of activation marker CD69 and secreted significantly higher levels of IFN-? than the unarmed ATC counterpart. In addition, compared with anti?HER2 mAb (Herceptin®) or unarmed ATC, HER2Bi-armed ATCs showed significant suppression against colorectal carcinoma cells. In colorectal tumor cell xenograft mice, infusion of HER2Bi-armed ATCs successfully inhibited the growth of Colo205-luc cells. The HER2Bi-armed ATCs with anti-tumor effects may provide a promising immunotherapy for colorectal carcinoma in the future. PMID:25242665

  1. Monoclonal antibodies against human cancer stem cells.

    PubMed

    Naujokat, Cord

    2014-01-01

    Cancer stem cells (CSCs) are a subpopulation of tumor cells that display self-renewal and tumor initiation capacity and the ability to give rise to the heterogenous lineages of cancer cells that comprise the tumor. CSCs exhibit intrinsic mechanisms of resistance to modern cancer therapeutics, allowing them to survive current cancer therapies and to initiate tumor recurrence and metastasis. Various cell surface and transmembrane proteins expressed by CSCs, including CD44, CD47, CD123, EpCAM (CD326), CD133, IGF receptor I, and proteins of the Notch and Wnt signaling pathways have been identified. Recently, monoclonal antibodies and antibody constructs raised against these CSC proteins have shown efficacy against CSCs in human cancer xenograft mice, and some of them have demonstrated antitumor activity in clinical studies. Since current cancer therapies fail to eliminate CSCs, leading to cancer recurrence and progression, selective targeting of CSCs with monoclonal antibodies and antibody constructs may represent a novel therapeutic strategy against cancer. PMID:24762074

  2. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin (San Ramon, CA); Stanker, Larry H. (Livermore, CA); Watkins, Bruce E. (Livermore, CA); Bailey, Nina R. (Berkley, CA)

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  3. Alzheimer neurofibrillary tangles: Monoclonal antibodies to inherent antigen(s)

    Microsoft Academic Search

    G. P. Wang; I. Grundke-Iqbal; R. J. Kascsak; K. Iqbal; H. M. Wisniewski

    1984-01-01

    Using isolated Alzheimer neurofibrillary tangles as the immunogen, nine mouse hybridomas were generated which produced antibodies to the tangles as tested in both tissue sections and isolated neurons from Alzheimer brain. Extraction of isolated neurofibrillary tangles with 2% SDS could not remove the antigen(s) with which these monoclonal antibodies reacted. Immunocytochemical study revealed that each of the monoclonal antibodies reacted

  4. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  5. Generation of med28 specific monoclonal antibodies.

    PubMed

    Yu, Min A; Cho, Jin Gu; Kim, Kwang-Il; Jo, Yeong Joon; Sung, Jong-Hyuk; Yang, Ho Bin; Park, Sang Gyu

    2015-02-01

    Med28 plays a role in transcription, signal transduction, and cell proliferation. The overexpression of med28 is associated with tumor progression in in vitro and in vivo models. Recently it has been reported that the elevated expression of med28 is associated with poor outcome in women with breast cancer. The expression level of med28 in in vitro and in vivo was examined by using anti-rabbit polyclonal antibody in previous reports. In this study, we report for the first time the generation and characterization of four monoclonal antibodies against med28 through immunoblotting, immunofluorescence microscopy, immunoprecipitation, and immunohistochemical analyses. These antibodies will be useful in detecting med28 in in vitro and in vivo. PMID:25723281

  6. Leukocyte analysis using monoclonal antibodies in human glomerulonephritis

    Microsoft Academic Search

    David H Hooke; David C Gee; Robert C Atkins

    1987-01-01

    Leukocyte analysis using monoclonal antibodies in human glomerulonephritis. The leukocyte subpopulations were analyzed within both the glomeruli and the interstitium in renal biopsies from 145 patients with various forms of glomerulonephritis. Cells were identified by monoclonal antibodies to leukocyte cell–surface antigens and immunoper-oxidase labelling. Leukocytes, as defined by a monoclonal antibody to the leukocyte common antigen (PHM1), were present in

  7. The production of monoclonal antibodies to Babesia microti

    E-print Network

    Ruwe, Kathryn Lyn

    1986-01-01

    17 18 18 20 22 24 25 29 45 48 49 VITA 55 LIST OF TABLES Table Page 1. Derivation of monoclonal antibodies to Babesia microti. 2. IFA titers of individual monoclonal antibody preparations to Babesia microti. 3. Properties of FITC... with an adjuvant into cattle provided protection against B. bovis challenge infections. Monoclonal antibodies have also been produced to many other protozoan parasites including ~7p 6 d '* (36(, '6* *pl ~d' ' (3(, Trypanosoma cruzi (4), and Plasmodium chabaudi...

  8. Comparison of physical chemical properties of llama V HH antibody fragments and mouse monoclonal antibodies

    Microsoft Academic Search

    R. H. J. van der Linden; L. G. J. Frenken; B. de Geus; M. M. Harmsen; R. C. Ruuls; W. Stok; L. de Ron; S. Wilson; P. Davis; C. T. Verrips

    1999-01-01

    Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and

  9. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. (Albert Einstein College of Medicine-Montefiore Medical Center, Bronx, NY (USA))

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  10. Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.

    PubMed

    Xu, Liming; Zhang, Yu; Wang, Qiuying; Zhao, Jingzhuang; Liu, Miao; Guo, Mo; Jiang, Yuanyuan; Cao, Hongwei; Li, Qingcui; Ren, Guiping; Li, Deshan

    2015-02-01

    Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1? (hIL-1?) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1? and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development. PMID:25526913

  11. Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications

    PubMed Central

    Compte, Marta; Álvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Sainz-Pastor, Noelia; Blanco-Toribio, Ana; Pescador, Nuria; Sanz, Laura; Álvarez-Vallina, Luis

    2014-01-01

    Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells. PMID:25057445

  12. Phylogenetic study of transcortin using monoclonal antibodies.

    PubMed

    Faict, D; De Moor, P

    1986-08-14

    We produced monoclonal antibodies that recognise three distinct epitopes of human transcortin. These epitopes are present on transcortin of humans with normal and altered transcortin levels, as well as on a variant with lower affinity for cortisol. One epitope is present on transcortin of Old World Monkeys and apes, the others are only present on transcortin of apes. The epitopes are not present on transcortin of other species. These results indicate that human transcortin contains a highly evolved and a more conserved part. PMID:2428359

  13. Monoclonal antibody disulfide reduction during manufacturing

    PubMed Central

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1? > IgG1? > IgG2? > IgG2?. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  14. NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  15. NCI Requests Targets for Monoclonal Antibody Production and Characterization

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  16. 75 FR 3244 - Prospective Grant of Exclusive License: Monoclonal Antibodies Against Smallpox/Orthopoxviruses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ...Grant of Exclusive License: Monoclonal Antibodies Against Smallpox/Orthopoxviruses AGENCY...4, Purcell et al., ``Monoclonal Antibodies Against Orthopoxviruses'', United...of use may be limited to monoclonal antibodies against orthopoxviruses...

  17. Structure and specificity of lamprey monoclonal antibodies

    PubMed Central

    Herrin, Brantley R.; Alder, Matthew N.; Roux, Kenneth H.; Sina, Christina; Ehrhardt, Götz R. A.; Boydston, Jeremy A.; Turnbough, Charles L.; Cooper, Max D.

    2008-01-01

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8–10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the ?-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region “arms” with antigen-binding “hands.” Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses. PMID:18238899

  18. Structure and specificity of lamprey monoclonal antibodies.

    PubMed

    Herrin, Brantley R; Alder, Matthew N; Roux, Kenneth H; Sina, Christina; Ehrhardt, Götz R A; Boydston, Jeremy A; Turnbough, Charles L; Cooper, Max D

    2008-02-12

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses. PMID:18238899

  19. DETECTION OF ROTAVIRUS IN HUMAN STOOLS BY USING MONOCLONAL ANTIBODY

    EPA Science Inventory

    A monoclonal antibody, 3F7, which reacts with the common rotavirus antigen on the sixth viral gene product was prepared. It was used in a direct monoclonal antibody radioimmunoassay (RIA) as a diagnostic reagent for detection in 3.5 hours of rotavirus in human pediatric stool spe...

  20. Production of a monoclonal antibody specific for seminomas and dysgerminomas.

    PubMed Central

    Bailey, D; Baumal, R; Law, J; Sheldon, K; Kannampuzha, P; Stratis, M; Kahn, H; Marks, A

    1986-01-01

    A monoclonal antibody (M2A, IgG2a) was produced against a cultured human ovarian epithelial adenocarcinoma cell line, HEY. Monoclonal antibody M2A reacted with a glycoprotein of molecular weight 40,000 on the surface of HEY cells. The affinity constant of the monoclonal antibody M2A for HEY cells was 10(9) M-1, and the number of binding sites on HEY cells was 2 X 10(4) per cell. The monoclonal antibody produced positive immunoperoxidase staining of fetal (but not adult) testis and of seminomas and dysgerminomas but did not stain various normal adult tissues or other gonadal or extragonadal tumors. Monoclonal antibody M2A may be useful for confirming a histological diagnosis of seminoma and dysgerminoma. Images PMID:3523489

  1. Characterization of monoclonal antibodies against human lactoferrin.

    PubMed

    van Berkel, Patrick H C; van Veen, Harrie A; Geerts, Marlieke E J; Nuijens, Jan H

    2002-09-15

    The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties. PMID:12165435

  2. Complement in Monoclonal Antibody Therapy of Cancer

    PubMed Central

    Rogers, Laura M.; Veeramani, Suresh; Weiner, George J.

    2015-01-01

    Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs, and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes, and which mechanisms are most responsible for effective elimination of malignant cells remain unclear. In this review, we discuss the mAbs currently approved for cancer treatment, and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment. PMID:24906530

  3. Monoclonal antibodies against Xenopus greatwall kinase.

    PubMed

    Wang, Ling; Fisher, Laura A; Wahl, James K; Peng, Aimin

    2011-10-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  4. Complement in monoclonal antibody therapy of cancer.

    PubMed

    Rogers, Laura M; Veeramani, Suresh; Weiner, George J

    2014-08-01

    Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes. How the balance of such effects impacts on the clinical efficacy of mAb therapy remains unclear. In this review, we discuss the mAbs currently approved for cancer treatment and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment. PMID:24906530

  5. A monoclonal antibody produced against Naked2.

    PubMed

    Cao, Chang; Wang, Shengyu; Lv, Sha; Li, Zhe; Wang, Xianjiang; Zeng, Fanwei; Zhang, Haipeng; Dai, Yujuan; Dou, Xiaofeng; Chen, Xiaoli; Li, Xiudong; Luo, Lingli; Hu, Tianhui; Yan, Jianghua

    2013-08-01

    Naked2 (NKD2) is a member of the Naked family and negatively regulates canonical Wnt signaling. NKD2 may play a role in embryo development and tumor formation by affecting Wnt signaling. In the present study, we describe the establishment of a monoclonal antibody against NKD2 (anti-NKD2 MAb) through the hybridoma method. The purified anti-NKD2 MAb measured a titer of 2.56 × 10(5) against NKD2 by indirect ELISA. Western blot analysis, immunoprecipitation, and confocal microscope showed that the anti-NKD2 MAb can specifically combine NKD2 protein in SW480 and LOVO cells. Competitive inhibition assays of Western blot and indirect ELISA showed that the anti-NKD2 MAb can be blocked with NKD2(1-217) protein. The anti-NKD2 MAb would be helpful for further studies on the structure activity relationship, protein detecting, and cell-signaling pathway of NKD2. PMID:23909424

  6. Monoclonal antibodies in treatment of multiple sclerosis

    PubMed Central

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, UK

    2014-01-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing–remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing–remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  7. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ({sup 123}I, {sup 131}I, and {sup 111}In) and with another radionuclide,{sup 211}At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for {sup 111}In and {sup 123}I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches.

  8. Screening Panels of Monoclonal Antibodies Using Phage-Displayed Antigen

    Microsoft Academic Search

    H. R. Lijnen; I. Lasters; M. Verstreken; D. Collen; L. Jespers

    1997-01-01

    A procedure is described to screen panels of hybridomas or purified monoclonal antibodies using antigen displayed on the surface of filamentous bacteriophage. In this system, samples containing murine monoclonal antibodies are incubated with phage-displayed antigen in microtiter plates coated with rabbit anti-mouse IgG, and bound antibody–phage complex is detected with horseradish peroxidase–sheep anti-phage M13 conjugate. The assay has been validated

  9. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

    PubMed

    Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

    2010-03-01

    The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile. PMID:19924542

  10. Localization of malignant melanoma using monoclonal antibodies

    SciTech Connect

    Wasselle, J.; Becker, J.; Cruse, W.; Espinosa, C.; Cox, C.; Reintgen, D. (Univ. of South Florida, Tampa (USA))

    1991-04-01

    Finding a screening test to evaluate patients with cancer for occult metastatic disease, as well as imaging all known disease, is a goal of research efforts. Twenty-nine evaluable patients with deeply invasive (stage I), regional nodal (stage II), or systemic (stage III) melanoma underwent imaging by administration of a preparation of the antimelanoma antibody labeled with technetium 99m. Scan results indicated that 28 of 32 confirmed metastatic sites were imaged with this technique (88% sensitivity). Analysis of the individual positive sites revealed that nodal basins and visceral metastases accounted for the highest percentage of metastatic sites imaged, with 14 (88%) of 16 nodal basin metastases and all four visceral metastases being detected through imaging. Occult nodal disease was detected in the iliac nodal chain in two of the 29 patients. The imaging of benign tumors and nodal basins not containing disease accounted for a confirmed false-positive rate of 21%. Three (10%) of the 29 scan results were confirmed to be false-negative. In vivo tumor localization with monoclonal antibodies showed a sensitivity similar to that of other roentgenographic procedures for identifying metastatic disease and was useful in two of three patients in identifying occult iliac nodal disease, a region that is difficult to evaluate with physical examination and other imaging modalities.

  11. Monoclonal antibodies in animal production; their use in diagnostics and passive immunization

    Microsoft Academic Search

    P. Booman

    1989-01-01

    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal antibodies have been applied to biological and medical sciences.In animal production monoclonal antibodies are increasingly finding application in

  12. Monoclonal Antibodies—Therapeutic and Diagnostic Uses in Malignancy

    PubMed Central

    Lowder, James N.; Levy, Ronald

    1985-01-01

    Murine monoclonal antibodies represent an attractive type of antitumor therapy because of their potential for exquisite specificity, production in large, pure quantities and mediation of in vivo cytotoxic effects. With maturing monoclonal antibody technology has come the use of these antibodies in clinical studies in patients with malignancy. These trials have established that monoclonal antibodies can be safely administered in large doses, that their pharmacokinetics and tissue penetration can be predicted and that in some instances a therapeutic effect can be produced by their infusion. A number of problems have also been identified by these studies, including antigenic heterogeneity of the tumor, the presence of free serum antigen, the immunogenicity of the xenogeneic antibody, modulation of the surface antigen by the antibody and a finite capacity of human effector mechanisms to mediate cytotoxicity directed by murine antibodies. Other workers are concurrently investigating the use of monoclonal antibodies in the ex vivo elimination of cells from bone marrow, as probes for serum tumor marker antigens and as carriers for radioimaging agents or toxins. Although most of these endeavors are at the earliest stages, promising preliminary results presage an important role for native and altered monoclonal antibodies in the diagnosis and treatment of malignant conditions. ImagesFigure 1.Figure 1. PMID:3911594

  13. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Biology seeks parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  14. Human and Improved Murine Monoclonal Antibodies Against CD22

    Cancer.gov

    The National Cancer Institute's Nanobiology Program is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize human monoclonal antibodies expressed in types of lymphoma.

  15. Monoclonal Antibody-Mediated Tumor Regression by Induction of Apoptosis

    Microsoft Academic Search

    Bernhard C. Trauth; Christiane Klas; Anke M. J. Peters; Siegfried Matzku; Peter Moller; Werner Falk; Klaus-Michael Debatin; Peter H. Krammer

    1989-01-01

    To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation

  16. Monoclonal antibodies to malignant human gliomas.

    PubMed

    Wikstrand, C J; Fredman, P; Svennerholm, L; Humphrey, P A; Bigner, S H; Bigner, D D

    1992-10-01

    Operationally specific monoclonal antibodies (MAbs) reactive with tumor but not normal adult tissues offer great potential for diagnosis and therapy of CNS neoplasms. Two targets for specific MAb localization were chosen for this study: (1) glioma-associated gangliosides GM2 [II3NeuAc-GgOse3Cer], GD2 [II3(NeuAc)2-GgOse3Cer], GD3[II3(NeuAc)2-LacCer], 3'-isoLM1 [IV3NeuAc-LcOse4Cer], and 3',6'-isoLD1 [IV3NeuAc,III6NeuAc-LcOse4Cer] and (2) epidermal growth factor receptor (EGFR) variant molecules. Epitopic specificity of isolated ganglioside hybridomas was determined with FAB-MS defined ganglioside standards. All MAb are IgM. Assay of 14 cytologic specimens and 31 frozen sections of primary CNS neoplasms revealed staining with anti-GD3 (14/14, 31/31), anti-GM2 (9/14, 26/31), and anti-GD2 (6/14, 24/30), respectively. 3'-isoLM1 and 3',6' isoLD1, which exhibit a restricted oncofetal expression pattern and are not detectable in adult human brain, are present in 15/31 primary CNS neoplasms and in 1/8 human glioma xenografts, as detected by MAbs SL-50 and DMAb-14, respectively. EGFR proteins, the second target, have unique amino acid spans resulting from gene deletion in the amplified EGFR gene present in subsets of malignant human gliomas. Antibodies against EGFR deletion-mutant Type III show highly restricted activity with a subset of glioma biopsies (6/35) expressing the mutant EGFR. These reagents should be useful for in vitro and in vivo diagnosis and, potentially, for treatment of malignant brain tumors. PMID:1384525

  17. An evaluation of monoclonal antibodies for serum ferritin measurements.

    PubMed

    Skikne, B S; Linpisarn, S; Cook, J D

    1984-08-01

    Serum ferritin measurements have become an important laboratory parameter of iron status in prevalence surveys. Because the use of monoclonal antibodies may reduce variability and facilitate standardization, the present study was undertaken to determine whether their performance is comparable to conventional immunological reagents in an immunoradiometric assay for serum ferritin. When evaluated as the capture antibody for the solid phase, no differences in dose response curves were observed between monoclonal reagent and whole antiserum. When monoclonal reagent was used as the radiolabeled indicator antibody, the dose response curves were also similar to those obtained with affinity-purified polyclonal reagent although between-assay variability was somewhat higher with monoclonal antibody. In measurements of serum ferritin in patients with a wide range of iron status, no consistent differences were observed in the results from assays using monoclonal antibody and those of the conventional reagent. These preliminary studies indicate that the performance of monoclonal antibodies in a conventional ferritin assay is comparable to conventional immunological reagents. PMID:6465064

  18. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. (Univ. of Georgia, Athens (USA))

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  19. Monoclonal antibody to dengue capsid protein

    PubMed Central

    Vazquez, Y; Vazquez, S V; Capó; Torres, G; Caballero, Y; Sánchez, A; Limonta, D; Alvarez, M; Guzmán, MG

    2009-01-01

    Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection. PMID:20061827

  20. Characterization and utilization of a monoclonal antibody against pancreatic carcinoma

    SciTech Connect

    Kurtzman, S.H.; Sindelar, W.F.; Atcher, R.W.; Mitchell, J.B.; DeGraff, W.G.; Gamson, J.; Russo, A. [National Cancer Institute, Bethesda, MD (United States); Friedman, A.M.; Hines, J.J. [Argonne National Lab., IL (United States)

    1994-10-01

    A monoclonal antibody was produced against a human pancreatic adenocarcinoma line and was found to react with several different human carcinomas by immunoperoxidase staining of fixed tissues. The original cells used to generate the monoclonal antibody were treated with detergent to lyse the cell membrane. A membrane associated protein of molecular weight 35kD was isolated from this detergent lysed preparation and found to be recognized by the monoclonal antibody. The binding constant of the antigen antibody reaction on the cells is 5 x 10{sup {minus}5}. It was further determined that there are 700,000 binding sites per cell. Kinetics of the antigen-antibody reaction under several conditions were also explored.

  1. Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

    PubMed

    Macor, P; Secco, E; Mezzaroba, N; Zorzet, S; Durigutto, P; Gaiotto, T; De Maso, L; Biffi, S; Garrovo, C; Capolla, S; Tripodo, C; Gattei, V; Marzari, R; Tedesco, F; Sblattero, D

    2015-02-01

    The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications. PMID:24903480

  2. Inline protein a mass spectrometry for characterization of monoclonal antibodies.

    PubMed

    Prentice, Kenneth M; Wallace, Alison; Eakin, Catherine M

    2015-02-17

    Purification of antibodies is an important first step to produce material for in depth characterization of biotherapeutics. To reduce the resource burden incurred by protein purification, we developed a high throughput protein A affinity capture step coupled to inline mass spectrometry (PrA-MS). Our method enables both UV quantitation of antibodies and product characterization of an intact molecule with microgram quantities of material. When purification and analysis are coupled along with the low material demand, PrA-MS is widely applicable to protein characterization and is uniquely advantageous for moieties that rely on molecular stoichiometry. Two model systems were studied using PrA-MS and are presented here: (a) bispecific antibodies (bsAb) and (b) glycan engineered antibodies. In the bsAb samples, hetero- and homodimer species, along with partial molecule, were readily identified and quantified directly from harvested cell culture fluid (HCCF). In the glycan engineered antibodies, fully afucosylated, as well as asymmetrically and symmetrically fucosylated, glycans were identified from HCCF in experiments that utilized a small molecule inhibitor of fucosyltrasferase. The PrA-MS method represents a high throughput alternative to offline purification and product characterization that may be leveraged across the product lifecycle of engineered antibodies to enable rapid development and production of important therapeutics. PMID:25647041

  3. MONOCLONAL ANTIBODY TO FENBENDAZOLE: UTILITY IN RESIDUE STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody-based ELISA was developed for fenbendazole, a widely used benzimidazole anthelmintic, with approved uses in cattle and other food animals. The antibody was elicited using as hapten 2-succinamido-5(6)-phenylthiobenzimidazole, which was conjugated with bovine serum albumin to pro...

  4. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  5. Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy.

    PubMed

    Cheng, Ming; Ahmed, Mahiuddin; Xu, Hong; Cheung, Nai-Kong V

    2015-01-15

    Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy. PMID:24895182

  6. A perspective of monoclonal antibodies: Past, present, and future

    SciTech Connect

    DeLand, F.H. (Veterans Administration Medical Center, Syracuse, NY (USA))

    1989-07-01

    In 1975, the development of the technique to produce monoclonal antibodies revolutionized the approach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater complexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to attain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming. 58 references.

  7. Preparation of a monoclonal antibody against human monocyte lineage

    Microsoft Academic Search

    Seishin Maruyama; Tsutomu Naito; Haruhiko Kakita; Susumu Kishimoto; Yuichi Yamamura; Tadamitsu Kishimoto

    1983-01-01

    Preparation and characterization of a monoclonal antibody termed M206 directed to a human monocyte lineage are described. The antibody was produced by somatic cell hybridization between BALB\\/c spleen cells primed with human histiocytic lymphoma cell line U937 and murine myeloma cell line P3U1. The antibody reacted intensely with human peripheral blood monocytes as well as U937 but did not react

  8. Second Generation Anti-MUCl Peptide Monoclonal Antibodies

    Microsoft Academic Search

    Pei-xiang Xing; Julie Prenzoska; Kaylene Quelch; Ian F. C. McKenzie

    Second generation antibodies to mammary mucins were produced by immunizing mice with a peptide with a sequence deduced from that of the MUC1 complementary DNA sequence (PAHGVTSAPDTRPAPGS- TAP). Four monoclonal antibodies (BCP7-10) were produced which gave different reactions. BCP8 was similar in tissue reactivity (by immunoper- oxidase staining) to anti-breast cancer or anti-human milk fat globule membranes (HMFG) antibodies and

  9. Use of monoclonal antibodies in a radioimmunoassay for human transcortin.

    PubMed

    Faict, D; De Moor, P

    1984-03-01

    We describe the production of monoclonal antibodies to human transcortin and their use in a radioimmunoassay (RIA). A high-affinity antibody (Ka = 4 X 10(10) L/mol) made possible a sensitive RIA for transcortin (detection limit = 0.23 ng per tube), whereas use of an antibody of moderate affinity (Ka = 5 X 10(8) L/mol) was more suitable for the routine measurement of transcortin in serum, only a 25-fold dilution of the sample being required instead of 1500-fold. The correlation was good between both RIAs (r = 0.959) and between each of the RIAs and radial immunodiffusion (r = 0.955 and 0.976 for the methods with high- and low-affinity antibody, respectively). Although monoclonal antibodies were used in the RIAs and polyclonal ones in the radial immunodiffusion procedure, similar values were obtained by all techniques. PMID:6421509

  10. Characterization of a monoclonal antibody prepared against plant actin.

    PubMed

    Andersland, J M; Fisher, D D; Wymer, C L; Cyr, R J; Parthasarathy, M V

    1994-01-01

    Anti-actin monoclonal antibodies were prepared using phalloidin-stabilized actin that was purified from pea roots by DNase I affinity chromatography. One monoclonal antibody, designated mAb3H11, bound plant actin in preliminary screenings and was further analyzed. Immunoblot analysis showed that this antibody had a high affinity for plant actin in crude and purified preparations but a low affinity for rabbit muscle actin. In immunoblots of plant extracts separated on two-dimensional gels it appeared to bind all actin isoforms recognized by the JLA20 anti-chicken actin antibody. Using immunofluorescent cytochemistry, the antibody was used to observe actin filaments in aldehyde-fixed and methanol-treated tobacco protoplasts. These results indicate that mAb3H11 should be a useful reagent for the study of plant actins. PMID:7859296

  11. Identification and typing of herpes simplex viruses with monoclonal antibodies.

    PubMed Central

    Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D; Lavery, C; Rawls, W E

    1982-01-01

    Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited number of ulcerative lesions revealed that indirect immunofluorescence and virus isolation were comparable in detecting herpes simplex virus. The results indicate that monoclonal antibodies can be used to accurately identify and type isolates of herpes simplex virus. PMID:6286719

  12. Rat monoclonal antibody specific for MyoD.

    PubMed

    Harada, Akihito; Ohkawa, Yasuyuki; Ao, Shinpei; Odawara, Jun; Okada, Seiji; Azuma, Masayuki; Nishiyama, Yuko; Nakamura, Mako; Tachibana, Taro

    2010-06-01

    Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation. PMID:20569002

  13. Human regulatory T cells kill tumor cells through granzyme-dependent cytotoxicity upon retargeting with a bispecific antibody.

    PubMed

    Choi, Bryan D; Gedeon, Patrick C; Herndon, James E; Archer, Gary E; Reap, Elizabeth A; Sanchez-Perez, Luis; Mitchell, Duane A; Bigner, Darell D; Sampson, John H

    2013-09-01

    A major mechanism by which human regulatory T cells (T(regs)) have been shown to suppress and kill autologous immune cells is through the granzyme-perforin pathway. However, it is unknown whether T(regs) also possess the capacity to kill tumor cells using similar mechanisms. Bispecific antibodies (bscAbs) have emerged as a promising class of therapeutics that activate T cells against tumor antigens without the need for classical MHC-restricted TCR recognition. Here, we show that a bscAb targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, redirects human CD4(+)CD25(+)FoxP3(+) T(regs) to kill glioblastoma (GBM) cells. This activity was significantly abrogated by inhibitors of the granzyme-perforin pathway. Notably, analyses of human primary GBM also displayed diffuse infiltration of granzyme-expressing FoxP3(+) T cells. Together, these data suggest that despite their known suppressive functions, tumor-infiltrating T(regs) possess potent cytotoxic mechanisms that can be co-opted for efficient tumor cell lysis. PMID:24570975

  14. Simulations of site-specific target-mediated pharmacokinetic models for guiding the development of bispecific antibodies.

    PubMed

    Chudasama, Vaishali L; Zutshi, Anup; Singh, Pratap; Abraham, Anson K; Mager, Donald E; Harrold, John M

    2015-02-01

    Bispecific antibodies (BAbs) are novel constructs that are under development and show promise as new therapeutic modalities for cancer and autoimmune disorders. The aim of this study is to develop a semi-mechanistic modeling approach to elucidate the disposition of BAbs in plasma and possible sites of action in humans. Here we present two case studies that showcase the use of modeling to guide BAb development. In case one, a BAb is directed against a soluble and a membrane-bound ligand for treating systemic lupus erythematosus, and in case two, a BAb targets two soluble ligands as a potential treatment for ulcerative colitis and asthma. Model simulations revealed important differences between plasma and tissues, when evaluated for drug disposition and target suppression. Target concentrations at tissue sites and type (soluble vs membrane-bound), tissue-site binding, and binding affinity are all major determinants of BAb disposition and subsequently target suppression. For the presented case studies, higher doses and/or frequent dosing regimens are required to achieve 80 % target suppression in site specific tissue (the more relevant matrix) as compared to plasma. Site-specific target-mediated models may serve to guide the selection of first-in-human doses for new BAbs. PMID:25559227

  15. Targeting Cytomegalovirus-Infected Cells Using T Cells Armed with Anti-CD3 × Anti-CMV Bispecific Antibody

    PubMed Central

    Lum, Lawrence G.; Ramesh, Mayur; Thakur, Archana; Mitra, Subhashis; Deol, Abhinav; Uberti, Joseph P.; Pellett, Philip E.

    2013-01-01

    Human cytomegalovirus (CMV) reactivation and infection can lead to poor outcomes after allogeneic stem cell transplantation. We hypothesized that anti-CD3 activated T cells (ATCs) armed with chemically heteroconjugated anti-CD3 × polyclonal anti-CMV bispecific antibody (CMVBi) will target and eliminate CMV-infected cells. Arming doses of CMVBi as low as 0.01 ng/106 ATCs was able to mediate specific cytotoxicity (SC) directed at CMV-infected target cells significant above unarmed ATCs at mutiplicities of infection (MOI) between 0.01 and 1. At effector-to-target ratios (E:T) of 25:1, 12.5:1, 6.25:1, and 3.125:1, armed ATCs significantly enhanced killing of CMV-infected targets compared with unarmed ATCs. At an MOI of 1.0, the mean % SC directed at CMV-infected targets cells for CMVBi-armed ATCs at E:T of 3.12, 6.25, and 12.5 were 79%, 81%, and 82%, respectively; whereas the mean % SC for unarmed ATCs at the same E:T were all <20%. ATCs, Cytogam®, or CMVBi alone did not lyse uninfected or CMV-infected targets. Co-cultures of CMVBi-armed ATCs with CMV-infected targets induced cytokine and chemokine release from armed ATCs. This nonmajor histocompatibility complex restricted strategy for targeting CMV could be used to prevent or treat CMV infections after allogeneic stem cell transplantation or organ transplantation. PMID:22313635

  16. Effect of Small Molecule Binding Affinity on Tumor Uptake In Vivo: A Systematic Study Using a Pretargeted Bispecific Antibody

    PubMed Central

    Orcutt, Kelly Davis; Rhoden, John J; Ruiz-Yi, Benjamin; Frangioni, John V; Wittrup, K Dane

    2014-01-01

    Small molecule ligands specific for tumor-associated surface receptors have wide applications in cancer diagnosis and therapy. Achieving high-affinity binding to the desired target is important for improving detection limits and for increasing therapeutic efficacy. However, the affinity required for maximal binding and retention remains unknown. Here, we present a systematic study of the effect of small molecule affinity on tumor uptake in vivo with affinities spanning a range of three orders of magnitude. A pretargeted bispecific antibody with different binding affinities to different DOTA-based small molecules is used as a receptor proxy. In this particular system targeting carcinoembryonic antigen, a small-molecule binding affinity of 400 pM was sufficient to achieve maximal tumor targeting, and an improvement in affinity to 10 pM showed no significant improvement in tumor uptake at 24 h post-injection. We derive a simple mathematical model of tumor targeting using measurable parameters that correlates well with experimental observations. We use relations derived from the model to develop design criteria for the future development of small molecule agents for targeted cancer therapeutics. PMID:22491799

  17. Blinatumomab, a Bi-Specific Anti-CD19/CD3 BiTE® Antibody for the Treatment of Acute Lymphoblastic Leukemia: Perspectives and Current Pediatric Applications

    PubMed Central

    Hoffman, Lindsey M.; Gore, Lia

    2014-01-01

    Leukemia is the most common childhood malignancy and acute lymphoblastic leukemia (ALL) represents the largest sub-type. Despite remarkable improvements over the last 40?years, standard therapy fails in 10–20% of newly diagnosed patients. Survival for children with relapsed ALL is poor, and the development and implementation of novel therapeutic strategies in pediatric ALL are critical to further advancements. Immunotherapeutic approaches have been central to more novel ALL therapies. However, more recent innovation in antibody engineering has improved potency and efficacy, and antibody–drug conjugates (ADCs) are an especially attractive option in severely immunocompromised patients. An even more sophisticated antibody design is that of bi-specific T-cell engaging or BiTE® antibodies, which directly recruit effector T cells to augment the anti-neoplastic effect. This review focuses on blinatumomab, a bi-specific anti-CD19/CD3 antibody that has shown efficacy in adult patients with precursor B-ALL and is currently being evaluated in the pediatric setting. PMID:24744989

  18. Freezing-induced perturbation of tertiary structure of monoclonal antibody

    E-print Network

    Liu, Lu; Kueltzo, L. A.; Jones, L. S.; Carpenter, J. F.

    2006-10-25

    Freezing-induced Perturbation of Tertiary Structure of Monoclonal Antibody Lu Liu, LaToya S. Jones, John F. Carpenter Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, School of Pharmacy, University... Temperature (C) -30 -20 -10 0 10 20 0.0 5.0e+5 1.0e+6 1.5e+6 2.0e+6 2.5e+6 Reference [1] Reichert, J.M., et al., Monoclonal antibody successes in the clinic. Nat Biotechnol, 2005. 23(9): p. 1073-8. [2] Gabellieri, E. and G.B. Strambini, Perturbation...

  19. MONOCLONAL ANTIBODIES IDENTIFY CONSERVED EPITOPES ON THE POLYHEDRIN OF 'HELIOTHIS ZEA' NUCLEAR POLYHEDROSIS VIRUS

    EPA Science Inventory

    Recent advances in monoclonal antibody techniques have provided an opportunity to simplify the procedures of serological identification of microorganisms. Because monoclonal antibodies are raised against individual antigenic determinants (epitopes), they can be used to screen wit...

  20. Diverse immunostaining patterns of mineralocorticoid receptor monoclonal antibodies.

    PubMed

    Gomez-Sanchez, Celso E; Warden, Mary; Gomez-Sanchez, Miriam T; Hou, Xu; Gomez-Sanchez, Elise P

    2011-12-20

    The mineralocorticoid receptor (MR) is a widely distributed ligand activated nuclear transcription factor that is bound by various chaperone proteins that alter its conformation depending upon its location in the cell and whether it is ligand-bound. We describe the development and characterization of new monoclonal antibodies produced against a rat recombinant protein corresponding to aminoacids 5-550 of the MR to produce antibodies that recognize the receptor in specific conformations. Most of the resulting monoclonal antibodies studied were similar to those we produced by immunization with peptide isotopes, however two detected a single band at the appropriate molecular mass as the MR and had distinct immunostaining characteristics in neurons. One labeled cytosolic MR, the other labeled membranes and cytosol, including axons. These antibodies will permit study of the subcellular localization of the MR under various physiological and pathological conditions. We have also confirmed that the MR is highly unstable and requires special handling. PMID:21945398

  1. Monoclonal antibody specific for Dhx9/NDHII/RHA.

    PubMed

    Kotani, Manato; Harada, Akihito; Odawara, Jun; Azuma, Masayuki; Okada, Seiji; Nishiyama, Yuko; Nakamura, Mako; Tachibana, Taro; Ohkawa, Yasuyuki

    2010-06-01

    Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression. PMID:20569003

  2. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  3. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

    PubMed Central

    Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

    2013-01-01

    The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

  4. The safety and side effects of monoclonal antibodies

    Microsoft Academic Search

    Harald Kropshofer; Thomas Singer; Jane A. Mitchell; Andrew J. T. George; Trevor T. Hansel

    2010-01-01

    Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases, as well as a range of new indications. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness and the generation of antibodies. In addition, there are numerous adverse effects of mAbs that are related to their

  5. 78 FR 7438 - Prospective Grant of Exclusive License: Development of Human Monoclonal Antibodies Against DR4

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-01

    ...License: Development of Human Monoclonal Antibodies Against DR4 AGENCY: National Institutes...entitled ``Agonistic Human Monoclonal Antibodies Against DR4'' (HHS Ref. No. E-158-2010...development of two human monoclonal antibodies (mAbs) that bind to death...

  6. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies

    Microsoft Academic Search

    F. T. Hufert; W. Lüdke; H. Schmitz

    1989-01-01

    Summary Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd,

  7. Rabbit Monoclonal Antibodies: Generating a Fusion Partner to Produce Rabbit-Rabbit Hybridomas

    Microsoft Academic Search

    Helga Spieker-Polet; Periannan Sethupathi; Pi-Chen Yam; Katherine L. Knight

    1995-01-01

    During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma

  8. A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

    PubMed

    Schanzer, Juergen M; Wartha, Katharina; Croasdale, Rebecca; Moser, Samuel; Künkele, Klaus-Peter; Ries, Carola; Scheuer, Werner; Duerr, Harald; Pompiati, Sandra; Pollman, Jan; Stracke, Jan; Lau, Wilma; Ries, Stefan; Brinkmann, Ulrich; Klein, Christian; Umana, Pablo

    2014-07-01

    In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats. PMID:24841203

  9. DEVELOPMENT AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO CHICKEN INTERLEUKIN-15

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chicken IL-15 gene was recently cloned and shown to encode a polypeptide with T cell growth factor activity similar to IL-2. To further characterize the chemical and biological properties of chicken IL-15, we generated a panel of monoclonal antibodies against bacterially expressed protein and c...

  10. Production and characterization of monoclonal antibodies against Taylorella equigenitalis

    E-print Network

    Boyer, Edmond

    strains of T equigenitalis isolated in France. They showed no cross-reaction with bacterial strains with previously reported antigenic cross-reactivity, nor did they react with other bacteria commonly found in genital flora. The epitopes recognized by eight of the monoclonal antibodies were situated in proteins

  11. Canine lymphoma-associated antigens defined by murine monoclonal antibodies

    Microsoft Academic Search

    Zenon Steplewski; K. Ann Jeglum; Carlos Rosales; Nancy Weintraub

    1987-01-01

    Lymphoma in dogs resembles human non-Hodgkin's lymphoma in pathological presentation, immunophenotype, and response to therapy, thus representing a good model for comparative studies with human disease. Monoclonal antibodies (MAbs) were derived from mice immunized with a dog lymphoma cell line. Three MAbs were selected for further application in immunophenotyping and immunotherapy. The binding specificities, antigen characterization, and isotypes for these

  12. Characterization of monoclonal antibodies produced against Avian metapneumovirus Sybtype C

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/Minnesota/turkey/1a/97). Six MAbs were selected based on ELISA activities and characterized by isotyping, neutralization test, Western blot analysis, and immunohistochemistry (IHC) assay. The results show...

  13. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  14. A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

    PubMed

    Blaudszun, André-René; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

    2015-01-10

    Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. PMID:25449805

  15. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    PubMed Central

    Gdowski, Andrew; Ranjan, Amalendu; Mukerjee, Anindita; Vishwanatha, Jamboor

    2015-01-01

    Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells. PMID:25690029

  16. Development of biodegradable nanocarriers loaded with a monoclonal antibody.

    PubMed

    Gdowski, Andrew; Ranjan, Amalendu; Mukerjee, Anindita; Vishwanatha, Jamboor

    2015-01-01

    Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%-22% and antibody loading of 0.3%-1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells. PMID:25690029

  17. Use of monoclonal antibodies in the assay of hepatitis B core antigen and antibody.

    PubMed

    Furuya, Y; Inoue, M; Yoshihara, N

    1984-08-01

    Hybridoma cells secreting antibody against hepatitis B core antigen (HBc Ag) were prepared. BALB/c mice were immunized with 0.2 ml of purified HBc Ag, and their spleen cells were fused with mouse myeloma (P3U1) cells by means of polyethylene glycol 1000. Activities of antibodies against HBc Ag (anti-HBc) were tested by the immune adherence hemagglutination (IAHA) and reverse passive hemagglutination inhibition (RPHI) techniques. Hybridoma cells found to contain antibodies accounted for 26.5% by IAHA and 52.1% by RPHI, respectively. Among 32 monoclonal anti-HBc antibodies, 18 were found to be positive by both IAHA and RPHI, and the remaining 14 positive by RPHI only. After cloning, they were injected intraperitoneally into ascitic mice. The highest anti-HBc activity with an IAHA titer of 1:4 X 10(6) and with an RPHI titer of 1:1 X 10(5) was detected in this ascitic fluid. Enzyme immunoassay (EIA) and RPHI with monoclonal antibody containing the highest anti-HBc activity were developed. All the sera in which anti-HBc was detected by IAHA and RPHI with polyclonal antibody were positive in EIA. RPHI titers obtained with monoclonal antibody were in good agreement with usual IAHA and RPHI titers obtained with polyclonal antibody. These results indicate that monoclonal antibody can be used in the HBc Ag and anti-HBc assay system. PMID:6396424

  18. High efficiency iodination of monoclonal antibodies for radiotherapy

    SciTech Connect

    Mather, S.J.; Ward, B.G.

    1987-06-01

    A technique for labeling monoclonal antibodies (MoAb) with large activities of radioiodine using the reagent N-bromosuccinimide is described. This technique has been used to label three tumor-associated antibodies with an average labeling efficiency of greater than 90%. No significant damage to the antibody could be detected by enzyme linked immunosorbent assay (ELISA), performed immediately after the labeling procedure. Because high radiochemical purity can be achieved without the need postlabeling purification, the procedure is rapid and results in very low radiation doses to staff.

  19. Monoclonal Antibody Cross-Reactions between Drosophila and Human Brain

    NASA Astrophysics Data System (ADS)

    Miller, Carol A.; Benzer, Seymour

    1983-12-01

    A panel of 146 monoclonal antibodies (MAbs), obtained with Drosophila melanogaster tissue as primary immunogen, was tested for cross-reactivity with the human central nervous system. Sites examined included spinal cord, cerebellum, hippocampus, and optic nerve. Nonnervous tissues tested were liver and lymph node. Approximately half of the antibodies reacted with one or more sites in the human central nervous system, identifying regional, cell class, and subcellular antigens. Some recognized neuronal, glial, or axonal subsets. Immunoblot analysis revealed that some antibodies reacted with similar antigen patterns in both species.

  20. Experimental Tumoricidal Effects of Monoclonal Antibody against Solid Breast Tumors

    NASA Astrophysics Data System (ADS)

    Capone, Patrick M.; Papsidero, Lawrence D.; Croghan, Gary A.; Chu, T. Ming

    1983-12-01

    Two distinct monoclonal antibodies (mAbs) were effective in the therapy of breast carcinomas of human origin established and growing in nude mice. Passive administration of either of the antibodies produced very rapid (less than 1 week) and significant reduction of in vivo tumor volume. Each of the mAbs showed in vivo targeting of the tumors. Histological analysis of mAb-treated tumors revealed extensive cellular necrosis. Each of the antibodies in vitro was effective in complement-mediated cytolysis at a concentration <1 ng/ml. The tumoricidal responses show that this is a useful model for passive human immunotherapy using mAbs.

  1. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    PubMed

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-12-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. PMID:6783353

  2. Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies

    E-print Network

    Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two

  3. Adsorption of monoclonal antibodies to glass microparticles.

    PubMed

    Hoehne, Matthew; Samuel, Fauna; Dong, Aichun; Wurth, Christine; Mahler, Hanns-Christian; Carpenter, John F; Randolph, Theodore W

    2011-01-01

    Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension. PMID:20575075

  4. Proteomic Identification of Monoclonal Antibodies from Serum

    PubMed Central

    2015-01-01

    Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between “true” and “false” identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases. PMID:24684310

  5. Monoclonal antibody production in dialyzed continuous suspension culture.

    PubMed

    Linardos, T I; Kalogerakis, N; Behie, L A; Lamontagne, L R

    1992-03-01

    Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitutes passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuously replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations. PMID:18600976

  6. Production of a rat monoclonal antibody specific for Myf5.

    PubMed

    Harada, Akihito; Okada, Seiji; Odawara, Jun; Kumamaru, Hiromi; Saiwai, Hirokazu; Aoki, Mayumi; Nakamura, Mako; Nishiyama, Yuko; Ohkawa, Yasuyuki

    2010-02-01

    Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function. PMID:20199153

  7. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  8. Production of monoclonal antibodies by glycoengineered Pichia pastoris.

    PubMed

    Potgieter, Thomas I; Cukan, Michael; Drummond, James E; Houston-Cummings, Nga Rewa; Jiang, Youwei; Li, Fang; Lynaugh, Heather; Mallem, Muralidhar; McKelvey, Troy W; Mitchell, Teresa; Nylen, Adam; Rittenhour, Alissa; Stadheim, Terrance A; Zha, Dongxing; d'Anjou, Marc

    2009-02-23

    The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities. PMID:19162096

  9. MAbs . Author manuscript Bispecific antibodies for cancer therapy: the light at the end of the tunnel?

    E-print Network

    Paris-Sud XI, Université de

    that was approved in the European Union in April 2009. This review describes the most recent advances and clinical ; immunology ; therapeutic use ; Antibody-Dependent Cell Cytotoxicity ; genetics ; immunology ; Drug Approval ; Europe ; Genetic Engineering ; Humans ; Immunoglobulin Fc Fragments ; genetics ; immunology ; Neoplasms

  10. Production and characterization of monoclonal antibodies against aflatoxin B1.

    PubMed

    Soukhtanloo, Mohammad; Talebian, Elham; Golchin, Mehdi; Mohammadi, Mojgan; Amirheidari, Bagher

    2014-01-01

    In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×10(11) M(-1) and 6×10(9) M(-1), respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits. PMID:24350626

  11. Detection of retroviral particles in hybridomas secreting monoclonal antibodies.

    PubMed

    Bartal, A H; Feit, C; Erlandson, R A; Hirshaut, Y

    1986-01-01

    Electronmicroscopy of hybridoma clones derived by fusing BALB/c mouse spleen cells with P3U1 mouse plasmacytoma cells to generate monoclonal antibodies against human sarcoma antigens, revealed the presence of large number of viral particles. These particles were also seen budding from the cell surfaces. The intracytoplasmic particles were intracisternal and resembled type-A oncornavirus, while the budding and extracellular forms, with a centrally located nucleoid, resembled mature type-C oncornaviruses. Cells of the parental P3U1 palsmacytoma cell line and of the NS-1 myeloma cell line contained morphologically identical viral structures. The scientific and medical communities engaged in hybridoma research should be alert to the possible presence of viruses in hybridomas and their products. The question is raised as to whether it is safe to use mouse monoclonal antibodies for clinical purposes, both diagnostic and therapeutic. PMID:3951394

  12. Monoclonal antibodies for the detection of trace chemicals

    SciTech Connect

    Vanderlaan, M.; Van Emon, J.; Watkins, B.; Stanker, L.

    1986-08-15

    Problems in analytical chemistry may limit monitoring for trace organic residues by traditional chromatographic methods. For example, the cost and analysis time per sample may preclude adequate sampling, making the development of alternative technologies desirable. Immunoassays are one such alternative, with the potential for cost reduction by automation and parallel sample processing. A particularly significant advance in the past decade has been the development of monoclonal antibodies, which offer greater selectivity and reproducibility than conventional antisera. Immunoassays can be developed that use simple, field-portable instrumentation, give rapid results, and have detection limits of less than a part-per-billion. This paper reviews the general technology for developing monoclonal antibodies to small organic molecules using the immunoassay of 2,3,7,8-tetrachlorodibenzodioxin (2,3,7,8-TCDD) as an example. 18 refs., 2 figs., 1 tab.

  13. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

  14. Monoclonal antibodies defining distinctive human T cell surface antigens

    Microsoft Academic Search

    P. C. Kung; G. Goldstein; E. L. Reinherz; S. F. Schlossman

    1979-01-01

    Three novel monoclonal antibodies (designated OKT1, OKT3, and OKT4) were generated against surface determinants of human peripheral T cells. Both OKT1 and OKT3 reacted with all human peripheral T cells and 5 to 10 percent of thymocytes but differed in their reactivities with T cell lines. By contrast, OKT4 reacted with 55 percent of human peripheral T cells and 80

  15. Alzheimer paired helical filaments: Identification of polypeptides with monoclonal antibodies

    Microsoft Academic Search

    I. Grundke-Iqbal; G. P. Wang; K. Iqbal; Y.-C. Tung; H. M. Wisniewski

    1985-01-01

    Paired helical filaments (PHF) were isolated from autopsied brain of cases of Alzheimer dementia, and their polypeptides were identified with monoclonal antibodies to PHF by Western blots. The PHF polypeptide profile consisted of several bands with a size difference of less than 5 kilodalton (kDa) between adjacent bands; the most prominent bands were in the 45–62 kDa region. These PHF

  16. Monoclonal antibodies for the identification of herpesvirus simiae (B virus)

    Microsoft Academic Search

    L. M. Cropper; D. N. Lees; R. Patt; I. R. Sharp; D. Brown

    1992-01-01

    Summary To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus,

  17. Monoclonal Antibody Specific for an Activated RAS Protein

    Microsoft Academic Search

    W. P. Carney; D. Petit; P. Hamer; C. J. der; T. Finkel; G. M. Cooper; M. Lefebvre; H. Mobtaker; R. Delellis; A. S. Tischler; Y. Dayal; H. Wolfe; H. Rabin

    1986-01-01

    Activated RAS transforming genes that encode proteins (p21s) with amino acid substitutions at positions 12, 13, or 61 have been detected in 10-20% of human neoplasms. This report describes a monoclonal antibody (DWP) raised against a synthetic peptide corresponding to amino acids 5-16 of a mutated RAS gene encoding Val instead of Gly at position 12. DWP reacted in competition

  18. Establishment of a novel monoclonal antibody against LGR5

    Microsoft Academic Search

    Yuka Sasaki; Hiromichi Kosaka; Katsuaki Usami; Hiroe Toki; Hironori Kawai; Norihiko Shiraishi; Toshio Ota; Kazuyasu Nakamura; Akiko Furuya; Mitsuo Satoh; Kazumasa Hasegawa; Kazuhiro Masuda

    2010-01-01

    LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for

  19. Monoclonal antibody drug immunoconjugates for targeted treatment of cancer

    Microsoft Academic Search

    Pamela A. Trail; Dalton H. King; Gene M. Dubowchik

    2003-01-01

    Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy.

  20. Preparation of a monoclonal antibody against human monocyte lineage.

    PubMed

    Maruyama, S; Naito, T; Kakita, H; Kishimoto, S; Yamamura, Y; Kishimoto, T

    1983-01-01

    Preparation and characterization of a monoclonal antibody termed M206 directed to a human monocyte lineage are described. The antibody was produced by somatic cell hybridization between BALB/c spleen cells primed with human histiocytic lymphoma cell line U937 and murine myeloma cell line P3U1. The antibody reacted intensely with human peripheral blood monocytes as well as U937 but did not react with peripheral blood T and B cells. Granulocytes were weakly stained with the antibody by indirect immunofluorescence. M206 also reacted intensely with the immature leukemic cells from patients with acute monocytic leukemia but was unreactive with other types of leukemic cells except cells from chronic myelogenous leukemia which showed varied patterns of reactivities. M206 reacted with a single polypeptide chain with a molecular weight of 180,000 on the surface of U937 cells. PMID:6338027

  1. Myeloid differentiation antigen defined by a monoclonal antibody IF10.

    PubMed

    Yamada, K; Okabe, N; Saito, H; Suzuki, R; Kumagai, K

    1983-09-01

    A monoclonal antibody of IgM class that defines the antigen present on human peripheral blood granulocytes was produced and characterized. This monoclonal antibody (IF10) was made from a single fusion between P3-X63-Ag8-U1 (P3U1) myeloma cells and splenocytes from a BALB/c mouse immunized against human cultured monocytoid cell line THP-1 cells. IF10-defined antigen(s) was expressed on the cells of granulocyte lineage such as peripheral blood granulocytes and metamyelocytes, myelocytes, promyelocytes, and a part of myeloblasts in the normal bone marrow, whereas it was not detected on resting and activated T lymphocytes, B lymphocytes, adherent monocytes, and thymocytes. The IF10-defined antigen was also expressed on cultured monocytoid cell lines as well as myeloid and myeloid/erythroid (K-562) cell lines. T lymphoblastoid cell lines, particularly those representing the T cells at an early thymocyte level, and a part of null cell lines were also reactive to IF10. Therefore, IF10 may be a unique monoclonal antibody that defines an antigen(s) which is expressed on almost whole stages of granulocytes and early stages of macrophages and T-cell lineages, and possibly during very early stages of erythroid lineage. PMID:6412543

  2. Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors.

    PubMed Central

    Young, N S; Hwang-Chen, S P

    1981-01-01

    The K562 leukemia cell has properties of self-renewal and pluripotency similar to those of the hematopoietic stem cell. Monoclonal antibodies to K562 cells have been produced by using hybridoma technology. By radioimmunoassay, some anti-K562 cell antibodies also bind to erythrocyte antigens or peripheral blood mononuclear cells; others are more specific for K562 cells. Antibody binding to hematopoietic progenitors was assayed by using the ability of these cells to form colonies in vitro. After exposure of human bone marrow cells to anti-K562 antibodies and complement, myeloid or erythroid colony formation was inhibited. Some of the inhibitory antibodies showed little binding to mature blood cells by radioimmunoassay, immunofluorescence, and complement cytotoxicity, suggesting that they recognize antigens specific to undifferentiated cells. With the fluorescence-activated cell sorter, one inhibitory antibody was shown to stain only 3% of bone marrow cells. Inhibitory anti-K562 antibodies also bind to myelogenous leukemia cells and virus-transformed lymphocytes. Thus, these antibodies appear to recognize antigens shared by normal hematopoietic progenitors, leukemic cells, and transformed lymphocytes. Images PMID:7031668

  3. Comparison of methods used to radiolabel monoclonal antibodies

    SciTech Connect

    Otsuka, F.L.; Welch, M.J.

    1985-05-01

    A model system designed for use in evaluating the binding capacity and biodistribution of radiolabeled monoclonal antibodies has been described. Using this system HDP-1 monoclonal antibodies labeled by iodination have been compared with those labeled with /sup 111/In. The /sup 111/In was bound to the antibodies after the attachment of deferoxamine, 1-(para-bromocetamidobenzyl)EDTA (BrEDTA) or diethylenetriamine pentaacetic acid (DTPA). Similar comparisons have been done with HDP-1 F(ab)/sub 2/ or Fab fragments labeled by the same methods. All labelled antibodies and fragments retain their ability to bind to the DNP beads when tested in the model system. The amount of /sup 125/I(HDP-1) or /sup 125/I(HDP-1) F(ab)/sub 2/ fragments in the lungs reaches maximum levels at approximately 24 hours after injection of the antibodies while maximum uptake is observed at 4 hours for the /sup 125/I(HDP-1) Fab fragments. The levels of radioactivity in the liver and kidney decline over the course of the experiment such that by 48 hours the % ID/g values in these regions are quite low. In contrast, high levels of /sup 111/In-labled HDP-1 are observed in the lungs over the course of the experiment; no decrease is observed. For the liver, however, the % ID/g values increase dramatically over the course of the experiment when /sup 111/In-labled HDP-1 is used. Similar increases are observed for the kidney when /sup 111/In-labeled fragments are used. The use of DTPA coupled whole antibody or fragments that have been reaffinity purified after attachment of the chelates does not alter these results. Overall, the authors' results suggest the labeling technique using BrEDTA is the preferred method for radiolabeling antibodies.

  4. Modulation of desmin intermediate filament assembly by a monoclonal antibody

    PubMed Central

    1988-01-01

    We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys- 324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right- handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody. PMID:2450097

  5. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  6. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Microsoft Academic Search

    Pietro Paolo Sanna; R. Anthony Williamson; Alessandro de Logu; Floyd E. Bloom; Dennis R. Burton

    1995-01-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage

  7. Methods for the generation of chicken monoclonal antibody fragments by phage display

    Microsoft Academic Search

    Jennifer Andris-Widhopf; Christoph Rader; Peter Steinberger; Roberta Fuller; Carlos F Barbas III

    2000-01-01

    Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be

  8. Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases.

    PubMed Central

    Zeitlin, L.; Cone, R. A.; Whaley, K. J.

    1999-01-01

    Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale. PMID:10081672

  9. Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment

    NASA Astrophysics Data System (ADS)

    Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.

    1991-04-01

    We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine ? globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  10. Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats

    Microsoft Academic Search

    Kelly A Byrnes-Blake; Elizabeth M Laurenzana; F. Ivy Carroll; Philip Abraham; W. Brooks Gentry; Reid D Landes; S. Michael Owens

    2003-01-01

    Our studies examined pharmacokinetic mechanisms involved in high-affinity (Kd?11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg\\/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg\\/kg

  11. Monoclonal antibody analysis of Perkinsus marinus extracellular products.

    PubMed

    Earnhart, Christopher G; Gauthier, David T; Vogelbein, Wolfgang K; Kaattari, Stephen L

    2005-02-01

    The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner. PMID:15710438

  12. Characterization of new Alternaria alternata--specific rat monoclonal antibodies.

    PubMed

    Denis, Olivier; Van Cauwenberge, Anne; Treutens, Greta; Es Saadi, Bouazza; Symoens, Françoise; Popovic, Nathalie; Huygen, Kris

    2012-03-01

    In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass. PMID:21892786

  13. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES, APTAMERS AND SINGLE CHAIN ANTIBODIES TO MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis (MAP) was identified as an unmet need at the 7th International Colloquium on Paratuberculosis in Bilbao, Spain. To fill this gap in Johne’s disease research, monoclonal antibodies (mAbs) against MAP were produced from BALB/c mice immunized with sonicated MAP extracts or recombinant...

  14. Labeling of cerebral amyloid in vivo with a monoclonal antibody.

    PubMed

    Walker, L C; Price, D L; Voytko, M L; Schenk, D B

    1994-07-01

    We assessed the ability of a murine monoclonal antibody to bind selectively to beta-amyloid in the brains of living nonhuman primates. To circumvent the blood-brain barrier, we injected unlabeled antibody 10D5 (murine whole IgG1 and/or Fab fragments) into the cerebrospinal fluid of the cisterna magna in three aged monkeys. A control animal was given an intracisternal injection of nonimmune mouse whole IgG plus Fab. Twenty-four hours later, the animals were perfused and prepared for immunohistochemical detection of bound murine immunoglobulin in brain. All three experimental animals showed selective binding of 10D5 to approximately 5-15% of amyloid deposits in cerebral cortex, primarily near the cortical surface. There was no labeling in the control animal. In vivo-labeled deposits were confirmed to be beta-amyloid by electron microscopy and by in vitro immunohistochemistry in adjacent sections. The animals tolerated the injection well, although some polymorphonuclear leukocytes infiltrated portions of the subarachnoid space and superficial neocortex. These results provide the first demonstration that it may be feasible to selectively direct a tagged monoclonal antibody to beta-amyloid in the brain for therapeutic or diagnostic purposes. With enhancement of labeling efficiency, the method also may be useful for studying the progression of beta-amyloidosis in experimental animals using emission tomography. PMID:8021711

  15. Monoclonal Antibody Therapy and Renal Transplantation: Focus on Adverse Effects

    PubMed Central

    Zaza, Gianluigi; Tomei, Paola; Granata, Simona; Boschiero, Luigino; Lupo, Antonio

    2014-01-01

    A series of monoclonal antibodies (mAbs) are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft), to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52). Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications). Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications. PMID:24590384

  16. Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis.

    PubMed

    Liu, Meng-yuan; Hu, Xue-ping; Xie, Mian; Jiang, Si-jing; Li, Lu-jun; Liu, Dong-xu; Yang, Xiao-song

    2014-12-01

    Specific targeting of tumor necrosis factor (TNF)-? antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-? and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-? and B-FN and neutralize TNF-? action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-?-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-? scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications. PMID:25129049

  17. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

  18. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Microsoft Academic Search

    ZhanHui Wang; LinLi Cheng; WeiMin Shi; SuXia Zhang; JianZhong Shen

    2010-01-01

    A fluorescence polarization immunoassay (FPIA) for the determination of salinomycin (SAL) was developed by using anti-SAL\\u000a monoclonal antibodies (mAb). Fluorescein labeled SAL (tracer) was synthesized by the N-hydroxysuccinimide active ester method\\u000a and purified using thin layer chromatography (TLC). The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng\\/mL\\u000a with an IC50 value of 33.2 ng\\/mL and

  19. Isolation of a monoclonal antibody which blocks vaccinia virus infection.

    PubMed Central

    Chang, W; Hsiao, J C; Chung, C S; Bair, C H

    1995-01-01

    We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry. PMID:7527087

  20. Development of Monoclonal Antibodies in China: Overview and Prospects

    PubMed Central

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Ung, Carolina Oi Lam; Wang, Yi-Tao

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Development and research trends of mAbs are analyzed to provide a future perspective of mAbs as therapeutic agents in China.

  1. Network biology in development of monoclonal antibody therapeutics.

    PubMed

    Ovacik, Ayse Meric

    2015-02-01

    Monoclonal antibodies (mAbs) are large glycoproteins that recognize and remove/neutralize a specific target. Inflammation and inflammatory diseases are often treated with mAb-based therapeutics. Mathematical modeling is widely used in development of mAbs. Bioinformatics and structural modeling is used for humanization of mAbs and PK/PD modeling is extensively used in preclinical and clinical development. The objective of this commentary is to introduce systems biology-based modeling that can accelerate and improve development of mAbs. PMID:25311982

  2. Monoclonal antibody based immunoassays for cooking-induced meat mutagens

    SciTech Connect

    Vanderlaan, M.; Hwang, M.; Knize, M.G.; Watkins, E.; Felton, J.S.

    1989-06-29

    We report here new monoclonal antibodies (Mabs) numbered AIA-8 through AIA-12 produced using the same methods used to produce AIA-1, and a new Mab, IQ-7, produced with the same methods used for IQ-1. Our motivation in seeking these new clones was to increase the repertoire of available Mabs to insure adequate coverage of all known AIAs. Also, the mice used to produce these new hybridoma clones had been immunized about six months longer than those used to generate the first clones. Longer immunization is often associated with higher affinity Mabs and therefore more sensitive competition immunoassays. 15 refs., 2 figs., 2 tabs.

  3. Two human monoclonal antibodies reacting with the major gangliosides of human melanomas and comparison with corresponding mouse monoclonal antibodies.

    PubMed

    Furukawa, K; Yamaguchi, H; Oettgen, H F; Old, L J; Lloyd, K O

    1989-01-01

    The fine specificity analysis of two human monoclonal antibodies (AbFCM1 and AbHJM1) reacting with gangliosides is described and their specificities are compared with analogous mouse monoclonal antibodies (mAbs). These two antibodies were generated from lymphocytes of melanoma patients by Epstein-Barr virus transformation followed by fusion with mouse myeloma NS-1. Using a wide variety of gangliosides, including N-glycolylneuraminic acid (NeuGc)-containing compounds, the precise structures recognized by these two antibodies were elucidated by enzyme-linked immunosorbent assay and immunostaining of thin-layer chromatograms. AbFCM1 reacted with N-acetylneuraminic acid (NeuAc)-type GM3, GD1a, sialylparagloboside, and GT1b in decreasing order of intensity. This antibody also reacted with (NeuAc-NeuGc-)-GD3 and -disialylparagloboside, but did not react with NeuGc-type GM3, GM2, sialylparagloboside, (NeuGc)2-GD3 and -disialylparagloboside. The main epitope structures recognized by AbFCM1 are, therefore, NeuAc alpha 2----3Gal beta 1- and NeuAc alpha 2----8NeuGc alpha 2----Gal beta 1-. These results are similar to the specificity of mouse mAb M2590. AbHJM1 reacted with NeuAc-type GD3 and disialylparagloboside, GD2, GD1b, GM3, and GT1b, in decreasing order of intensity. Among NeuGc-type gangliosides, this antibody reacts with (NeuAc-NeuGc-)-GD3 and -disialylparagloboside, but did not react with gangliosides containing only NeuGc. Consequently the epitope structure recognized by AbHJM1 is probably (R)-(NeuAc alpha 2----8Sialic acid alpha 2----3)Gal beta 1-. Mouse anti-GD3 mAbR24, in contrast, showed strong reactivity only with GD3 and -disialylparagloboside among NeuAc-type gangliosides, but showed a similar pattern to AbHJM1 in its reactivity with NeuGc-containing gangliosides. Although these two human monoclonal antibodies are not highly restricted in their specificities, they reacted best with the major gangliosides, GM3 and GD3, present in the majority of human melanomas. PMID:2908845

  4. Mass Spectrometry for the Biophysical Characterization of Therapeutic Monoclonal Antibodies

    PubMed Central

    Zhang, Hao; Cui, Weidong; Gross, Michael L.

    2014-01-01

    Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecular drugs (150-600 Da) that have rigid structures, mAbs (~150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes. PMID:24291257

  5. Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies

    SciTech Connect

    Fisher, D.R.; Durham, J.S.; Hui, T.E.; Hill, R.L.

    1990-11-01

    In gauging the efficacy of radiolabeled monoclonal antibodies in cancer treatment, it is important to know the amount of radiation energy absorbed by tumors and normal tissue per unit administered activity. This paper describes methods for estimating absorbed doses to human tumors and normal tissues, including intraperitoneal tissue surfaces, red marrow, and the intestinal tract from incorporated radionuclides. These methods use the Medical Internal Radiation Dose (MIRD) scheme; however, they also incorporate enhancements designed to solve specific dosimetry problems encountered during clinical studies, such as patient-specific organ masses obtained from computerized tomography (CT) volumetrics, estimates of the dose to tumor masses within normal organs, and multicellular dosimetry for studying dose inhomogeneities in solid tumors. Realistic estimates of absorbed dose are provided within the short time requirements of physicians so that decisions can be made with regard to patient treatment and procurement of radiolabeled antibodies. Some areas in which further research could improve dose assessment are also discussed. 16 refs., 3 figs.

  6. Glycoengineered Pichia-based expression of monoclonal antibodies.

    PubMed

    Zha, Dongxing

    2013-01-01

    Currently, mammalian cells are the most commonly used hosts for the production of therapeutic monoclonal antibodies (mAbs). These hosts not only secrete mAbs with properly assembled two heavy and two light chains but also deliver mAbs with a glycosylation profile that is compatible with administration into humans. GlycoFi, a wholly owned subsidiary of Merck & Co., Inc., humanized the Pichia glycosylation pathway which allows it to express glycoproteins with a human-like glycan profile. This offers an alternative mAb production platform similar to mammalian hosts and in some cases it even provides more homogenous product and better efficacy, such as enhanced effector function. This chapter describes a protocol for using glycoengineered Pichia to produce full-length mAbs. It covers a broad spectrum of mAb expression technologies in yeast including expression vector construction, yeast transformation, high-throughput strain selection to fermentation, and antibody purification. PMID:23475712

  7. Characterization of Bispecific T-cell Engager (BiTE®) Antibodies with a High-Capacity T-cell Dependent Cellular Cytotoxicity (TDCC) Assay.

    PubMed

    Nazarian, Aaron A; Archibeque, Ivonne L; Nguyen, Yen H; Wang, Paul; Sinclair, Angus M; Powers, David A

    2014-12-01

    The Bispecific T-cell Engager (BiTE®) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors. PMID:25477202

  8. Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients

    Microsoft Academic Search

    Stephanie Br Andlein; Judith Lorenz; Nele Ruoff; Frank Hensel; Ines Beyer; Justus M Uller; Konrad Neukam; Bertram Illert; Matthias Eck; Hans Konrad M Uller-hermelink; H. Peter Vollmers

    Abstract. Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross- linking) and low-mutated IgM antibodies (less immunogenic),are believed to be the most effective weapons,against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also

  9. Monoclonal antibodies: a target therapy for multiple sclerosis.

    PubMed

    Lorefice, Lorena; Fenu, Giuseppe; Frau, Jessica; Coghe, Giancarlo; Marrosu, Maria Giovanna

    2014-01-01

    Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system. It is characterized by a proinflammatory and neurodegenerative process that results in neuroaxonal damage. Over the last two decades, a wide range of immunomodulatory and immunosuppressive treatments have been used for the management of MS. Several treatments have been developed or are under evaluation for reducing relapses, disease progression and long-term MS-related disability. Recently, a growing interest has emerged for therapeutics with very selective actions, particularly monoclonal antibodies, to target several biological pathways involved in MS. To date, only Natalizumab (Tysabri(®)) has been approved for the treatment of active MS forms. Its therapeutic mechanism is the blockade of the a4-integrin molecule of many leukocytes, which leads to a decrease of immune cells migration, in particular of lymphocytes, across the blood-brain barrier. Furthermore, other promising molecules are under study in clinical trials. In this review, we summarize and discuss the history, pharmacodynamics and safety of monoclonal antibodies that have been approved or are under evaluation for the selective treatment of MS. PMID:24479836

  10. Monoclonal antibodies directed to fucoidan preparations from brown algae.

    PubMed

    Torode, Thomas A; Marcus, Susan E; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S; Hervé, Cécile; Knox, J Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance. PMID:25692870

  11. Bothropic antivenom based on monoclonal antibodies, is it possible?

    PubMed

    Frauches, Thiago S; Petretski, Jorge H; Arnholdt, Andrea C V; Lasunskaia, Elena B; de Carvalho, Eulógio C Q; Kipnis, Thereza L; da Silva, Wilmar D; Kanashiro, Milton M

    2013-09-01

    Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future. PMID:23732123

  12. Monoclonal Antibodies Directed to Fucoidan Preparations from Brown Algae

    PubMed Central

    Torode, Thomas A.; Marcus, Susan E.; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S.; Hervé, Cécile; Knox, J. Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance. PMID:25692870

  13. Intracavitary use of two radiolabeled tumor-associated monoclonal antibodies

    SciTech Connect

    Malamitsi, J.; Skarlos, D.; Fotiou, S.; Papakostas, P.; Aravantinos, G.; Vassilarou, D.; Taylor-Papadimitriou, J.; Koutoulidis, K.; Hooker, G.; Snook, D.

    1988-12-01

    Six patients with metastatic breast cancer and malignant pleural effusions and 13 patients with known or suspected ovarian cancer, underwent immunoscintigraphy after intracavitary (intrapleural or intraperitoneal) administration of iodine-131-(131I) or indium-111-(111In) labeled tumor associated monoclonal antibodies HMFG2 and H17E2. This method proved to be sensitive and specific with a true-positive result in 13 out of 14 patients with tumor and a true-negative result in five out of five patients without tumor. At any one time, 65%-80% of the whole-body radioactivity was closely associated with the cavity into which the radiolabeled antibody was administered while the radioactivity in the blood was always low, (approximately 4 X 10(-3) of administered dose/ml of blood). Concentrations of radiolabeled antibody (per gram of tumor tissue) ranged from 0.02%-0.1% of the injected dose in intracavitary tumors, but only 0.002% in a retroperitoneal metastasis. The specificity of this approach was documented in four control patients with benign ovarian cysts and in two patients who were imaged using both specific and nonspecific radiolabeled antibody. We conclude that the intracavitary administration of 131I- or 111In-labeled HMFG2 and H17E2 is a favorable route of administration and offers significant advantages over previously reported intravenous administration for the localization of breast or ovarian metastases confined to the pleural or peritoneal cavities.

  14. Screening individual hybridomas by microengraving to discover monoclonal antibodies

    PubMed Central

    Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

    2014-01-01

    The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

  15. Strategies in the development of human monoclonal antibodies.

    PubMed

    Jahn, S; Grunow, R; Kiessig, S T; Settmacher, U; Von Baehr, R

    1990-01-01

    A high-efficiency, HAT-sensitive heteromyeloma fusion line CB-F7 has been developed from an 8-Azaguanin-treated Ig-non-secreting human X mouse heterohybridoma. The use of this line allowed us to produce human hybridomas more successfully by fusion of cell material from blood, lymph node or spleen. A polyspecific repertoire of IgM isotype was detected among the hybridomas obtained from the spleen. These IgM antibodies reacted with autoantigens as well as with foreign material. This naturally occurring repertoire may be of interest since it has anti-bacterial activity. The frequency of the occurrence of polyspecific antibody-producing hybridomas was high in the spleen. Apart from the detection of polyspecific IgM antibodies we did not find IgG-secreting hybridomas with anti-bacterial reactivity among thousands of initial lines derived from non-immunized persons. We therefore tried to fuse lymphocytes from donors, who were boosted with Tetanus Toxoid (TTd). A short and limited optimum time period at which the blood should be taken from the donors after boosting, was detected. Seven days after in vivo immunization high yields of IgG-anti-TTd-producing lines (239 out of 731 IgG-producers) were found. The methods of developing more efficient production of human monoclonal antibodies of a pre-defined specificity were discussed. PMID:2401386

  16. Examination of HER3 targeting in cancer using monoclonal antibodies.

    PubMed

    Gaborit, Nadège; Abdul-Hai, Ali; Mancini, Maicol; Lindzen, Moshit; Lavi, Sara; Leitner, Orith; Mounier, Lucile; Chentouf, Myriam; Dunoyer, Sai; Ghosh, Manjusha; Larbouret, Christel; Chardès, Thierry; Bazin, Hervé; Pèlegrin, André; Sela, Michael; Yarden, Yosef

    2015-01-20

    The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients. PMID:25564668

  17. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    DOEpatents

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  18. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

  19. Affinity Precipitation of a Monoclonal Antibody From an Industrial Harvest Feedstock Using an

    E-print Network

    Chen, Wilfred

    Affinity Precipitation of a Monoclonal Antibody From an Industrial Harvest Feedstock Using an ELP Steven M. Cramer1 1 Department of Chemical and Biological Engineering, Center for Biotechnology affinity precipi- tation process is developed for monoclonal antibody (mAb) purification from industrial

  20. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

    1994-08-02

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

  1. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, Larry H. (Livermore, CA); Vanderlaan, Martin (Danville, CA); Watkins, Bruce E. (Livermore, CA)

    1994-01-01

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

  2. Monoclonal antibody against human ovarian tumor-associated antigens

    SciTech Connect

    Poels, L.G.; Peters, D.; van Megen, Y.; Vooijs, G.P.; Verheyen, R.N.; Willemen, A.; van Niekerk, C.C.; Jap, P.H.; Mungyer, G.; Kenemans, P.

    1986-05-01

    Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by /sup 125/I-labeled OV-TL 3 antibodies.

  3. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    SciTech Connect

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F. (Columbia Univ. College of Physicians and Surgeons, New York, NY (USA))

    1990-11-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.

  4. Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia.

    PubMed Central

    Pennington, J E; Small, G J; Lostrom, M E; Pier, G B

    1986-01-01

    A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations. PMID:3093385

  5. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology.

    PubMed

    Berry, Jody D; Gaudet, Ryan G

    2011-09-01

    Antibody preparations have a long history of providing protection from infectious diseases. Although antibodies remain the only natural host-derived defense mechanism capable of completely preventing infection, as products, they compete against inexpensive therapeutics such as antibiotics, small molecule inhibitors and active vaccines. The continued discovery in the monoclonal antibody (mAb) field of leads with broadened cross neutralization of viruses and demonstrable synergy of antibody with antibiotics for bacterial diseases, clearly show that innovation remains. The commercial success of mAbs in chronic disease has not been paralleled in infectious diseases for several reasons. Infectious disease immunotherapeutics are limited in scope as endemic diseases necessitate active vaccine development. Also, the complexity of these small markets draws the interest of niche companies rather than big pharmaceutical corporations. Lastly, the cost of goods for mAb therapeutics is inherently high for infectious agents due to the need for antibody cocktails, which better mimic polyclonal immunoglobulin preparations and prevent antigenic escape. In cases where vaccine or convalescent populations are available, current polyclonal hyperimmune immunoglobulin preparations (pIgG), with modern and highly efficient purification technology and standardized assays for potency, can make economic sense. Recent innovations to broaden the potency of mAb therapies, while reducing cost of production, are discussed herein. On the basis of centuries of effective use of Ab treatments, and with growing immunocompromised populations, the question is not whether antibodies have a bright future for infectious agents, but rather what formats are cost effective and generate safe and efficacious treatments to satisfy regulatory approval. PMID:21473942

  6. Monoclonal antibodies to mineralized matrix molecules of the avian eggshell.

    PubMed

    Dennis, J E; Carrino, D A; Yamashita, K; Caplan, A I

    2000-12-01

    The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly. PMID:11102757

  7. Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila.

    PubMed Central

    Sampson, J S; Plikaytis, B B; Aloisio, C H; Carlone, G M; Pau, C P; Stinson, A R

    1991-01-01

    Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays. Images PMID:1890189

  8. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies.

    PubMed

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-03-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the ?/? TCR. Replacing CH1 with C? and CL with C?, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of C? with a canonical ?-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and C?/C? domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases. PMID:25611120

  9. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    PubMed

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  10. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  11. Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.

    PubMed

    Asano, Ryutaro; Shimomura, Ippei; Konno, Shota; Ito, Akiko; Masakari, Yosuke; Orimo, Ryota; Taki, Shintaro; Arai, Kyoko; Ogata, Hiromi; Okada, Mai; Furumoto, Shozo; Onitsuka, Masayoshi; Omasa, Takeshi; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Umetsu, Mitsuo; Kumagai, Izumi

    2014-01-01

    One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies. PMID:25517309

  12. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-17

    ...Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human Cancers and...239 entitled ``Human Monoclonal Antibodies Specific for CD22'' [HHS Ref. E-080-2008...Human and Improved Murine Monoclonal Antibodies Against CD22'' [HHS Ref....

  13. Monoclonal Antibodies to the S1 Spike and Membrane Proteins of Avian Infectious Bronchitis Coronavirus Strain Massachusetts M41

    Microsoft Academic Search

    A. P. ADRIAN MOCKETT; DAVID CAVANAGH; T. D. K. Brown

    1984-01-01

    SUMMARY We have established four murine hybridoma cell lines which secrete specific antibody to avian infectious bronchitis virus (IBV) strain Massachusetts M41. Two monoclonal antibodies reacted with the spike protein and two reacted with the membrane protein. The specificity of the monoclonal antibodies for the external structural proteins was detected by immunoprecipitations using radiolabelled virus. The reactions of the monoclonal

  14. Induction of albuminuria in mice: Synergistic effect of two monoclonal antibodies directed to different domains of aminopeptidase A

    Microsoft Academic Search

    Stef Mentzel; Jacco P. H. F. Van Son; Henri B. P. M. Dijkman; Jack F. M. Wetzels; Karel J. M. Assmann

    1999-01-01

    Induction of albuminuria in mice: Synergistic effect of two monoclonal antibodies directed to different domains of aminopeptidase A.BackgroundAminopeptidase A is an enzyme that is present on podocytes and is involved in the degradation of angiotensin II. In previous studies in mice, we administered single monoclonal antibodies directed against aminopeptidase A. We observed that only monoclonal antibodies that inhibited aminopeptidase A

  15. Characterization of murine monoclonal antibodies to Escherichia coli J5.

    PubMed Central

    Miner, K M; Manyak, C L; Williams, E; Jackson, J; Jewell, M; Gammon, M T; Ehrenfreund, C; Hayes, E; Callahan, L T; Zweerink, H

    1986-01-01

    Twenty-eight independently derived monoclonal antibodies (MAb) directed against Escherichia coli J5 endotoxin were produced and characterized. Each MAb exhibited a specific titer by both radioimmunoassay and passive hemagglutination assay. Most of the MAb were of the immunoglobulin G isotype; however, several immunoglobulin M antibodies and one immunoglobulin A antibody were produced. When characterized for their capacity to cross-react with purified endotoxin preparations from several gram-negative bacteria, 22 MAb exhibited no cross-reactivity; 6 demonstrated a limited capacity to cross-react with other endotoxin preparations. When characterized for their capacity to react with the intact organism instead of the purified endotoxin the pattern of cross-reactivity was quite different. Most of the MAb were able to react with Salmonella minnesota Re595. Eighteen were able to react with E. coli O111:B4 (the parent strain of E. coli J5), 13 MAb reacted weakly with Pseudomonas aeruginosa, and 3 reacted weakly with Klebsiella pneumonia. The data imply that MAb generated against E. coli J5 endotoxin demonstrate greater cross-reactivity when assayed against the whole bacterium than when assayed against the corresponding purified endotoxin. We were unable to demonstrate that any of the 28 MAb could passively protect mice against lethal endotoxin challenge. PMID:3514463

  16. Anti-idiotype monoclonal antibody carrying the internal image of ganglioside GM3.

    PubMed

    Yamamoto, S; Yamamoto, T; Saxton, R E; Hoon, D S; Irie, R F

    1990-11-21

    Murine anti-idiotype monoclonal antibodies were generated against a human IgM monoclonal antibody (L612) that recognizes ganglioside GM3 on human melanoma. Hybridomas secreting antibodies that bound specifically to L612 were selected by enzyme-linked immunosorbent assay using L612 and three negative control human IgMs, including monoclonal anti-GM2 and anti-GD2 antibodies, as well as purified serum IgM, as antigen sources. GM3-binding inhibition and cell-binding inhibition assays were used to identify seven anti-idiotype monoclonal antibodies that recognized determinants located within the antigen-combining sites of L612. To determine whether these anti-idiotype monoclonal antibodies possessed the internal image of the original antigen, we immunized syngeneic BALB/c mice with one of the anti-idiotype monoclonal antibodies, 4C10, coupled with keyhole limpet hemocyanin. Sera from the immunized mice reacted strongly with an antigen-positive M12 melanoma cell line and with purified GM3. Because L612 detects and kills melanoma tumor cells in vitro and in vivo in the presence of complement without affecting normal tissues, anti-idiotype monoclonal antibodies carrying the internal image of GM3 may be an effective tool for active specific immunotherapy in patients with melanoma. PMID:1700134

  17. Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity.

    PubMed

    Sampei, Zenjiro; Igawa, Tomoyuki; Soeda, Tetsuhiro; Funaki, Miho; Yoshihashi, Kazutaka; Kitazawa, Takehisa; Muto, Atsushi; Kojima, Tetsuo; Nakamura, Satoshi; Hattori, Kunihiro

    2015-01-01

    While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell. PMID:25524207

  18. Pyroglutamate and O-Linked Glycan Determine Functional Production of Anti-IL17A and Anti-IL22 Peptide-Antibody Bispecific Genetic Fusions

    PubMed Central

    Zhong, Xiaotian; Kieras, Elizabeth; Sousa, Eric; D'Antona, Aaron; Baber, J. Christian; He, Tao; Desharnais, Joel; Wood, Lauren; Luxenberg, Deborah; Stahl, Mark; Kriz, Ronald; Lin, Laura; Somers, Will; Fitz, Lori J.; Wright, Jill F.

    2013-01-01

    Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule. PMID:23184956

  19. Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

    NASA Astrophysics Data System (ADS)

    Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

    1987-05-01

    A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

  20. Monoclonal antibody to N protein of porcine epidemic diarrhea virus.

    PubMed

    Pan, Xi; Kong, Ning; Shan, Tongling; Zheng, Hao; Tong, Wu; Yang, Shen; Li, Guoxin; Zhou, Enmin; Tong, Guangzhi

    2015-02-01

    The N gene of porcine epidemic diarrhea virus (PEDV) was amplified by RT-PCR using specific primers, and inserted into the expression vector pCold-I to construct a recombinant plasmid pCold-I-N. The recombinant plasmid was expressed in Escherichia coli BL21 (DE3) under IPTG induction. Then, female BALB/c mice were immunized with the purified recombinant N protein and one strain of hybridoma cells named 2B8 secreting anti-N protein monoclonal antibodies (MAb) was obtained by hybridoma technique. The MAb was specifically reacted with PEDV and identified by Western blot and indirect immunofluorescence assays. This work indicated that the MAb would be a valuable tool as a specific diagnostic reagent for PEDV epidemiological surveys and diagnosis in the future. PMID:25723284

  1. Development of HPLC analysis methods for therapeutic monoclonal antibodies.

    PubMed

    Todoroki, Kenichiro

    2015-01-01

      Therapeutic monoclonal antibody (mAb) preparations are produced from cultured cells; therefore, detailed and multidimensional analyses of their heterogeneities are required. We analyzed five commercially available mAb preparations by high-temperature reversed-phase LC using a wide-pore core-shell column for pluralistic quality assessment. At a highly elevated column temperature, isopropanol with high eluotropic strength coefficients and a wide-pore core-shell type octyl column showed good peak resolution of the investigated mAbs and their related constituents. We used this method to estimate the residual rate of intact mAbs after a heat aggregation treatment and conducted fragmentation analysis by analyzing their pepsin digests. Each peak component was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All results were compared with those of reversed-phase and size exclusion analyses. PMID:25747215

  2. Rapid identification of smooth Brucella species with a monoclonal antibody.

    PubMed Central

    Roop, R M; Preston-Moore, D; Bagchi, T; Schurig, G G

    1987-01-01

    A colony blot enzyme-linked immunosorbent assay was developed for the rapid identification of smooth Brucella species, i.e., Brucella abortus, B. melitensis, and B. suis. Bacterial colonies from plates were blotted onto nitrocellulose disks, lysed by immersion in chloroform, and reacted with BRU 38, a rat monoclonal antibody with specificity for the O side chain of B. abortus. Reaction with anti-rat immunoglobulin G conjugated to horseradish peroxidase and development in 4-chloro-1-naphthol resulted in colonies of naturally occurring smooth Brucella species staining purple. Results could be obtained within 4 h after colonies were visible on plates and individual colonies could be detected. Yersinia enterocolitica serovar O:9 strains were the only other organisms tested which showed cross-reaction by using this procedure. Because of its speed, sensitivity, and specificity, this technique should be very useful for identifying smooth Brucella strains in diagnostic laboratories. Images PMID:3693540

  3. Infectious Complications Associated with Monoclonal Antibodies and Related Small Molecules

    PubMed Central

    Salvana, Edsel Maurice T.; Salata, Robert A.

    2009-01-01

    Summary: Biologics are increasingly becoming part of routine disease management. As more agents are developed, the challenge of keeping track of indications and side effects is growing. While biologics represent a milestone in targeted and specific therapy, they are not without drawbacks, and the judicious use of these “magic bullets” is essential if their full potential is to be realized. Infectious complications in particular are not an uncommon side effect of therapy, whether as a direct consequence of the agent or because of the underlying disease process. With this in mind, we have reviewed and summarized the risks of infection and the infectious disease-related complications for all FDA-approved monoclonal antibodies and some related small molecules, and we discuss the probable mechanisms involved in immunosuppression as well as recommendations for prophylaxis and treatment of specific disease entities. PMID:19366915

  4. [Monoclonal antibodies for the treatment of multiple sclerosis].

    PubMed

    Sánchez-Seco, Victoria Galán; Casanova Peño, Ignacio; Arroyo González, Rafael

    2014-12-01

    Until the mid 1990s, with the appearance of interferon beta and glatiramer acetate, there was no treatment for multiple sclerosis (MS). However, due to their moderate therapeutic potential in some patients, a broad search was continued to find new and more effective treatment strategies, largely concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for the treatment of MS, was approved at the end of 2004, representing a major advance in the field of neuroimmunology. Today, there is broad experience with natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab, ofatumumab and anti-lingo-1) that are pending commercialization or are under phase II or III of development with promising results. The present review analyzes the efficacy and safety results of all these drugs. PMID:25732947

  5. Neutralization of Yellow Fever Virus Studied Using Monoclonal and Polyclonal Antibodies

    Microsoft Academic Search

    A. Buckley; E. A. Gould

    1985-01-01

    SUMMARY Monoclonal and polyclonal antibodies with known specificity for either the 54K envelope glycoprotein or the 48K non-structural glycoprotein of yellow fever (YF) virus-infected cells were studied in plaque reduction neutralization tests. Viruses employed in the tests comprised wild-type and vaccine strains of YF and a selection of other flaviviruses. Of 17 monoclonal antibodies examined, six of the 54K-specific antibodies

  6. Vasculogenesis in the early quail blastodisc as studied with monoclonal-antibody recognizing endothelial-cells

    E-print Network

    Pardanaud, L.; Altmann, C.; Kitos, Paul A.; Dieterlenlievre, F.; Buck, C. A.

    1987-06-01

    Development 100, 339-349 (1987) Printed in Great Britain ? The Company of Biologists Limited 1987 339 Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells LUC PARDANAUD 1 , CURTIS ALTMANN 2... embryo treated with the monoclonal antibody QH1 and subsequently with fluorescein-labelled goat anti- mouse IgG. The antibody can be seen to react essentially with two cell types, the endothelial cells lining the dorsal aorta, common cardinal vein...

  7. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A

    PubMed Central

    Yoshihashi, Kazutaka; Takeda, Minako; Kitazawa, Takehisa; Soeda, Tetsuhiro; Igawa, Tomoyuki; Sampei, Zenjiro; Kuramochi, Taichi; Sakamoto, Akihisa; Haraya, Kenta; Adachi, Kenji; Kawabe, Yoshiki; Nogami, Keiji; Shima, Midori; Hattori, Kunihiro

    2014-01-01

    ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required. PMID:25274508

  8. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A.

    PubMed

    Muto, Atsushi; Yoshihashi, Kazutaka; Takeda, Minako; Kitazawa, Takehisa; Soeda, Tetsuhiro; Igawa, Tomoyuki; Sampei, Zenjiro; Kuramochi, Taichi; Sakamoto, Akihisa; Haraya, Kenta; Adachi, Kenji; Kawabe, Yoshiki; Nogami, Keiji; Shima, Midori; Hattori, Kunihiro

    2014-11-13

    ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required. PMID:25274508

  9. Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process.

    PubMed

    Hall, Troii; Wilson, Joseph J; Brownlee, Tammy J; Swartling, James R; Langan, Sarah E; Lambooy, Peter K

    2015-01-15

    During the purification development of a bispecific antibody, cation-exchange chromatography was screened for its ability to separate a prominently expressed (>12%) mis-formed disulfide bond variant, termed MAb-diabody, and aggregate from the product of interest. The influence of pH, product load (g of product per liter of resin) and linear velocity on the separations were evaluated for the strong cation-exchange resins SP Sepharose HP and POROS(®) HS50. Cation-exchange chromatography is commonly operated distant to the isoelectric point of a molecule, generally leading to acidic conditions for antibody purification. However, the results herein demonstrated improved removal of MAb-diabody with increasing pH, resulting in reduction of MAb-diabody content greater than 12-fold when operating near the alkaline pI of the product. This approach was successful over a range of linear velocities and g/L of resin loading. Aggregate removal was less affected by pH and was effectively reduced from 10.9% to less than 3% for each condition. Furthermore, this method was successfully scaled to a 60 cm diameter column using SP Sepharose HP resin. PMID:25462105

  10. Exceptionally Potent and Broadly Cross-Reactive, Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains

    PubMed Central

    Feng, Yang; Prabakaran, Ponraj; Ying, Tianlei; Wang, Yanping; Sun, Jianping; Macedo, Camila D. S.; Zhu, Zhongyu; He, Yuxian; Polonis, Victoria R.

    2014-01-01

    Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus. PMID:24198429

  11. Characterization of new monoclonal antibodies identifying avian T lymphocyte antigens.

    PubMed

    Knabel, M; Cihak, J; Lösch, U

    1993-08-01

    Six monoclonal antibodies (mAb) were produced to identify and characterize surface antigens of chicken T cells. Determination of their reactivity with different lymphatic cells using immunofluorescence analysis demonstrates that mAb KH8, NA6, PD4 and TH8 stained 32-43% blood lymphocytes, 72-77% thymocytes and 19-27% spleen cells, mAb OC5 approximately 99% thymocytes and 55% blood and spleen lymphocytes each, and mAb OC2 36% blood lymphocytes, 79% thymocytes and 62% spleen cells. The KH8, NA6, PD4 and TH8 antibodies immunoprecipitated from lysates of surface-labeled chicken thymocytes a polypeptide of M(r) 60,000 under non-reducing conditions and the OC5 antibody a glycoprotein of M(r) 68,000 under reducing conditions. MAb OC2 precipitated a single polypeptide of M(r) 40,000 under both conditions. The mAb KH8, NA6, PD4, TH8 and OC2 inhibited ConA-induced proliferative responses of blood T cells in vitro. However, sepharose-bound or soluble OC5 antibody was able to increase DNA synthesis significantly. These results indicate that (a) the mAb KH8, NA6, PD4 and TH8 identify the avian homologue of the mammalian CD4 molecule, (b) the mAb OC2 detects the avian CD2 antigen, and (c) the mAb OC5 recognizes the putative avian CD5 homologue. PMID:8244446

  12. Monoclonal antibodies specific for human monocytes, granulocytes and endothelium.

    PubMed Central

    Hogg, N; MacDonald, S; Slusarenko, M; Beverley, P C

    1984-01-01

    Four monoclonal antibodies against antigens of human myeloid cells have been produced and thoroughly characterized in terms of their reactions with peripheral blood cells, cell lines, nine lymphoid and non-lymphoid tissues and the polypeptides with which they react. UCHM1 and SmO identify antigens present on the majority of blood monocytes and a variable, but lower, proportion of tissue macrophages. From their morphology and location in tissues, these cells appear to be recirculating monocytes. SMO antigen is also present on platelets. In addition, both antibodies stained endothelial cells, SMO in all tissues examined and UCHM1 variably. Biochemical investigation indicated that the UCHM1 antigen is a protein of 52,000 MW while the SMO antigen could not be indentified. The antibodies TG1 and 28 identify antigens mainly present on granulocytes. While mAb 28 reacted with neutrophils, TG1 also stained eosinophils and stained strongly a proportion of monocytes. TG1 also reacted variably with some non-haemopoietic cell lines. Both antibodies reacted predominantly with granulocytes in tissue sections. MAb TG1 precipitated a single polypeptide of 156,000 MW from monocytes and granulocytes, while mAb 28 precipitated non-convalently associated polypeptides of 83,000 and 155,000 MW from granulocytes but only a single molecule from monocytes, corresponding to the lower MW chain of 83,000. The epitope with which mAb 28 reacts appears not to be exposed on the surface of intact monocytes. This suggests that a similar or identical 83,000 MW molecule is made by both neutrophils and monocytes, but that its expression differs according to cell type. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6389324

  13. Serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the microimmunofluorescence test.

    PubMed Central

    Andersen, A A

    1991-01-01

    A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chlamydia psittaci was prepared. The panel included one monoclonal antibody to each of the 10 serovars. Monoclonal antibodies were selected for their specificity in the indirect microimmunofluorescence test. Each of the monoclonal antibodies had a titer of 1:1,280 or higher to the homologous strain, with only two showing any cross-reactivity at a dilution of 1:10. Chlamydial antigen derived from organisms growing in tissue culture of one well of a 96-well multiwell dish was usually sufficient for the serotyping of an isolate. Infected yolk sac preparations were also suitable for serotyping. The panel of monoclonal antibodies was used to serotype 55 mammalian and avian strains. All except five of the strains were successfully serotyped; these five strains are presumed to represent at least two additional serovars. The use of a panel of monoclonal antibodies in the indirect microimmunofluorescence test provides a rapid and reliable method for serotyping new isolates. Monoclonal antibodies to new serovars can easily be added to the panel. PMID:1890172

  14. The history of monoclonal antibody development - Progress, remaining challenges and future innovations.

    PubMed

    Liu, Justin K H

    2014-12-01

    As medicine progresses into a new era of personalised therapy, the use of monoclonal antibodies to treat a wide range of diseases lies at the heart of this new forefront. Since the licencing of the first monoclonal antibody for clinical use 30 years ago, the monoclonal antibody industry has expanded exponentially and is now valued at billions of dollars. With major advances in genetic sequencing and biomedical research, much research into monoclonal antibodies now focuses on identifying new targets for development and maximising their efficacy for use in clinical practice. However, a balance has to be struck with regards to reducing numbers of side-effects and overall economic cost, which arguably somewhat blighted their early clinical and commercial successes. Nowadays, there are approximately 30 monoclonal antibodies that have been approved for use in clinical practice with many more currently being tested in clinical trials. Some of the current major limitations include: the use of inefficient models for generation, a lack of efficacy and issues of cost-effectiveness. Some of the current research focuses on ways to improve the efficacy of existing monoclonal antibodies through optimising their effects and the addition of beneficial modifications. This review will focus on the history of monoclonal antibody development - how it has increasingly moved away from using laborious animal models to a more effective phage display system, some of the major drawbacks from a clinical and economical point of view and future innovations that are currently being researched to maximise their effectiveness for future clinical use. PMID:25568796

  15. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    SciTech Connect

    Srivastava, S.C.; Buraggi, G.L.

    1986-01-01

    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

  16. Monoclonal antibody-based therapies for microbial diseases

    PubMed Central

    Saylor, Carolyn; Dadachova, Ekaterina; Casadevall, Arturo

    2009-01-01

    The monoclonal antibody (mAb) revolution that currently provides many new options for the treatment of neoplastic and inflammatory diseases has largely bypassed the field of infectious diseases. Only one mAb is licensed for use against an infectious disease, although there are many in various stages of development. This situation is peculiar given that serum therapy was one of the first effective treatments for microbial diseases and that specific antibodies have numerous antimicrobial properties. The underdevelopment and underutilization of mAb therapies for microbial diseases has various complex explanations that include the current availability of antimicrobial drugs, small markets, high costs and microbial antigenic variation. However, there are signs that the climate for mAb therapeutics in infectious diseases is changing given increasing antibiotic drug resistance, the emergence of new pathogenic microbes for which no therapy is available, and development of mAb cocktail formulations. Currently, the major hurdle for the widespread introduction of mAb therapies for microbial diseases is economic, given the high costs of immunoglobulin preparations and relatively small markets. Despite these obstacles there are numerous opportunities for mAb development against microbial diseases and the development of radioimmunotherapy provides new options for enhancing the magic bullet. Hence, there is cautious optimism that the years ahead will see more mAbs in clinical use against microbial diseases. PMID:20006139

  17. Anti-bacterial monoclonal antibodies: back to the future?

    PubMed

    Oleksiewicz, Martin B; Nagy, Gábor; Nagy, Eszter

    2012-10-15

    Today's medicine has to deal with the emergence of multi-drug resistant bacteria, and is beginning to be confronted with pan-resistant microbes. This worsening inadequacy of the antibiotics concept, which has ruled infectious medicine in the last six decades creates an increasing unmet medical need that can be addressed by passive immunization. While past experience from the pre-antibiotic era with serum therapy was in many cases encouraging, antibacterial monoclonal antibodies have so far suffered high attrition rates in the clinic, generally from lack of efficacy. Yet, we believe that recent developments in a number of areas such as infectious disease pathogenesis research, translational medicine, mAb engineering, mAb manufacturing and rapid bedside diagnostics are converging to make the medium-term future permissive for antibacterial mAb development. Here, we review antibacterial mAb-based approaches that are or were in clinical development, and may potentially act as paradigms with regards to molecular targets, antibody formats and mode-of-action, pre-clinical validation and selection of most relevant patient populations, in order to increase the likelihood of successful product development in this field. PMID:22705202

  18. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    PubMed

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D

    2013-10-15

    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics. PMID:24006877

  19. Macrophages eliminate circulating tumor cells after monoclonal antibody therapy.

    PubMed

    Gül, Nuray; Babes, Liane; Siegmund, Kerstin; Korthouwer, Rianne; Bögels, Marijn; Braster, Rens; Vidarsson, Gestur; ten Hagen, Timo L M; Kubes, Paul; van Egmond, Marjolein

    2014-02-01

    The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (Fc?RI) and the low-affinity IgG-binding Fc receptor (Fc?RIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity. PMID:24430180

  20. Development of Monoclonal Antibodies Specific for Glycated Prion Protein

    PubMed Central

    Dvorakova, Eva; Prouza, Marek; Janouskova, Olga; Panigaj, Martin; Holada, Karel

    2011-01-01

    Transmissive spongiform encephalopathies (TSE) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSE. As deposits of PrPTSE remain in the body for long periods, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have potential to serve as a diagnostic marker. Monoclonal antibodies specific for carboxymethyl lysine/arginine-modified prion protein were prepared. Recombinant human prion protein (rhPrP) was bacterially expressed and purified by affinity chromatography. rhPrP was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 6 mice, and 960 hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of four promising clones. One of them (EM-31) strongly reacts with human and mouse recombinant PrP-CML, and three other clones react also with CML in vitro modified human and mouse brain PrP. Besides possible implication in TSE diagnostics, the antibodies may serve as tolls to advance our knowledge regarding the role of glycation in the prion pathophysiology. PMID:22043908

  1. Monoclonal antibody against Mycoplasma fermentans-specific aminoglycoglycerolipid.

    PubMed

    Matsuda, K; Li, J L; Ichinose, S; Harasawa, R; Saito, M; Yamamoto, N

    2000-01-01

    Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholine-containing glycoglycerolipids: GGPL-I and GGPL-III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF-III-1) specific to GGPL-III (phosphocholine-containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin-layer chromatography immunostaining. The MF-III-1 antibody specifically recognized M. fermentans attached to the surface of HTLV-I-infected human helper T-cells, and it did not cross-react with other lipids nor with human T-cell antigens. Since MF-III-1 distinguishes GGPL-III from GGPL-I, the binding site may include a serinol (2-amino-1,3-propanediol) residue of GGPL-III. MF-III-1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases. PMID:11021400

  2. Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody

    SciTech Connect

    Oster, Z.H.; Srivastava, S.C.; Som, P.; Meinken, G.E.; Scudder, L.E.; Yamamoto, K.; Atkins, H.L.; Brill, A.B.; Coller, B.S.

    1985-05-01

    An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with In or STI is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein IIb/IIIa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with STI. With el camera, visualization of venous and arterial thrombi as well as sites of initial injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted.

  3. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

    PubMed

    Collins, S P; Comis, A; Marshall, M; Hartwick, R F; Howden, M E

    1993-09-01

    1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions. PMID:8104761

  4. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST THE AH RECEPTOR

    EPA Science Inventory

    Six hybridomas secreting monoclonal antibodies that are specific for the N-terminal peptide sequence of the murine Ah receptor were isolated. hese antibodies bind with high specificity to the Al receptor on protein blots of Hepa 1c1c7 cytosol. hree IgG1 antibodies (Rpt1, 2, and 3...

  5. Development of Oligodendrocytes and Schwann Cells Studied with a Monoclonal Antibody against Galactocerebroside

    Microsoft Academic Search

    B. Ranscht; P. A. Clapshaw; J. Price; M. Noble; W. Seifert

    1982-01-01

    We have generated a hybridoma cell line secreting a monoclonal antibody that specifically binds to the surfaces of oligodendrocytes and Schwann cells, the cells involved in myelin formation in the central and peripheral nervous systems, respectively. Binding studies using purified sphingolipids showed that this antibody reacts strongly with galactocerebroside (GalC), the major galactosphingolipid of myelin. The antibody was used in

  6. Detection of Macrophage Migration Inhibitory Factor by Monoclonal Antibody in Sézary Syndrome

    Microsoft Academic Search

    Christine Neumann; Renate Schlegel; Friedhelm Steckel; Clemens Sorg

    1987-01-01

    We have reported previously on the generation of a monoclonal antibody against human macrophage migration inhibitory factor (MIF), which is a mediator of cellular immunity. Macrophage migration inhibitory factor activity in the migration assay was closely correlated with antibody reactivity. Using this antibody called 1C5\\/B, we are now able to study the expression of MIF in situ. Here, we report

  7. Tumor-associated hyaluronan limits efficacy of monoclonal antibody therapy.

    PubMed

    Singha, Netai C; Nekoroski, Tara; Zhao, Chunmei; Symons, Rebecca; Jiang, Ping; Frost, Gregory I; Huang, Zhongdong; Shepard, H Michael

    2015-02-01

    Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HA(high)) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER2(3+) primary breast tumors and approximately 40% of EGFR(+) head and neck squamous cell carcinomas are HA(high), and hypothesized that HA(high) tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HA(high) tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HA(high) tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HA(high) tumors, resulting in enhanced trastuzumab- and NK cell-mediated tumor growth inhibition in vivo. These results suggest that HA(high) matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HA(high) tumors. PMID:25512619

  8. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces. PMID:25119992

  9. 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

  10. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.

    PubMed

    Woods, A; Sherwin, T; Sasse, R; MacRae, T H; Baines, A J; Gull, K

    1989-07-01

    The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting. PMID:2606940

  11. One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles.

    PubMed

    Kao, Chien-Han; Wang, Jaw-Yuan; Chuang, Kuo-Hsiang; Chuang, Chih-Hung; Cheng, Ta-Chun; Hsieh, Yuan-Chin; Tseng, Yun-Long; Chen, Bing-Mae; Roffler, Steve R; Cheng, Tian-Lu

    2014-12-01

    Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR(+) and HER2(+) cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy. PMID:25212525

  12. Fully human HER2/cluster of differentiation 3 bispecific antibody triggers potent and specific cytotoxicity of T lymphocytes against breast cancer.

    PubMed

    Zhou, Yan; Gou, Lan-Tu; Guo, Zhi-Hui; Liu, Hai-Rong; Wang, Jiang-Man; Zhou, Shu-Xian; Yang, Jin-Liang; Li, Xiao-An

    2015-07-01

    The use of a bispecific antibody (BsAb) is a promising and highly specific approach to cancer therapy. In the present study, a fully human recombinant single chain variable fragment BsAb against human epidermal growth factor receptor (HER)2 and cluster of differentiation (CD)3 was constructed with the aim of developing an effective treatment for breast cancer. HER2/CD3 BsAb was expressed in Chinese hamster ovary cells and purified via nickel column chromatography. Flow cytometry revealed that the HER2/CD3 BsAb was able to specifically bind to HER2 and CD3?positive cells. HER2/CD3 BsAb was able to stimulate T-cell activation and induce the lysis of cultured SKBR?3 and BT474 cells in the presence of unstimulated T lymphocytes. HER2/CD3 BsAb efficiently inhibited the growth of breast cancer tissue by activating and inducing the proliferation of tumor tissue infiltrating lymphocytes. Therefore, HER2/CD3 BsAb is a potent tool which may be a suitable candidate for the treatment of breast cancer. PMID:25760691

  13. Phase I Study of Anti-CD3 x Anti-Her2 Bispecific Antibody in Metastatic Castrate Resistant Prostate Cancer Patients

    PubMed Central

    Thakur, Archana; Rathore, Ritesh; Kouttab, Nicola; Lum, Lawrence G.

    2015-01-01

    Background. New nontoxic targeted approaches are needed for patients with castrate resistant prostate cancer (CRPC). Our preclinical studies show that activated T cells (ATC) armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi) kill prostate cancer cells lines, induce a Th1 cytokine pattern upon engagement of tumor cells, prevent the development of prostate tumors, and retard tumor growth in immunodeficient mice. These studies provided strong rationale for our phase I dose-escalation pilot study to test ATC armed with Her2Bi (aATC) for safety in men with CRPC. Methods. Seven of 8 men with CRPC were evaluable after receiving two infusions per week for 4 weeks. The men received 2.5, 5 or 10 × 109 aATC per infusion with low dose interleukin-2 and granulocyte-macrophage colony stimulating factor. Results. There were no dose limiting toxicities, and there was 1 partial responder and 3 of 7 patients had significant decreases in their PSA levels and pain scores. Immune evaluations of peripheral blood mononuclear cells in 2 patients before and after immunotherapy showed increases in IFN-? EliSpot responses and Th1 serum cytokines. Conclusions. These results provide a strong rationale for developing phase II trials to determine whether aATC are effective for treating CRPC. PMID:25802762

  14. Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody*

    E-print Network

    Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody allergen Bla g 2 and the Fab fragment of a monoclonal anti- body 7C11 was solved at 2.8-A° resolution. Bla of an -helix and a helical turn from each allergen monomer, exhibiting a novel dimerization

  15. Monoclonal Antibody 14F7, Which Recognizes a Stage-Specific Immature Oligodendrocyte

    E-print Network

    Kawato, Suguru

    Monoclonal Antibody 14F7, Which Recognizes a Stage-Specific Immature Oligodendrocyte Surface Molecule, Inhibits Oligodendrocyte Differentiation Mediated in Co-Culture With Astrocytes Kazunori Institute of Medical Science, Tokyo, Japan Cells at an intermediate stage of oligodendrocyte lineage

  16. Preparation of species-specific murine monoclonal antibodies against the yeast phase of Paracoccidioides brasiliensis.

    PubMed Central

    Figueroa, J I; Hamilton, A J; Bartholomew, M A; Harada, T; Fenelon, L; Hay, R J

    1990-01-01

    A panel of four murine monoclonal antibodies showing species specificity for the yeast phase of the pathogenic dimorphic fungus Paracoccidioides brasiliensis was produced by using a modification of the standard monoclonal antibody technology. This involved the use of the immunosuppressive drug cyclophosphamide to suppress the immune response of test animals to fungi showing cross-reactivity, i.e., to Histoplasma capsulatum. One monoclonal antibody, P4, which had a high titer by enzyme-linked immunosorbent assay, was shown to recognize a linear antigenic epitope of P. brasiliensis at a molecular size of 70,000 to 75,000 daltons by Western blot (immunoblot) analysis. The potential use of these monoclonal antibodies, which are the first species-specific probes to P. brasiliensis that have been produced, in the field of serodiagnosis is discussed. Images PMID:2394802

  17. Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels production

    E-print Network

    California at Riverside, University of

    Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels to the cellulose a Chemical and Environmental Engineering Department, University of California, Riverside wall structure and thereby allow hydrolyzing enzymes better access to the cellulose core. However, few

  18. DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES

    EPA Science Inventory

    Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

  19. Comparison of the growth and monoclonal antibody production of suspended mammalian cells in three perfusion systems

    E-print Network

    Hufford, Kathy (Kathy E.)

    2007-01-01

    The purpose of this thesis was to provide a broad survey of bioprocess options for typical drug production vehicles in the biotechnology industry. This goal was accomplished by comparing the growth and monoclonal antibody ...

  20. Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

    E-print Network

    Wang, Ying

    We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant ...

  1. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

  2. Potent effects of the monoclonal antibody-mitomycin C conjugate on human colon cancers

    Microsoft Academic Search

    Hitoshi Kotanagi; Ken Fukuda; Naoto Ogata; Masahiro Takahashi; Tetsuzo Masuda; Kenji Koyama; Toshio Takahashi

    1987-01-01

    The effects of the monoclonal antibody-mitomycin C conjugate against human colon cancers were studied,in vitro andin vivo. Mitomycin C (MMC) was conjugated with the human colon cancer-specific monoclonal antibody, using a cyanogen bromide method.\\u000a The effect of the conjugate and free MMC,in vitro, was measured by incubation with human colon cancer SW1116 cells, The MMC concentration of the conjugate and

  3. Radiolabeled Monoclonal Antibody Indium 111-Labeled CYT356 Localizes Extraprostatic Recurrent Carcinoma After Prostatectomy

    Microsoft Academic Search

    Peter E Levesque; Peter T Nieh; Leonard N Zinman; David W Seldin; John A Libertino

    1998-01-01

    Objectives. The sites of recurrent carcinoma of the prostate were localized with radiolabeled monoclonal antibody, and these sites were correlated with the response of patients treated with pelvic radiation after prostatectomy.Methods. Radionuclide scans were performed with indium 111-labeled CYT-356, a monoclonal antibody that binds to prostate epithelial cells, in 48 men diagnosed with recurrent carcinoma detected by prostate-specific antigen (PSA)

  4. Characterization of antigenic structures on arabis mosaic virus with monoclonal antibodies

    Microsoft Academic Search

    Ralf G. Dietzgen

    1986-01-01

    Summary Seven different epitopes on arabis mosaic virus (ArMV) were discerned. Neo-, crypto-, and epitopes exposed on the virion and isolated coat protein were differentiated by their reactivity with monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. The monoclonal antibodies, producedin vitro andin vivo, were of IgGl, IgM and IgA isotype. No epitope exclusively specific for one isolate was found.

  5. Radioimmunolocalization of human pancreatic carcinoma xenograft by 99m Tc-labeled YPC3 monoclonal antibody

    Microsoft Academic Search

    Qikui Chen; Shizhen Yuan

    1994-01-01

    Monoclonal antibodies specific to tumor cells may be a useful tool for the diagnosis and treatment of carcinomas. In this study, YPC3 mAb, a specific monoclonal antibody against the human pancreatic carcinoma cell line Capan-2, was labeled directly with99mTc after being pretreated with 2-mercaptoethanol. The radiolabeling efficiency was found to be high (88%–95%) and the immunoreactivity retained, as assessed by

  6. Assignment of a fibronectin gene to human chromosome 2 using monoclonal antibodies

    Microsoft Academic Search

    G. A. Koch; R. C. Schoen; R. J. Klebe; T. B. Shows

    1982-01-01

    The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin. Both monoclonal antibodies demonstrate a highly concordant association

  7. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    NASA Astrophysics Data System (ADS)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  8. Investigation of the functional characteristics of antibodies to therapeutic anti-human tumor necrosis factor alpha monoclonal antibody

    Microsoft Academic Search

    Aysegul Yucel; Arzu L. Aral; Bilkay Basturk; Resul Karakus; Canan Aybay; Cemalettin Aybay

    2007-01-01

    Humanized antibody-based treatment modalities represent an active area of investigation. Included in these strategies are passive administrations of monoclonal antibodies, which recognize tumor necrosis factor alpha (TNF-?). However, several problems associated with these types of treatment strategies have been reported in the literature. We attempted to address the issue related to unresponsiveness to infliximab that might be induced by anti-idiotype

  9. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  10. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

    PubMed Central

    Joly, J R; McKinney, R M; Tobin, J O; Bibb, W F; Watkins, I D; Ramsay, D

    1986-01-01

    A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed. PMID:3517064

  11. The Preparation And Application Of Porphyrin-Monoclonal Antibodies For Cancer Therapy

    NASA Astrophysics Data System (ADS)

    Steele, Kevin J.; Liu, Daniel; Davis, Noelle; Deal, Heather; Levy, Julia G.

    1989-06-01

    Monoclonal antibodies with reactivity to T suppressor cells and T suppressor factors, conjugated to hematoporphyrin, when injected intravenously into mice bearing either the P815 mastocytoma or L1210 thymoma were able to reject a significant number of palpable tumors, in comparison to appropriate controls. This effect is attributed to immune modulation by selective elimination of T suppressor cells. Investigation of novel methodology for reliable linkage of photosensitizers to antibodies indicate that derivatives of polyvinyl alcohol provide suitable carriers for photosensitizers. Such carriers can relatively easily be conjugated to monoclonal antibodies without jeopardizing the biological activity of the antibody or the photosensitizer, or the solubility of the complex.

  12. Identification of a microsporidian polar tube protein reactive monoclonal antibody.

    PubMed

    Keohane, E M; Takvorian, P M; Cali, A; Tanowitz, H B; Wittner, M; Weiss, L M

    1996-01-01

    The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analysis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents. PMID:8563706

  13. Human monoclonal antibodies broadly neutralizing against influenza B virus.

    PubMed

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-02-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  14. Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin

    SciTech Connect

    Stanker, L.H.; Branscomb, E.; Vanderlaan, M.; Jensen, R.H.

    1986-06-01

    Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated ..cap alpha.. and ..beta.. globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at ..beta..5, threonine at ..beta..13, glutamine at ..beta..125, and leucine at ..cap alpha..68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the ..beta.. globin gene, whereas the amino acid required for Rh-2 binding could only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

  15. Analysis of C5a receptor by monoclonal antibody.

    PubMed

    Watanabe, H; Kuraya, M; Kasukawa, R; Yanagisawa, H; Yanagisawa, M; Fujita, T

    1995-09-11

    We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the human C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk- cells transfected with cDNA of human C5aR (Ltk-/C5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the IgM kappa subclass, detected a 43 kDa band on cell lysates of Ltk-/C5aR by immunoblotting analysis. Flow cytometry revealed that 4C8 specifically bound to Ltk-/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracellular Ca2+ increase in PMN stimulated by C5a and 125I-C5a binding to C5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibited the binding of C5a to C5aR on PMN and Ltk-/C5aR. Therefore, it seems likely that this mAb does not recognize the C5aR active site but sterically inhibits the binding of C5a to its receptor. Using this mAb, we detected a 50 kDa band of C5aR on cell lysates of PMN, monocytes and platelets by immunoblotting. C5aR was expressed on PMN and monocytes as determined by flow cytometry, whereas it was not demonstrated on the surface of platelets. Based on these results, this mAb should be useful for analysis of C5aR expression in various immunological conditions and inflammatory diseases. PMID:7665898

  16. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  17. Human monoclonal antibody with dual GM2/GD2 specificity derived from an immunized melanoma patient.

    PubMed

    Yamaguchi, H; Furukawa, K; Fortunato, S R; Livingston, P O; Lloyd, K O; Oettgen, H F; Old, L J

    1990-05-01

    GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207. PMID:2159145

  18. Production of a Chaetomium globosum Enolase Monoclonal Antibody

    PubMed Central

    Nayak, Ajay P.; Lemons, Angela R.; Rittenour, William R.; Hettick, Justin M.; Beezhold, Donald H.

    2014-01-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  19. Epitope of 24G7 anti-bilirubin monoclonal antibody.

    PubMed

    Yamaguchi, T; Shioji, I; Sugimoto, A; Komoda, Y; Nakajima, H

    1996-02-01

    We examined an antigenic epitope recognized by an anti-bilirubin monoclonal antibody designated 24G7 (Shimizu, S., Izumi, Y., Yamazaki, M., Shimizu, K., Yamaguchi, T., and Nakajima, H. (1988) Biochim. Biophys. Acta 967, 255-260). The reactivity of bilirubin-IX alpha, its analogues (III alpha, XIII alpha, and mesobilirubin-IX alpha) and related azo compounds, with 24G7 was compared by means of an enzyme-linked immunosorbent assay (ELISA). The order of reactivity was as follows: (a) aniline azopigment > ethyl anthranilate azopigment > sulfanilic acid azopigment, (b) bilirubin-XIII alpha > bilirubin-IX alpha > bilirubin-III alpha, (c) bilirubin-IX alpha = mesobilirubin-IX alpha. These findings indicated that the epitope is present in (a) the dipyrrolic moiety of the endovinyl (correspond to N-21 and N-22 of bilirubin), or exovinyl types (N-23 and N-24); (b) the dipyrrolic moiety of the endovinyl type, which contains (c) a methyl group (C-2) but not a vinyl group (C-3) in the dipyrrolic moiety of endovinyl type. Therefore, we concluded that the epitope was the region containing the oxo group at C-1, the methyl group at C-2, C-(4,5,6,9), and the N-21 and -22 of bilirubin-IX alpha. PMID:8605219

  20. Antigenic heterogeneity in Mycoplasma iowae demonstrated with monoclonal antibodies.

    PubMed

    Panangala, V S; Gresham, M M; Morsy, M A

    1992-01-01

    Western blots of proteins of 14 Mycoplasma iowae strains and isolates resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with three monoclonal antibodies (MAbs), MI6, MI7, and MI8. MAb MI6 reacted with one or more antigens with apparent molecular weights of 60,000, 70,000, and 94,000. In three strains (N-PHN-D13, R-D2497, and K 1805), antigens located on a single peptide band were recognized, while in others additional epitopes at different molecular-weight positions were revealed. A similar pattern was observed with MAb MI7, although it reacted with fewer antigens than did MAb MI6 and failed to recognize antigens in strains N-PHN-D13 and R-D2497. MAb MI8 reacted with an antigen at an apparent molecular-weight position of 28,000 in four of the 14 strains and isolates. The diverse reaction patterns observed with the MAbs in the 14 M. iowae strains and isolates confirms the occurrence of antigenic variation within this species. Antigenic variation in M. iowae may be pivotal in determining host-parasite interactions, pathogenesis, and the outcome of disease. PMID:1373600

  1. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

  2. Production of a Chaetomium globosum enolase monoclonal antibody.

    PubMed

    Green, Brett J; Nayak, Ajay P; Lemons, Angela R; Rittenour, William R; Hettick, Justin M; Beezhold, Donald H

    2014-12-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  3. Monoclonal antibodies for the identification of herpesvirus simiae (B virus).

    PubMed

    Cropper, L M; Lees, D N; Patt, R; Sharp, I R; Brown, D

    1992-01-01

    To differentiate between B virus and HSV isolates from monkeys and man monoclonal antibodies (mabs) were produced to herpesvirus simiae (B virus) and herpes simplex type 1 and 2 (HSV-1 and HSV-2). Mabs were tested by indirect immunofluorescence (IFAT) for reactivity against herpesviruses from Asiatic monkeys (B virus), African monkeys (SA 8 virus), and man (HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus). Mabs could be divided into groups A-E displaying specific reactivity for B virus (A); reactivity with both B virus and SA 8 but not HSV (B); reactivity with B virus, SA 8 virus and HSV strains (C); specific reactivity with HSV-1 (D); and specific reactivity with HSV-2 (E). Two of the B virus specific mabs were able to differentiate between cynomolgus and rhesus strains of B virus. None of the mabs reacted with human varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. A panel of mabs for the unequivocal identification of B virus isolates from monkey or man is proposed. PMID:1314049

  4. Characterization of a new monoclonal antibody against mercury (II)

    SciTech Connect

    Marx, A.; Hock, B. [Technische Univ. Muenchen, Freising (Germany). Dept. of Botany

    1998-07-01

    Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunized with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 {micro}g/L HgCl{sub 2} (= 2.1 {micro}g/L Hg{sup 2+}) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).

  5. Characterization of binding epitopes of CA125 monoclonal antibodies.

    PubMed

    Marcos-Silva, Lara; Narimatsu, Yoshiki; Halim, Adnan; Campos, Diana; Yang, Zhang; Tarp, Mads A; Pereira, Pedro J B; Mandel, Ulla; Bennett, Eric P; Vakhrushev, Sergey Y; Levery, Steven B; David, Leonor; Clausen, Henrik

    2014-07-01

    The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies. PMID:24850311

  6. Production of monoclonal antibody to herbicide fenoxaprop-ethyl.

    PubMed

    Cui, Yongliang; Nan, Tiegui; Tan, Guiyu; Li, Qing X; Wang, Baomin; Liu, Shangzhong

    2011-10-01

    Fenoxaprop-ethyl is a selective aryloxyphenoxypropionate herbicide used widely to control annual and perennial grasses. A monoclonal antibody (MAb) against fenoxaprop-ethyl (FE), designated as 3E6B9C, was produced and had very low cross-reactivity with some of its structural analogs, such as clodinafop-propargyl, diclofop-methyl, lactofen, and quizalofop-p-ethyl. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The concentration of R-(+)-fenoxaprop-ethyl (R-FE) producing 50% of inhibition (IC(50)) and the working range of icELISA were 3.1?ng/mL and 0.6-29?ng/mL, respectively. This assay is also sensitive to R-fenoxaprop, S-(-)-fenoxaprop-ethyl, and metamifop with IC(50) of 3.4, 2.7, and 3.5?ng/mL, respectively. The recoveries of R-FE in soil samples with the icELISA were 86-102%. PMID:22008074

  7. Trial Watch: Immunostimulatory monoclonal antibodies in cancer therapy.

    PubMed

    Aranda, Fernando; Vacchelli, Erika; Eggermont, Alexander; Galon, Jerome; Fridman, Wolf Hervé; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2014-01-01

    Immunostimulatory monoclonal antibodies (mAbs) exert antineoplastic effects by eliciting a novel or reinstating a pre-existing antitumor immune response. Most often, immunostimulatory mAbs activate T lymphocytes or natural killer (NK) cells by inhibiting immunosuppressive receptors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1 (PDCD1, best known as PD-1), or by engaging co-stimulatory receptors, like CD40, tumor necrosis factor receptor superfamily, member 4 (TNFRSF4, best known as OX40) or TNFRSF18 (best known as GITR). The CTLA4-targeting mAb ipilimumab has been approved by the US Food and Drug Administration for use in patients with unresectable or metastatic melanoma in 2011. The therapeutic profile of ipilimumab other CTLA4-blocking mAbs, such as tremelimumab, is currently being assessed in subjects affected by a large panel of solid neoplasms. In the last few years, promising clinical results have also been obtained with nivolumab, a PD-1-targeting mAb formerly known as BMS-936558. Accordingly, the safety and efficacy of nivolumab and other PD-1-blocking molecules are being actively investigated. Finally, various clinical trials are underway to test the therapeutic potential of OX40- and GITR-activating mAbs. Here, we summarize recent findings on the therapeutic profile of immunostimulatory mAbs and discuss clinical trials that have been launched in the last 14 months to assess the therapeutic profile of these immunotherapeutic agents. PMID:24701370

  8. Adverse events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Baldo, Brian A

    2013-01-01

    Fifteen monoclonal antibodies (mAbs) are currently registered and approved for the treatment of a range of different cancers. These mAbs are specific for a limited number of targets (9 in all). Four of these molecules are indeed directed against the B-lymphocyte antigen CD20; 3 against human epidermal growth factor receptor 2 (HER2 or ErbB2), 2 against the epidermal growth factor receptor (EGFR), and 1 each against epithelial cell adhesion molecule (EpCAM), CD30, CD52, vascular endothelial growth factor (VEGF), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, best known as RANKL), and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Collectively, the mAbs provoke a wide variety of systemic and cutaneous adverse events including the full range of true hypersensitivities: Type I immediate reactions (anaphylaxis, urticaria); Type II reactions (immune thrombocytopenia, neutopenia, hemolytic anemia); Type III responses (vasculitis, serum sickness; some pulmonary adverse events); and Type IV delayed mucocutaneous reactions as well as infusion reactions/cytokine release syndrome (IRs/CRS), tumor lysis syndrome (TLS), progressive multifocal leukoencephalopathy (PML) and cardiac events. Although the term “hypersensitivity” is widely used, no common definition has been adopted within and between disciplines and the requirement of an immunological basis for a true hypersensitivity reaction is sometimes overlooked. Consequently, some drug-induced adverse events are sometimes incorrectly described as “hypersensitivities” while others that should be described are not. PMID:24251081

  9. Her2 Monoclonal Antibodies, Antibody Drug Conjugates, and Site Specific Antibody Conjugate Methods

    Cancer.gov

    Antibody drug conjugates (ADC) can demonstrate high efficacy as cancer therapeutics, however, much more can be done to improve their efficacy and safety profile. Site-specific antibody drug conjugation is a promising way to do this.

  10. Monoclonal antibodies to human interferon-gamma. I. Antibodies to a synthetic carboxyl-terminal peptide.

    PubMed

    Ichimori, Y; Kurokawa, T; Honda, S; Suzuki, N; Wakimasu, M; Tsukamoto, K

    1985-06-12

    Two types of hybridomas secreting monoclonal antibodies (MAB) against human interferon-gamma (HuIFN-gamma) were obtained by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with a conjugate of a synthetic carboxyl-terminal peptide (residues 131-146) of HuIFN-gamma and bovine thyroglobulin. One of the antibodies bound to recombinant HuIFN-gamma produced in E. coli as well as to natural HuIFN-gamma, while the others bound only to recombinant HuIFN-gamma. These 2 types of MAB did not neutralize the anti-viral activity of HuIFN-gamma. They were useful for effectively purifying recombinant HuIFN-gamma and quantitatively determining it by an enzyme-linked immunosorbent assay. PMID:3925018

  11. Characterization of murine monoclonal antibodies against serogroup B salmonellae and application as serotyping reagents.

    PubMed Central

    Tsang, R S; Chan, K H; Lau, N W; Choi, D K; Law, D K; Ng, M H

    1991-01-01

    Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other O serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory. PMID:1774314

  12. [Application of monoclonal antibodies to diagnosis and treatment of small cell lung cancers].

    PubMed

    Fujisawa, M; Okabe, T

    1988-04-01

    Recently we developed monoclonal antibodies against small cell lung cancer (SCLC) and applied them for diagnosis using histology and imaging, and treatment using SCLC transplanted into nude mice. One monoclonal antibody designated TFS-2 is cytotoxic to SCLC cells with complement. This antibody seems to be useful for purging tumor cells from the bone marrow in vitro when autologous bone marrow transplantation is applied. The other monoclonal antibody, TFS-4, has a high specificity to SCLC. This antibody was useful for diagnosis of SCLC using histology, and for the detection of metastatic tumor cells in the bone marrow. It was possible using imaging by radioisotope-labelled TFS-4 to reveal the localization of SCLC transplanted into nude mice. As for treatment with TFS-4, we injected 500 mu Ci of 131I-labeled TFS-4 into nude mice bearing SCLC, and recognized a reduction in tumor size of about 60%. PMID:2839119

  13. Preparation and characterization of an anti-DNA monoclonal antibody showing size selectivity toward DNA fragments.

    PubMed

    Onishi, Yoshiaki; Kato, Megumi; Hanyu, Yoshiro

    2004-10-01

    Anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. Balb/c mouse splenocytes were immunized with HeLa cell nuclear extract in the presence of N-acetylmuramyl-L-alanyl-D-isoglutamine and fused with P3U1 myeloma cells using PEG 4000. After HAT selection and ELISA using fragmented HeLa genomic DNA, an anti-DNA monoclonal antibody was obtained. The monoclonal antibody D-1-1, whose isotype was IgM, interacted with a variety of double-stranded DNA. The antibody reacted only with DNA fragments longer than 0.8 kbp, and its apparent dissociation constant for a 1.0-kbp DNA fragment was 34 nM. This antibody will be a helpful tool for the detection of DNA structures. PMID:15672610

  14. Development of an antigen microarray for high throughput monoclonal antibody selection

    PubMed Central

    Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J.

    2014-01-01

    Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host. PMID:24472540

  15. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    PubMed

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

  16. Monoclonal Anti-Idiotypic Antibodies as Functional Internal Images of Enzyme Active Sites: Production of a Catalytic Antibody with a Cholinesterase Activity

    Microsoft Academic Search

    Ladan Izadyar; Alain Friboulet; Marie Helene Remy; Alberto Roseto; Daniel Thomas

    1993-01-01

    Monoclonal antibody 9A8 was selected by immunizing mice with AE-2, a monoclonal antibody directed against the active site of acetylcholinesterase. In accordance with the idiotypic network theory, monoclonal anti-idiotypic antibody 9A8 displayed internal-image properties of the original immunogen, the acetylcholinesterase active site. Hydrolysis of acetylthiocholine and related esters of thiocholine by 9A8 follows saturation kinetics and kinetic parameters were determined.

  17. Monoclonal antibodies as developmental markers to characterize pea floral homeotic transformations

    Microsoft Academic Search

    Luis A. Cañas; Roudeïna Essid; María D. Gómez; José Beltrán

    2002-01-01

    Monoclonal antibodies (mAbs) specific for the different floral organs of the garden pea ( Pisum sativum L.) were raised using two different types of immunization procedures. These antibodies were powerful tools to study the functionality of floral organ identity genes in pea homeotic mutants. The mAbs were used extensively as developmental markers for the immunohistochemical characterization of two pea floral

  18. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

  19. Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus

    Microsoft Academic Search

    Mark Throsby; Cecile Geuijen; Jaap Goudsmit; Arjen Q. Bakker; Jehanara Korimbocus; R. Arjen Kramer; Marieke Clijsters-van der Horst; Maureen de Jong; Mandy Jongeneelen; Sandra Thijsse; Renate Smit; Therese J. Visser; Nora Bijl; Wilfred E. Marissen; Mark Loeb; David J. Kelvin; Wolfgang Preiser; J. ter Meulen; J. de Kruif

    2006-01-01

    Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred

  20. Improved radioimmunolocalization of human tumor xenografts following subcutaneous delivery of monoclonal antibodies

    Microsoft Academic Search

    Richard L. Wahl; Linda Laino; Susan Fisher; Miriam Schteingart; William H. Beierwaltes

    1988-01-01

    The localization of a radiolabeled murine monoclonal antibody reactive with choriocarcinomas to human choriocarcinoma xenografts following intravenous and subcutaneous injection was evaluated by gamma scanning and tissue sampling. Tumor xenografts were established in the popliteal node region of athymic nude mice after repeated innoculations of the hind foot pads with BEWO choriocarcinoma cells. In dual label specific antibody studies, tumor\\/non

  1. Monoclonal antibody against E selectin attenuates subarachnoid hemorrhage–induced cerebral vasospasm

    Microsoft Academic Search

    Chih-Lung Lin; Aaron S. Dumont; Tarkan Calisaneller; Aij-Lie Kwan; Shen-Long Hwong; Kevin S. Lee

    2005-01-01

    BackgroundIncreasing evidence indicates that inflammatory responses are implicated in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). However, the role of adhesion molecules in SAH-induced vasospasm is less clear. This study was designed to examine the effect of a highly specific antibody, monoclonal anti–E-selectin antibody, on cerebral vasospasm in a new murine SAH model.

  2. Prophylaxis and therapy of influenza pneumonia in mice by intratracheal instillation of monoclonal antibody

    SciTech Connect

    Ratcliffe, D.R.

    1985-01-01

    This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs for a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.

  3. Mesenteric vascular occlusion: a new diagnostic method using a radiolabeled monoclonal antibody reactive with platelets

    Microsoft Academic Search

    Z. H. Oster; P. Som; P. O. Zamora

    1989-01-01

    A new method for diagnosing mesenteric vaso-occlusive bowel disease with the use of radioimmunoscintigraphy was developed and tested in experimental models of arterial and venous disease, as well as in a model simulating bowel strangulation. The method involves the use of a monoclonal antibody fragment mixture that binds to platelets. The antibody was labeled with technetium-99m, and imaging was performed

  4. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  5. Monoclonal antibodies distinguish between storage and secreted forms of eosinophil cationic protein

    Microsoft Academic Search

    Po-Chun Tai; Christopher J. F. Spry; Christer Peterson; Per Venge; Inge Olsson

    1984-01-01

    The toxic effects of eosinophils on parasites1 and cells2 are due largely to the secretion of various granule proteins, following stimulation3. In order to study this secretory process (degranulation) further, we have raised mouse monoclonal antibodies against both human eosinophil granule extracts and secretion products. From immunocytochemical studies it appears that one antibody, EG1, recognized both the storage and secreted

  6. The monoclonal antibody GB 42 — a useful marker for the differentiation of myofibroblasts

    Microsoft Academic Search

    Gaby Kohnen; Mario Castellucci; Bae-Li Hsi; Chang-Jing G. Yeh; Peter Kaufmann

    1995-01-01

    The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commerical antibodies

  7. A Monoclonal Antibody Against Kinesin Inhibits Both Anterograde and Retrograde Fast Axonal Transport in Squid Axoplasm

    Microsoft Academic Search

    Scott T. Brady; K. Kevin Pfister; George S. Bloom

    1990-01-01

    One of our monoclonal antibodies against the heavy chain of bovine kinesin (H2) also recognized the heavy chain of squid kinesin. The immunofluorescence pattern of H2 in axoplasm was similar to that seen in mammalian cells with antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membrane-bounded organelles. Although kinesin is assumed to

  8. CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE GLAUCOMA-ASSOCIATED PROTEIN MYOCILIN

    PubMed Central

    Ezzat, Mohamed-Karim; Howell, Kyle G.; Bahler, Cindy K.; Beito, Thomas G.; Loewen, Nils; Poeschla, Eric M.; Fautsch, Michael P.

    2010-01-01

    Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33–214), two antibodies recognize the middle third of the protein (amino acids 215–368), and one antibody recognizes the C-terminus (amino acids 369–504). Isotyping revealed all antibodies are of the IgG1? class except one, which is IgG2b?. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells. PMID:18674535

  9. PRODUCTION OF MONOCLONAL ANTIBODIES TO 'LEGIONELLA PNEUMOPHILA' SEROGROUPS 1 AND 6

    EPA Science Inventory

    To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, were produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common anti...

  10. Measurement of cross linked fibrin derivatives in plasma: an immunoassay using monoclonal antibodies

    Microsoft Academic Search

    A N Whitaker; M J Elms; P P Masci; P G Bundesen; D B Rylatt; A J Webber; I H Bunce

    1984-01-01

    Fibrinogen degradation, fibrin polymerisation, and the insertion of cross links into fibrin by fibrin stabilising factor lead to the appearance of new antigenic determinants. Antibodies against these antigenic sites may react specifically with the derivatives but not with the parent molecules. We have utilised a monoclonal antibody, which interacts with the cross linked fragment D dimer and related high molecular

  11. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    NASA Astrophysics Data System (ADS)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  12. Characterization of immunoregulatory T lymphocytes during ageing by monoclonal antibodies.

    PubMed Central

    Mascart-Lemone, F; Delespesse, G; Servais, G; Kunstler, M

    1982-01-01

    Monoclonal antibodies of the OKT series were used to identify T lymphocytes (OKT3+) and their inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets in the peripheral blood mononuclear cells (PBMC) of 32 healthy old-aged people more than 70 years old (16 men and 16 women) compared to 47 adults (29 men, 18 women) less than 40 years old. The absolute lymphocyte count in the peripheral blood was not significantly influenced by age or sex. Both the proportions and the absolute numbers of T3+ and T4+ cells were significantly lower in aged than in young participants. The proportions but not the absolute counts of OKT8+ cells were higher in the elderly. Most interesting is the influence of sex and these parameters. Old women have normal numbers and proportions of T3+, T4+ and T8+ cells when compared to young women. The latter have a significantly higher proportion of T8+ cells than young adult males. Old men have a striking reduction of both the numbers and proportions of OKT3+ and OKT4+ cells when compared with young men and with women. In addition, old men have an elevated proportion, but a normal absolute number, of OKT8+ cells. The responses of PBMC to phytohaemagglutinin extent (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are reduced to the same extent in ageing male and female subjects when compared to young adults. In the older group, the magnitude of the lymphocyte response to PHA and Con A but not to PWM is negatively correlated with the proportions of OKT8+ cells. Surprisingly, these correlations are observed only in old women but not in old men. The latter finding excludes the possibility that the age-associated decline of the lymphocyte response to T cell mitogens is secondary to an imbalance between T4+ and T8+ lymphocytes. PMID:6211314

  13. CCR5 Monoclonal Antibodies for HIV-1 Therapy

    PubMed Central

    Olson, William C.; Jacobson, Jeffrey M.

    2009-01-01

    Purpose of review This report summarizes emerging clinical and preclinical data pertaining to the use of CCR5 monoclonal antibodies (mAbs) as therapies for HIV-1 infection. The epitope specificity of CCR5 mAbs is discussed in relation to its critical impact on antiviral activity and CCR5 antagonism. We compare and contrast mAbs and small-molecule CCR5 antagonists in terms of their binding and antiviral properties. Two CCR5 mAbs have entered clinical testing and have successfully completed proof-of-concept studies in HIV-infected individuals, providing initial information on the potential therapeutic utility of these agents. Recent findings New studies support the view that the most potently antiviral CCR5 mAbs recognize the second extracellular loop of CCR5 either exclusively or in combination with the amino terminus. Studies have revealed fundamental differences in how mAbs and small molecules bind CCR5 and inhibit HIV-1. CCR5 mAbs and small-molecule CCR5 antagonists have demonstrated consistent antiviral synergy and limited or no viral cross-resistance in independent studies. Single intravenous infusions of CCR5 mAbs significantly reduced HIV-1 RNA levels in infected individuals for 2–3 weeks without appreciable toxicity. Summary CCR5 mAbs have demonstrated broad and potent antiviral activity in vitro. Clinical studies have established CCR5 mAbs as potent antiretroviral agents with prolonged activity following a single dose. CCR5 mAbs represent both a distinct class of CCR5 inhibitor and a novel approach to HIV-1 therapy. PMID:19339948

  14. Response of a concentrated monoclonal antibody formulation to high shear.

    PubMed

    Bee, Jared S; Stevenson, Jennifer L; Mehta, Bhavya; Svitel, Juraj; Pollastrini, Joey; Platz, Robert; Freund, Erwin; Carpenter, John F; Randolph, Theodore W

    2009-08-01

    There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates between 20,000 and 250,000 s(-1) for between 5 min and 30 ms using a parallel-plate and capillary rheometer, respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s(-1) and 0.06 pN, respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20-150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases, air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production. PMID:19370772

  15. Response of a Concentrated Monoclonal Antibody Formulation to High Shear

    PubMed Central

    Bee, Jared S.; Stevenson, Jennifer L.; Mehta, Bhavya; Svitel, Juraj; Pollastrini, Joey; Platz, Robert; Freund, Erwin; Carpenter, John F.

    2009-01-01

    There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates of between 20,000 and 250,000 s-1 for between 5 minutes and 30 ms using a parallel-plate and capillary rheometer respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s-1 and 0.06 pN respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20 to 150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production. PMID:19370772

  16. Molecular simulations of the pairwise interaction of monoclonal antibodies.

    PubMed

    Lapelosa, Mauro; Patapoff, Thomas W; Zarraga, Isidro E

    2014-11-20

    Molecular simulations are employed to compute the free energy of pairwise monoclonal antibodies (mAbs) association using a conformational sampling algorithm with a scoring function. The work reported here is aimed at investigating the mAb-mAb association driven by weak interactions with a computational method capable of predicting experimental observations of low binding affinity. The simulations are able to explore the free energy landscape. A steric interaction component, electrostatic interactions, and a nonpolar component of the free energy form the energy scoring function. Electrostatic interactions are calculated by solving the Poisson-Boltzmann equation. The nonpolar component is derived from the van der Waals interactions upon close contact of the protein surfaces. Two mAbs with similar IgG1 framework but with small sequence differences, mAb1 and mAb2, are considered for their different viscosity and propensity to form a weak interacting dimer. mAb1 presents favorable free energy of association at pH 6 with 15 mM of ion concentration reproducing experimental trends of high viscosity and dimer formation at high concentration. Free energy landscape and minimum free energy configurations of the dimer, as well as the second virial coefficient (B22) values are calculated. The energy distributions for mAb1 are obtained, and the most probable configurations are seen to be consistent with experimental measurements. In contrast, mAb2 shows an unfavorable average free energy at the same buffer conditions due to poor electrostatic complementarity, and reversible dimer configurations with favorable free energy are found to be unlikely. Finally, the simulations of the mAb association dynamics provide insights on the self-association responsible for bulk solution behavior and aggregation, which are important to the processing and the quality of biopharmaceuticals. PMID:25350229

  17. Mapping broadly reactive norovirus genogroup I and II monoclonal antibodies.

    PubMed

    Crawford, Sue E; Ajami, Nadim; Parker, Tracy Dewese; Kitamoto, Noritoshi; Natori, Katsuro; Takeda, Naokazu; Tanaka, Tomoyuki; Kou, Baijun; Atmar, Robert L; Estes, Mary K

    2015-02-01

    Noroviruses are responsible for most acute nonbacterial epidemic outbreaks of gastroenteritis worldwide. To develop cross-reactive monoclonal antibodies (MAbs) for rapid identification of genogroup I and II (GI and GII) noroviruses (NoVs) in field specimens, mice were immunized with baculovirus-expressed recombinant virus-like particles (VLPs) corresponding to NoVs. Nine MAbs against the capsid protein were identified that detected both GI and GII NoV VLPs. These MAbs were tested in competition enzyme-linked immunosorbent assays (ELISAs) to identify common epitope reactivities to GI and GII VLPs. Patterns of competitive reactivity placed these MAbs into two epitope groups (groups 1 and 2). Epitopes for MAbs NV23 and NS22 (group 1) and MAb F120 (group 2) were mapped to a continuous region in the C-terminal P1 subdomain of the capsid protein. This domain is within regions previously defined to contain cross-reactive epitopes in GI and GII viruses, suggesting that common epitopes are clustered within the P1 domain of the capsid protein. Further characterization in an accompanying paper (B. Kou et al., Clin Vaccine Immunol 22:160-167, 2015, http://dx.doi.org/10.1128/CVI.00519-14) revealed that MAb NV23 (epitope group 1) is able to detect GI and GII viruses in stool. Inclusion of the GI and GII cross-reactive MAb NV23 in antigen detection assays may facilitate the identification of GI and GII human noroviruses in stool samples as causative agents of outbreaks and sporadic cases of gastroenteritis worldwide. PMID:25428246

  18. Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

    PubMed Central

    Morton, Laura Dill; Spindeldreher, Sebastian; Kiessling, Andrea; Allenspach, Roy; Hey, Adam; Muller, Patrick Y; Frings, Werner; Sims, Jennifer

    2010-01-01

    Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed. PMID:20421713

  19. Monoclonal Antibodies That Distinguish Antigenic Variants of Canine Parvovirus

    PubMed Central

    Nakamura, Masato; Nakamura, Kazuya; Miyazawa, Takayuki; Tohya, Yukinobu; Mochizuki, Masami; Akashi, Hiroomi

    2003-01-01

    Canine parvovirus (CPV) is classified as a member of the feline parvovirus (FPV) subgroup. CPV isolates are divided into three antigenic types: CPV type 2 (CPV-2), CPV-2a, and CPV-2b. Recently, new antigenic types of CPV were isolated from Vietnamese leopard cats and designated CPV-2c(a) or CPV-2c(b). CPV-2c viruses were distinguished from the other antigenic types of the FPV subgroup by the absence of reactivity with several monoclonal antibodies (MAbs). To characterize the antigenicity of CPV-2c, a panel of MAbs against CPV-2c was generated and epitopes recognized by these MAbs were examined by selection of escape mutants. Four MAbs were established and classified into three groups on the basis of their reactivities: MAbs which recognize CPV-2a, CPV-2b, and CPV-2c (MAbs 2G5 and 20G4); an MAb which reacts with only CPV-2b and CPV-2c(b) (MAb 21C3); and an MAb which recognizes all types of the FPV subgroup viruses (MAb 19D7). The reactivity of MAb 20G4 with CPV-2c was higher than its reactivities with CPV-2a and CPV-2b. These types of specificities of MAbs have not been reported previously. A mapping study by analysis of neutralization-resistant mutants showed that epitopes recognized by MAbs 21C3 and 19D7 belonged to antigenic site A. Substitution of the residues in site B and the other antigenic site influenced the epitope recognized by MAb 2G5. It was suggested that the epitope recognized by MAb 20G4 was related to antigenic site B. These MAbs are expected to be useful for the detection and classification of FPV subgroup isolates. PMID:14607871

  20. Immunocytochemical study on internalization of anti-carbohydrate monoclonal antibodies.

    PubMed

    Kusano, A; Ohta, S; Shitara, K; Hanai, N

    1993-01-01

    The fate of anti-tumor monoclonal antibodies (mAbs) after binding to the cell surface is one of the important factors for application of mAbs in therapy. For mAbs to remain on the cell surface for a long time is preferable character for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). On the other hand, quick internalization of mAbs into cytoplasm is advantageous for cytotoxic effects of toxins or cytotoxic drugs conjugated with mAbs. In this study we investigated the localization of anti-carbohydrate mAbs in vitro by immunocytochemical methods at the electron microscopic level. Anti-ganglioside GM3 mAb (mouse IgM) was localized both in cytoplasmic vesicles and on the cell surface membrane of mouse melanoma cells after incubation for 30 min at 37 degrees C. Anti-ganglioside GM2 mAb (mouse IgM), anti-ganglioside GD2 mAb (mouse IgG3) anti-ganglioside GD3 mAb (mouse IgG3) mainly existed on the cell surface of human small cell lung carcinoma cells, human neuroblastoma cells and human melanoma cells, respectively, after incubation for 30 min at 37 degrees C. On the other hand, anti-sialyl Lea mAb (mouse IgG1) was intensively incorporated into the cytoplasm of human colon tumor cells under the same conditions. Anti-ganglioside GD3 mAb and anti-sialyl Lea mAb were radiolabelled with 125Iodine and traced in in vitro culture. When the cells were incubated in the presence of the 125I-mAb for 90 min on ice, the ratio of intracellular counts to surface counts was 8/92 for anti-ganglioside GD3 mAb and 31/69 for anti-sialyl Lea mAb respectively. After the subsequent incubation for 60 min at 37 degrees C, the ratio altered to 13/87 for anti-ganglioside GD3 mAb and 54/46 for anti-sialyl Lea antigen mAb. This study suggested that internalization of anti-carbohydrate mAbs after the binding to the cell surface was different among the mAbs depending on the character of antigens. In conclusion, anti-ganglioside mAbs have beneficial characters for CDC or ADCC while anti-sialyl Lea mAb is suitable for immunoconjugates. PMID:8297135

  1. The development of therapeutic monoclonal antibodies: overview of the nonclinical safety assessment.

    PubMed

    Lansita, Janice A; Mounho-Zamora, Barbara

    2015-03-01

    Monoclonal antibodies (mAbs) represent a class of biotechnology-derived therapeutics for use in the treatment of various disease indications such as oncology, autoimmune, cardiovascular, and metabolic disorders. Monoclonal antibodies are immunoglobulin (Ig) proteins engineered to bind to specific antigens with high specificity. The concepts reviewed in this paper include 1) the regulatory procedures and guidelines that apply to mAbs, 2) the types of toxicology studies applicable to mAbs, and 3) the scientific challenges, such as the selection of a relevant animal species and the development of anti-drug antibodies, that can arise due to the unique properties of mAbs. PMID:25681157

  2. Highly tumor-reactive, internalizing, mouse monoclonal antibodies to Le(y)-related cell surface antigens.

    PubMed

    Hellström, I; Garrigues, H J; Garrigues, U; Hellström, K E

    1990-04-01

    Two monoclonal antibodies, designated BR64 (IgG1) and BR96 (IgG3), were generated that, according to immunohistology, bind selectively to carcinomas of the colon, breast, ovary, and lung. They have no or very low reactivity with normal human tissues, except for the fact that BR64 strains capillaries in the hearts from certain normal donors and that both monoclonal antibodies stain some epithelial cells from the gastrointestinal system, including stomach. Preliminary studies indicate that at least a portion of the epitope recognized by BR64 and BR96 is a Le(y) carbohydrate chain. Both monoclonal antibodies can be "internalized" by antigen-positive tumor cells, since immunoconjugates with ricin A-chain are cytotoxic. BR96 has the additional properties of being cytotoxic by itself, and it can mediate antibody-dependent cellular cytotoxicity and complement dependent cytotoxicity. PMID:1690595

  3. Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

    PubMed Central

    Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E

    1989-01-01

    Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770

  4. Rat monoclonal antibody specific for the chromatin remodeling factor, CHD1.

    PubMed

    Yoshimura, Saori; Harada, Akihito; Odawara, Jun; Azuma, Masayuki; Okada, Seiji; Nakamura, Mako; Ohkawa, Yasuyuki; Tachibana, Taro

    2010-06-01

    CHD1 is a subfamily member of the CHD family, which possesses a chromodomain, a helicase domain, and a DNA-binding domain. The CHD family regulates gene expression by contributing to ATP-dependent chromatin remodeling. CHD1 exists in the transcriptionally active region and alters the chromatin structure. Little is known about the function of endogenous CHD1, however, and studies have been hindered by the lack of an antibody specific for CHD1 in mammals. In the present study, we established a monoclonal antibody specifically against CHD1 using the rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody showed specific binding to CHD1, allowing us to identify the deduced full-length CHD1. In addition, cell immunostaining clearly revealed the nuclear localization of CHD1. This monoclonal antibody will be useful for further analysis of CHD1 function in mammals. PMID:20568999

  5. Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2

    Microsoft Academic Search

    Dan Lu; Helen Kotanides; Xenia Jimenez; Qinwei Zhou; Kris Persaud; Peter Bohlen; Larry Witte; Zhenping Zhu

    1999-01-01

    Bispecific antibody (BsAb) technology has been successfully used as a means to construct novel antibody (Ab) molecules with increased avidity for binding, by combining two Ab or their fragments directed against different epitopes within the same antigen. Using two single chain antibodies (scFv) isolated from a phage display library, we have constructed a bispecific diabody directed against two different epitopes

  6. Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab

    PubMed Central

    Schlegel, Patrick; Lang, Peter; Zugmaier, Gerhard; Ebinger, Martin; Kreyenberg, Hermann; Witte, Kai-Erik; Feucht, Judith; Pfeiffer, Matthias; Teltschik, Heiko-Manuel; Kyzirakos, Christina; Feuchtinger, Tobias; Handgretinger, Rupert

    2014-01-01

    We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 ?g/m2/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival. PMID:24727818

  7. Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies.

    PubMed Central

    Vockley, J; Meyer, L J; Harris, H

    1984-01-01

    Two forms of intestinal alkaline phosphatase have been recognized in humans. They are very similar in a number of biochemical and immunologic characteristics, but the exact genetic relationship between them remains unclear. To further study this problem, six monoclonal antibodies and a polyclonal rabbit antiserum to human fetal intestinal alkaline phosphatase have been produced. All of the monoclonal antibodies and the rabbit antiserum crossreact with adult intestinal alkaline phosphatase and with the intestinal-like alkaline phosphatase found in D98/AH-2 human tissue-culture cells. Four of the monoclonal antibodies and the rabbit antiserum crossreact with placental alkaline phosphatase, while none of the antibodies or the antiserum recognize liver or kidney alkaline phosphatase. Four of the monoclonal antibodies can distinguish between adult and fetal intestinal alkaline phosphatase in electrophoretic titration-binding studies, with the relative binding of adult enzyme being significantly greater than that of the fetal enzyme in each case. One of these antibodies, which also reacts with placental alkaline phosphatase, can distinguish the type 3 allelic variant of the placental enzyme from types 1 and 2. This indicates that the antibody detects a structural difference in the protein moiety of one of the allelic forms of the enzyme. These data suggest that adult and fetal intestinal alkaline phosphatases represent structurally distinct proteins, either encoded for by different genes or produced by differential processing of a common precursor molecule determined by a single gene. Images Fig. 1 Fig. 2 Fig. 4 PMID:6437214

  8. Targeting Cancer Micrometastases with Monoclonal Antibodies: A Binding-Site Barrier

    NASA Astrophysics Data System (ADS)

    Saga, Tsuneo; Neumann, Ronald D.; Heya, Toshiro; Sato, Jun; Kinuya, Seigo; Le, Nhat; Paik, Chang H.; Weinstein, John N.

    1995-09-01

    Monoclonal antibodies penetrate bulky tumors poorly after intravenous administration, in part because of specific binding to the target antigen. Experiments presented here demonstrate an analogous phenomenon in micrometastases; poor antibody penetration, attributable to a "binding-site barrier" phenomenon, can be seen in guinea pig micrometastases as small as 300 ?m in diameter. Increasing the dose of antibody can partially overcome this limitation, but at a cost in specificity.

  9. Immunofluorescence studies of chondroitin sulfate proteoglycan biosynthesis: the use of monoclonal antibodies.

    PubMed

    Vertel, B M; Barkman, L L

    1984-01-01

    Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L , which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D , and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan. PMID:6202457

  10. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    SciTech Connect

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-12-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 ..mu..g of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10/sup 4/ times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected.

  11. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    SciTech Connect

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  12. Role of monoclonal antibodies in the treatment of immune-mediated glomerular diseases.

    PubMed

    Manrique, Joaquín; Cravedi, Paolo

    2014-05-21

    Non-specific immunosuppressants have represented for decades the only therapies for patients with immune-mediated glomerular diseases. These treatments, however, are associated with high rates of no-response and are burdened by toxicities that frequently offset the benefits of proteinuria reduction. Monoclonal antibodies targeting selective cell populations or mediators implicated in the pathophysiology of glomerular diseases have recently become available. Rituximab, a chimeric monoclonal antibody against the CD20 antigen on B cells, safely reduced proteinuria in patients with nephrotic syndrome secondary to membranous nephropathy, minimal change disease, or focal segmental glomerulosclerosis. Its ability to reduce auto-antibody formation has been instrumental to treat also ANCA-associated vasculitis, lupus nephritis, and mixed cryoglobulinemia. Many reports have also documented the efficacy of the anti-C5 humanized monoclonal antibody Eculizumab to treat atypical hemolytic uremic syndrome, C3 nephropathy, and membranoproliferative glomerulonephritis. Thanks to these encouraging findings, monoclonals are becoming very helpful tools to treat patients with glomerular diseases. Moreover, thanks to their specific mechanism of action, these and other monoclonal antibodies are important in improving our understanding of the pathophysiology of glomerular diseases. Their still high costs, however, might represent a major hurdle for their widespread implementation for all patients in need. PMID:24798567

  13. 8th Annual European Antibody Congress 2012

    PubMed Central

    Beck, Alain; Carter, Paul J.; Gerber, Hans-Peter; Lugovskoy, Alexey A.; Wurch, Thierry; Junutula, Jagath R.; Kontermann, Roland E; Mabry, Robert

    2013-01-01

    The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:23493119

  14. Differentiation of mumps virus strains with monoclonal antibody to the HN glycoprotein.

    PubMed Central

    Server, A C; Merz, D C; Waxham, M N; Wolinsky, J S

    1982-01-01

    A hybridoma cell line secreting antibody of the immunoglobulin G3 isotype with kappa light chains and with activity against the HN glycoprotein of the Kilham strain of mumps virus was established. The antibody exhibited structural homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a microheterogeneous isoelectric spectrum characteristic of an antibody of monoclonal origin. The specificity of the monoclonal antibody, shown by immunoprecipitation performed with radiolabeled virus and infected cell lysates, was for the larger mumps virus glycoprotein. In functional assays the antibody inhibited the hemagglutinating and neuraminidase activities and neutralized the infectivity of the homologous Kilham strain of virus and clearly differentiated this strain from two heterologous strains, Enders and O'Take. The antibody was markedly less effective with the O'Take strain than with either the Kilham or Enders strain in inhibiting both hemagglutination and neuraminidase activity against the macromolecular substrate fetuin. The inhibition of the neuraminidase activity of the Kilham strain was independent of substrate size, the antibody inhibiting the hydrolysis of both fetuin and the trisaccharide neuraminlactose. By contrast, the antibody did not inhibit the hydrolysis of neuraminlactose by the two heterologous mumps strains. These results provide the first demonstration of antigenic differences between mumps virus strains and highlight the utility of monoclonal antibody in analyzing the structural basis underlying functional activities of the HN glycoproteins. Images PMID:6172379

  15. Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

    PubMed Central

    Ruf, J; Carayon, P; Sarles-Philip, N; Kourilsky, F; Lissitzky, S

    1983-01-01

    Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease. PMID:6196190

  16. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  17. West Nile virus: characterization and diagnostic applications of monoclonal antibodies

    PubMed Central

    2012-01-01

    Background Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. Methods Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. Results All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys ? Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser ? Ile) or E278 (Thr ? Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. Conclusions MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization. PMID:22500562

  18. Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum.

    PubMed

    Uzêda, R S; Schares, G; Ortega-Mora, L M; Madruga, C R; Aguado-Martinez, A; Corbellini, L G; Driemeier, D; Gondim, L F P

    2013-11-01

    Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum. PMID:23927916

  19. Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies

    Microsoft Academic Search

    Kang-Seuk Choi; Jin-Ju Nah; Cheong-Up Choi; Young-Joon Ko; Hyun-Joo Sohn; Genevieve Libeau; Shien-Young Kang; Yi-Seok Joo

    2003-01-01

    An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV

  20. Characterization of functional properties of C4-binding protein by monoclonal antibodies.

    PubMed

    Fujita, T; Kamato, T; Tamura, N

    1985-05-01

    We prepared mouse monoclonal antibodies to human C4-binding protein (C4-bp) by fusing spleen cells from mice immunized with purified C4-bp to the mouse myeloma line P3U1. Of four monoclonal antibodies that reacted with intact C4-bp, two were specific for a 48K fragment, one of the chymotryptic cleavage products of C4-bp, and one was specific for another fragment (160K). The fourth monoclonal antibody did not react with either fragment. One of the monoclonals that reacted with the 48K fragment blocked the binding of C4-bp to cell-bound C4b. This monoclonal antibody (TK3) also inhibited two other functions of C4-bp, serving as an essential cofactor for C3b/C4b inactivator (I) in the cleavage of fluid-phase C4b and accelerating the decay of C2a from the C4b,2a complex. The other monoclonals had little or no effect on these activities of C4-bp. In addition, we found that the 48K fragment lost the binding affinity for C4b. However, it can function as a cofactor for I and as a decay-accelerator, although its activities were about 200 times weaker than intact C4-bp on a molar basis. The monoclonal antibody TK3 completely inhibited these activities of the 48K fragment. On the basis of these findings, we conclude that the functionally active site of C4-bp is located on the 48K fragment. Probably, the cofactor and decay-accelerating activities of C4-bp result from the binding of C4-bp to C4b. PMID:3872332

  1. Monoclonal antibodies against Nereis virens brain homogenates as probes for isolation and in situ detection of new neurosecretions.

    PubMed

    Delaire-Hesdin, A; Bulet, P; Boilly-Marer, Y; Porchet-Hennere, E; Masson, M

    1992-01-01

    The brain of Nereis contains 26 ganglionic nuclei which produce numerous neurosecretions. Only a few of them have been characterized. The production of monoclonal antibodies was adopted as an approach to discover unknown neurosecretions. Monoclonal antibodies produced against Nereis virens brain homogenates were selected using stepwise ELISA tests first with brain homogenates, then with brain neurosecretions. Eight antibodies specific for Nereis neurosecretions were selected. The results are illustrated with one of these monoclonal antibodies which was directed against a major peak after HPLC purification of brain neurosecretions. This antibody was subsequently used for the in situ detection of recognized epitope(s) in the brain and ventral nerve cord cells. PMID:1473354

  2. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  3. The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

    NASA Technical Reports Server (NTRS)

    Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1997-01-01

    The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

  4. Monoclonal antibodies to Mycoplasma hyorhinis surface antigens: tools for analyzing mycoplasma-lymphoid cell interactions.

    PubMed Central

    Wise, K. S.; Watson, R. K.

    1983-01-01

    A library has been constructed of approximately 50 monoclonal antibodies that recognize antigens of Mycoplasma hyorhinis. Characteristics of six antibodies are discussed. Each reacts with a discrete determinant borne on a protein-containing molecule of distinct molecular size. Three of these respective antigens are expressed at the surface of mycoplasmas colonizing lymphoblastoid cells in culture. Of these three surface antigens, two are selectively expressed on strain GDL but not on strain BTS-7 of M. hyorhinis, thus defining strain-restricted immunological specificities within these species. One monoclonal antibody of the IgM class (mu, kappa) mediated marked complement-dependent growth inhibition of M. hyorhinis in broth culture. These monoclonal reagents should facilitate analysis of mycoplasma surface architecture, and the molecular interactions of these organisms with the host cell surface. Images FIG. 1 FIG. 2 PMID:6206658

  5. Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments

    Microsoft Academic Search

    Tomoko Yoda; Yoshitake Terano; Yasuhiko Suzuki; Kenji Yamazaki; Isao Oishi; Tsuyoshi Kuzuguchi; Hiroyoshi Kawamoto; Etsuko Utagawa; Koichi Takino; Hajime Oda; Tadayoshi Shibata

    2001-01-01

    BACKGROUND: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that

  6. Concentrations of EpCAM ectodomain as found in sera of cancer patients do not significantly impact redirected lysis and T-cell activation by EpCAM/CD3-bispecific BiTE antibody MT110

    PubMed Central

    Petsch, Silke; Gires, Olivier; Rüttinger, Dominik; Denzel, Sabine; Lippold, Sandra; Wolf, Andreas

    2011-01-01

    Ectodomains of target antigens for antibody-based therapies can be shed from the target cell surface and found in sera of patients. Shed ectodomains of therapeutic targets not only pose the risk of sequestering therapeutic antibodies but, in a multimeric form, of triggering T cell activation by bispecific antibodies binding to CD3 on T cells. Recently, epithelial cell adhesion molecule (EpCAM) has been shown to be activated by release of its ectodomain, called EpEX. Here, we show that only very low amounts of EpEX are detectable in sera of cancer patients. Among 100 cancer patient samples tested, only 17 (17%) showed serum levels of EpEX in excess of 0.05 ng/ml with highest EpEX concentrations of 5.29, 1.37 and 0.52 ng/ml. A recombinant form of human EpEX (recEpEX) was produced to assess its possible effect on redirected lysis and T cell activation by EpCAM/CD3-bispecific BiTE antibody MT110, currently being tested in patients with solid tumor malignancies. RecEpEX had a very minor effect on redirected lysis by MT110 with an approximate IC50 value of 3,000 ng/ml, which is a concentration close to three orders of magnitude higher than the highest EpEX concentration found in cancer patients. Concentrations of 30 ng/ml EpEX in combination with 250 ng/ml MT110 were minimally required to induce a detectable activation of CD4+ and CD8+ T cells. We conclude that soluble EpEX in sera of cancer patients is unlikely to pose an issue for the efficacy or safety of MT110, and perhaps other antibodies binding to N-terminal epitopes of EpCAM. PMID:21107020

  7. Concentrations of EpCAM ectodomain as found in sera of cancer patients do not significantly impact redirected lysis and T-cell activation by EpCAM/CD3-bispecific BiTE antibody MT110.

    PubMed

    Petsch, Silke; Gires, Olivier; Rüttinger, Dominik; Denzel, Sabine; Lippold, Sandra; Baeuerle, Patrick A; Wolf, Andreas

    2011-01-01

    Ectodomains of target antigens for antibody-based therapies can be shed from the target cell surface and found in sera of patients. Shed ectodomains of therapeutic targets not only pose the risk of sequestering therapeutic antibodies but, in a multimeric form, of triggering T cell activation by bispecific antibodies binding to CD3 on T cells. Recently, epithelial cell adhesion molecule (EpCAM) has been shown to be activated by release of its ectodomain, called EpEX. Here, we show that only very low amounts of EpEX are detectable in sera of cancer patients. Among 100 cancer patient samples tested, only 17 (17%) showed serum levels of EpEX in excess of 0.05 ng/ml with highest EpEX concentrations of 5.29, 1.37 and 0.52 ng/ml. A recombinant form of human EpEX (recEpEX) was produced to assess its possible effect on redirected lysis and T cell activation by EpCAM/CD3-bispecific BiTE antibody MT110, currently being tested in patients with solid tumor malignancies. RecEpEX had a very minor effect on redirected lysis by MT110 with an approximate IC 50 value of 3,000 ng/ml, which is a concentration close to three orders of magnitude higher than the highest EpEX concentration found in cancer patients. Concentrations of 30 ng/ml EpEX in combination with 250 ng/ml MT110 were minimally required to induce a detectable activation of CD4 (+) and CD8 (+) T cells. We conclude that soluble EpEX in sera of cancer patients is unlikely to pose an issue for the efficacy or safety of MT110, and perhaps other antibodies binding to N-terminal epitopes of EpCAM. PMID:21107020

  8. Novel monoclonal antibody inhibits tumor growth in breast cancer and angiosarcoma

    Cancer.gov

    A monoclonal antibody targeting a protein known as SFPR2 has been shown by researchers at the University of North Carolina and its Lineberger Comprehensive Cancer Center to inhibit tumor growth in pre-clinical models of breast cancer and angiosarcoma. In a paper published in the April 19 issue of Molecular Cancer Therapeutics, a team used a monoclonal antibody to target SFRP2 expressed in cells from triple-negative breast cancer and the aggressive blood-vessel malignancy angiosarcoma, reducing the rate of tumor growth.

  9. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    SciTech Connect

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  10. RIA of thyroglobulin using monoclonal antibodies: Minimal interference by anti-thyroglobulin autoantibodies

    SciTech Connect

    Nakashima, T.; Koizumi, M.; Sakahara, H.; Ohta, H.; Kohsaka, T.; Misaki, T.; Iida, Y.; Kasagi, K.; Endo, K.; Konishi, J.

    1985-05-01

    Thyroglobulin (Tg) is considered to be secreted from the thyroid gland with the stimulation of TSH and/or thyroid stimulating immunoglobulins. However its use as a prognostic marker for Graves' disease is hampered by anti-Tg autoantibodies in patients' serum. In order to resolve this drawback, the authors have developed monoclonal antibodies to human Tg with very little cross-reactivities with autoantiobodies. Nine monoclonal antibodies were produced by the immunization with Tg prepared from Graves' thyroid and one of them (IgGl), designated as 59A, showed the highest affinity to Tg (3.6 x 10/sup 40/M/sup -1/) and the least cross-reactivity with anti-Tg autoantibodies. The binding of I-125 labeled 59A to beads coated with Tg was not inhibited by the addition of purified IgG obtained from various thyroid diseases except a few Hashimoto's patients with very high titer of anti-Tg antibodies, although the binding of other monoclonal antibodies to Tg was greatly influenced even in the presence of Graves' IgG. The sensitivity of the assay using 59A was enough to detect 20ng Tg/ml and Tg concentrations, in patients with no detectable anti-Tg antibodies, were comparable to those determined by the conventional RIA kit (Eiken), using radioiodinated Tg and polyclonal rabbit anti-Tg antiserum. Further, the shelf-life of I-125 labeled monoclonal antibody was much longer than the radioiodinated Tg. These results indicated that RIA of Tg using monoclonal antibodies would be useful for measuring Tg values not only in patients with thyroid cancer but also in Graves' disease with anti-Tg autoantibodies.

  11. Monoclonal antibodies to type X collagen. Biosynthetic studies using an antibody to the amino-terminal domain.

    PubMed

    Summers, T A; Irwin, M H; Mayne, R; Balian, G

    1988-01-01

    Monoclonal antibodies to chick type X collagen have been used to study the structure, biosynthesis, and location of type X in cartilage. The antibodies were produced by injecting purified type X collagen into female SJL/J mice and then fusing their spleen cells with Sp2/0 myeloma cells. Hybridoma culture supernatants were screened for antibodies to type X collagen by enzyme-linked immunosorbent assay and Western blots. Positive supernatants did not cross-react with other collagen types (I, II, IX, XI) or with fibronectin. Three monoclonal antibodies were chosen for further characterization. Two of them (1A6 and 6F6) recognize a pepsin-sensitive domain of type X collagen. Rotary shadowing showed that 1A6 and 6F6 both recognize the same end of type X, probably the aminoterminal non-triple helical domain. Amino acid sequencing of the intact protein and of the epitope-containing peptide confirmed that the antibody recognition sites for 1A6 and 6F6 are within the amino-terminal domain. Monoclonal antibody 2B3 reacts with the pepsinized (45 kDa) and weakly with the nonpepsinized (59 kDa) forms of type X collagen. The monoclonal antibodies were used for immunolocalization of type X in hypertrophic chondrocytes and reacted only with tissue samples from areas undergoing endochondral ossification, e.g. growth plate and fracture callus. Antibody 6F6, when coupled to Sepharose, selectively binds to type X collagen from cell and organ cultures. In a pulse-chase experiment, no processing of the 59-kDa form of type X could be detected. Two components with molecular masses of approximately 70 and 85 kDa, arising from a disulfide-bonded aggregate, were synthesized by both the permanent and calcifying cartilage organ cultures but did not react with the antibody, suggesting that these proteins are not related to type X. In summary, the pulse-chase results and the immune precipitation with monoclonal antibody 6F6 did not detect biosynthetic precursors larger than 59 kDa or proteolytically processed forms of type X. PMID:2826450

  12. Polyclonal and monoclonal antibodies directed against SK & F 94461, a specific H1 histamine receptor ligand.

    PubMed

    Chatenoud, L; Villemain, F; Hoebeke, J; Garbarg, M; Korner, M; Gros, C; Ruat, M; Cazenave, P A; Ganellin, C R; Bach, J F

    1988-08-01

    SK & F 94461, an aminopentyl analogue of mepyramine, is a recently described H1 receptor antagonist. At variance with the other available H1 receptor ligands, SK & F 94461 offers the possibility of coupling to a protein carrier to render the molecule immunogenic. SK & F 94461 coupled to succinylated bovine serum albumin was used as an immunogen to raise polyclonal antibodies in rabbits and BALB/c mice. In parallel, spleen cells from immunized mice were used to produce hybridomas by somatic cell fusion. Thus, six different murine monoclonal antibodies sharing anti-SK & F 94461 specificity were selected for further detailed characterization of their binding properties. Pharmacologic studies of competitive inhibition using a set of 11 histaminergic agents allowed analysis of the fine specificity of anti-SK & F 94461 antibodies. Both polyclonal and monoclonal anti-SK & F 94461 antibodies showed very high affinity for the immunizing molecule (i.e., Ka values for monoclonal antibodies 8 and 12 were, respectively, 3 X 10(10) and 1.4 X 10(10) M-1). Both types of antibodies bound with high affinity (IC50 ranging from 10(-10) to 10(-12) M) to mepyramine, which has a chemical structure closely resembling that of SK & F 94461. Moreover, these antibodies displayed clear-cut stereoselectivity inasmuch as they bound the d-configuration of chlorpheniramine with significantly higher affinity than the l-form. Thus, all six monoclonal antibodies showed IC50 values 1 to 6 log units lower for d- than for l-chlorpheniramine. For some monoclonal antibodies, spectroscopic and fluorescence spectra studies showed that their different binding capacities correlated with their optical properties. Similarly, polyclonal anti-SK & F 94461 antibodies showed a 500-fold lower affinity for l- than for d-chlorpheniramine. All these results indicate that the polyclonal and the majority of monoclonal anti-SK & F 94461 antibodies recognized with high affinity structural configurations known to be important for the pharmacologic activity of H1 ligands, namely the presence of the dimethylaminoethyl side chain and, with stereochemical selectivity, the d-configuration of chlorpheniramine. These data extend for the first time to an H1 histamine receptor ligand results reported in other hormone systems. PMID:3412319

  13. Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

    SciTech Connect

    Ku, Hsing-Hsu.

    1988-01-01

    BALB/c mice were immunized with adenosine 6-aminocaproyl-BSA. Hybridoma cell lines that secreted anti-idiotypic antibodies were identified by their binding to rabbit anti-adenosine antibodies, but not to normal rabbit immunoglobulins. Two such monoclonal antibodies, AA18 and AA21, also inhibited the binding of ({sup 3}H)adenosine to the rabbit anti-adenosine antibodies. Therefore, both appeared to recognize idiotypic determinants on the rabbit anti-adenosine antibodies. The monoclonal antibodies AA18 and AA21 were established as being directed at adenosine receptors by the following criteria: (1) they bound to both rat and bovine brain membranes, and binding could be inhibited by CHA, an adenosine receptor agonist, (2) they inhibited the binding of ({sup 3}H)R-PIA, an adenosine receptor agonist, to rat brain membranes; and (3) they inhibited the adenylate cyclase of rat brain membranes. The monoclonal antibodies were used to screen cDNA libraries in lambda gt11.

  14. Identification with monoclonal antibodies of hemolysin produced by clinical isolates of Escherichia coli.

    PubMed Central

    Hugo, F; Arvand, M; Reichwein, J; Mackman, N; Holland, I B; Bhakdi, S

    1987-01-01

    Murine monoclonal antibodies were generated against the 107,000-dalton hemolysin encoded by the hemolytic determinant from Escherichia coli LE 2001, and colony blotting was used to assay for production of the hemolysin by 35 hemolytic strains of E. coli and other hemolytic members of the family Enterobacteriaceae of clinical origin. All hemolytic E. coli strains gave positive reactions with two monoclonal antibodies. In contrast, none of the hemolytic, non-E. coli isolates yielded positive colony blots. In addition, Western blotting showed that the hemolysins produced by all clinical E. coli isolates had a similar molecular weight of about 107,000. Discrete antigenic variation may occur in the molecule, since a third monoclonal antibody did not react with the hemolysin from a number of wild-type E. coli strains. Western blot analysis was used to assess the presence of immunoglobulin G (IgG), IgA, and IgM antibodies to E. coli hemolysin in human sera. All 20 of the tested sera from healthy adults contained antibodies to the toxin, with various constellations among the antibody classes. In contrast, sera from five of eight infants aged 8 to 36 months contained no antihemolysin antibodies. We conclude that the 107,000-dalton hemolysin of E. coli is a widespread immunogen that is produced by most or all hemolytic E. coli strains in the human host. Images PMID:3539994

  15. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  16. 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.

  17. Redirected T-cell killing of solid cancers targeted with an anti-CD3/Trop-2-bispecific antibody is enhanced in combination with interferon-?.

    PubMed

    Rossi, Edmund A; Rossi, Diane L; Cardillo, Thomas M; Chang, Chien-Hsing; Goldenberg, David M

    2014-10-01

    Trop-2 has limited presence on normal tissues but is highly expressed in diverse epithelial cancers. (E1)-3s is a T-cell-redirecting trivalent bispecific antibody (bsAb), comprising an anti-CD3 scFv covalently linked to a stabilized dimer of a Trop-2-targeting Fab using Dock-and-Lock. We show for the first time that bsAb-mediated bidirectional trogocytosis occurs between target and T cells and involves immunologic synapses. We studied the effects of interferon-? (INF?) on (E1)-3s-mediated T-cell killing of human gastric and pancreatic cancer cell lines. T-cell activation, cytokine induction, and cytotoxicity were evaluated ex vivo using peripheral blood mononuclear cells (PBMC) or T cells with NCI-N87 gastric cancer as target cells. In vivo activity was assayed with NCI-N87 and Capan-1 (pancreatic) xenografts. In the presence of target cells and PBMCs, (E1)-3s did not cause excess cytokine production. When combined with (E1)-3s, peginterferonalfa-2a--which alone did not increase T-cell activation or raise cytokine levels over baseline--increased CD69 expression but did not significantly increase cytokine induction. (E1) 3s mediated a highly potent T-cell lysis of NCI-N87 target cells in vitro. Inclusion of peginterferonalfa-2a or a more potent form of INF?, 20*-2b, significantly potentiated the activity of (E1)-3s by more than 2.5- or 7-fold, respectively. In vivo, combining peginterferonalfa-2a with (E1)-3s delayed Capan-1 growth longer than each single agent. Similarly, combination therapy delayed tumor proliferation of NCI-N87 compared with (E1)-3s or peginterferonalfa-2a single-treatment groups. (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancers, which was enhanced with INF?. PMID:25053819

  18. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    PubMed Central

    Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall’Acqua, William; Damschroder, Melissa; Hammond, Scott A.

    2012-01-01

    MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. PMID:22574157

  19. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

  20. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, Ronald H. (Livermore, CA); Vanderlaan, Martin (San Ramon, CA); Bigbee, William L. (Livermore, CA); Stanker, Larry H. (Livermore, CA); Branscomb, Elbert W. (Walnut Creek, CA); Grabske, Robert J. (Berkeley, CA)

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  1. Production of monoclonal antibody to human IgG allotype G3M T.

    PubMed

    Kimura, A; Tamaki, Y; Kishida, T; Fukuda, M; Tsuji, T

    1989-01-01

    An efficient method for the production of monoclonal anti-G3M T antibody is described. IgG3 protein of GM B3ST phenotype was isolated by affinity chromatography on Ricinus communis lectin I-agarose and used for immunization. A mouse hybridoma clone was obtained by fusion of popliteal lymph node cells and P3U1 myeloma cells. The antibody produced was tested for allotype specificity by hemagglutination inhibition and ELISA methods using 101 IgG-allotyping control sera. The antibody was neutralizable by all G3M T-positive sera and entirely nonneutralizable by G3M T-negative sera in the inhibition test, and reacted only with G3M T-positive IgG coats in the ELISA test. The results prove the antibody to be allotype-specific, and therefore practically establishes its monoclonality. PMID:2915127

  2. A rat monoclonal antibody against the chromatin remodeling factor CHD5.

    PubMed

    Yoshimura, Saori; Yoshimi, Tomohiko; Ohkawa, Yasuyuki; Azuma, Masayuki; Tachibana, Taro

    2010-02-01

    CHD5 (chromodomain/helicase/DNA-binding protein 5) is a member of the CHD subfamily of chromatin remodeling Swi/Snf proteins, and has been recently identified as a tumor suppressor in a diverse range of human cancers. We report here on the establishment of a hybridoma cell line for producing a monoclonal antibody against CHD5 by the rat medial iliac lymph node method. Immunoblotting analyses indicated that this antibody, MAb 5A10, specifically recognizes endogenous CHD5. In immunostaining using the antibody, a nuclear staining pattern was observed. The monoclonal antibody will be useful in immunoblotting and immunolocalization experiments in a variety of cells and tissues, as well as in further studies of the biological function and cellular dynamics of this protein. PMID:20199154

  3. Combined active and passive immunization against nicotine: Minimizing monoclonal antibody requirements using a target antibody concentration strategy

    PubMed Central

    Cornish, Katherine E.; Harris, Andrew C.; LeSage, Mark G.; Keyler, Dan E.; Burroughs, Danielle; Earley, Cathy; Pentel, Paul R.

    2011-01-01

    Nicotine vaccines have shown preliminary evidence of efficacy for enhancing smoking cessation rates, but the serum nicotine-specific antibody (NicAb) concentrations produced are highly variable and many subjects do not develop effective levels. As an alternative to vaccination, passive immunization with nicotine-specific monoclonal antibodies could produce more uniform serum NicAb concentrations, but its use is limited by their high cost and shorter elimination half-life. This study investigated supplementing vaccination with monoclonal antibodies in a targeted fashion to increase vaccine efficacy while minimizing the required monoclonal antibody dose. Rats were vaccinated and then given individualized supplemental doses of the nicotine-specific monoclonal antibody Nic311 to achieve a target total serum NicAb concentration known to be effective for blocking locomotor sensitization (LMS) to nicotine. Rats received vaccine, Nic311, both, or neither, followed by 0.3 mg/kg nicotine s.c. for 10 days to produce LMS. Combination immunotherapy completely blocked the development of LMS, while monotherapy with vaccine or Nic311 alone were only minimally effective. Lower brain nicotine levels were associated with reduced locomotor activity averaged over days 7-10. Despite its greater efficacy, combination immunotherapy did not reduce the variability in the resulting total serum NicAb concentrations. Variability in total serum NicAb concentrations was contributed to by both vaccine-generated antibody and by Nic311. These data show that combination immunotherapy, using a Nic311 dose that is by itself only minimally effective, can substantially enhance nicotine vaccine efficacy. However, variability in serum NicAb levels with combination immunotherapy may make translation of this approach challenging. PMID:21802529

  4. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

    PubMed Central

    1988-01-01

    We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

  5. Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205

    Microsoft Academic Search

    M. Goedert; R. Jakes; E. Vanmechelen

    1995-01-01

    Hyperphosphorylated microtubule-associated protein tau is the major component of the paired helical filament of Alzheimer's disease. Phosphorylation-dependent anti-tau antibodies are being used to identify specific amino acids that are phosphorylated in tau from normal brain and Alzheimer's disease brain. As such, monoclonal antibody AT8 is widely used. By a combination of site-directed mutagenesis of recombinant tau and in vitro phosphorylation,

  6. The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells

    SciTech Connect

    Addis, J.B. (Hospital for Sick Children, Toronto, Canada); Tisch, R.; Falk, J.A.; Letarte, M.

    1982-11-01

    The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens.The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10/sup 5/ molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90. Sequential immunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of /sup 125/I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.

  7. Separation of functional subsets of human t cells by a monoclonal antibody

    Microsoft Academic Search

    E. L. Reinherz; P. C. Kung; G. Goldstein; S. F. Schlossman

    1979-01-01

    A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55 to 60% of the peripheral blood T cell population (OKT4\\/sup +\\/) and unreactive with normal B cells, null cells, and macrophages. With cell-sorter separation of OKT4\\/sup +\\/ and OKT4⁻ cells, it was shown that these T

  8. Monoclonal antibody (UCHL1) that recognises normal and neoplastic T cells in routinely fixed tissues

    Microsoft Academic Search

    A J Norton; A D Ramsay; S H Smith; P C Beverley; P G Isaacson

    1986-01-01

    UCHL1 is a murine monoclonal antibody that recognises a 180-185 kD determinant on CD4 (72%) and CD8 (36%) positive T cells. This antibody is effective in formalin fixed and paraffin embedded tissues, using the immunoperoxidase method. One hundred and forty three cases of malignant lymphoma were examined. Neoplastic cells in 100% of cases of Mycosis fungoides (n = 10), 83%

  9. Specific and nonspecific imaging of localized Fisher immunotype 1 Pseudomonas aeruginosa infection with radiolabeled monoclonal antibody

    Microsoft Academic Search

    R. H. Rubin; L. S. Young; W. P. Hansen; M. Nedelman; R. Wilkinson; M. J. Nelles; R. Callahan; B. A. Khaw; H. W. Strauss

    1988-01-01

    To determine if radiolabeled specific antibodies directed against bacterial antigens could be used to detect sites of infection, gamma camera imaging studies were performed in animals infected with Pseudomonas aeruginosa. Murine monoclonal antibodies (Mabs) directed against Fisher Immunotype 1 Pseudomonas aeruginosa and a nonmicrobial, nonmammalian haptene, p-arsanilic acid, were labeled with 125I by the lodogen-Bead method. Unilateral, deep thigh infections

  10. Characterization of a Mouse Monoclonal Antibody Specific for O-Linked N-Acetylglucosamine

    Microsoft Academic Search

    Frank I. Comer; Keith Vosseller; Lance Wells; Mary Ann Accavitti; Gerald W. Hart

    2001-01-01

    ?-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in ?-O-glycosidic linkage to both

  11. MDR1 Gene-specific Monoclonal Antibody C494 Cross-Reacts with Pyruvate Carboxylase1

    Microsoft Academic Search

    Vallabhaneni V. Rao; Douglas C. Anthony; David Piwnica-Worms

    1994-01-01

    ABSTRACT Overexpression of P-glycoprotein, the plasma membrane protein prod uct of the MURI gene, is a major determinant in the development of resistance to a large number,of cancer chemotherapeutic,agents. A battery of antibodies, including the MURI gene-specific monoclonal antibody mi.\\\\ln C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we

  12. Inhibition of Fibrinogen Binding to Stimulated Human Platelets by a Monoclonal Antibody

    Microsoft Academic Search

    Joel S. Bennett; James A. Hoxie; Susan F. Leitman; Gaston Vilaire; Douglas B. Cines

    1983-01-01

    Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 mu M ADP, 10 mu M epinephrine,

  13. In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

    1983-05-01

    A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

  14. Further Characterization of Immunomodulation by a Monoclonal Antibody against Streptococcus mutans Antigen P1

    Microsoft Academic Search

    Nikki R. Rhodin; Marloes L. J. A. Van Tilburg; Monika W. Oli; William P. McArthur; L. Jeannine Brady

    2004-01-01

    We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also

  15. Monoclonal Antibody Raised by Sera of Athymic Mice Bearing Human Lung Cancer Xenografts

    Microsoft Academic Search

    Tesshi Yamada; Setsuo Hirohashi; Yukio Shimosato; Masahiko Watanabe; Shigenori Kamio; Tsukasa Kunii; Yoshihiro Hayata

    1987-01-01

    A monoclonal antibody, NCC-LU-165 (IgM, k), was obtained by somatic cell hybridization between mouse myeloma cells (P3-X63-Ag8-Ul) and spleen cells of an immunocompetent mouse (BALB\\/c, nu\\/ + ) immunized with sera of immunodeficient athymic mice (BALB\\/c, nu\\/nu) bearing human giant cell lung cancer xenografts (Lu65). Immunohistochemically, the antibody was reactive with most nonsmall cell carcinoma of the lung. It also

  16. Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of trichoderma reesei

    SciTech Connect

    Nieves, R.A.; Todd, R.J.; Ellis, R.P. (Colorado State Univ., Fort Collins (USA)); Himmel, M.E. (Solar Energy Research Institute, Golden, CO (USA))

    1990-04-01

    Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.

  17. Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria.

    PubMed

    Rossmann, Friederike S; Laverde, Diana; Kropec, Andrea; Romero-Saavedra, Felipe; Meyer-Buehn, Melanie; Huebner, Johannes

    2015-01-01

    Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 ?g/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials. PMID:25706415

  18. Preparation of monoclonal antibody of anti-feline calicivirus and establishment of double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

    PubMed

    Yuan, B; Ai, C-X; Yuan, L; Gao, W; Hu, J-P; Chen, J; Ren, W-Z

    2014-01-01

    This study aimed to prepare monoclonal antibody of feline calicivirus (FCV) and identify its basic biological characteristics. Saturated ammonium sulfate precipitation, combined differential centrifugation, and cesium chloride density gradient centrifugation were used for the purification of FCV. The purified FCV was used as antigen to immunize BALB/c mice. The hybridoma lines of anti-FCV monoclonal antibodies were established using cell fusion and hybridoma screening techniques. The subtypes of the monoclonal antibody were identified. The results showed that 3 strains of hybridoma cell lines stably secreted anti-FCV monoclonal antibody; they were named as D8, E5, and H4. The D8 and E5 were IgM subtype antibodies, and H4 was IgG2b subtype antibody. The monoclonal antibody obtained shared no cross-reactivity with feline parvovirus, canine parvovirus, and canine distemper virus. According to the different recognition sites of 2 monoclonal antibodies E5 and H4 to the FCV, they were used to coat microtiter plates and prepare 2 enzyme-labeled secondary antibodies to establish double-antibody sandwich enzyme-linked immunosorbent assay detecting method. PMID:25222237

  19. Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

    NASA Astrophysics Data System (ADS)

    Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

    1989-05-01

    We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

  20. Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy.

    PubMed

    Stork, Abraham C J; Jacobs, Bart C; Tio-Gillen, Anne P; Eurelings, Marijke; Jansen, Marc D; van den Berg, Leonard H; Notermans, Nicolette C; van der Pol, W-Ludo

    2014-03-15

    IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients. In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation. PMID:24529728

  1. B cell responses to HIV and the development of human monoclonal antibodies.

    PubMed Central

    Boyd, J E; James, K

    1992-01-01

    In this review B cell responses in HIV-infected individuals are summarized together with the techniques used to date to produce human monoclonals to HIV and the properties of these antibodies. Profound disturbances in B cell responses are apparent both in vivo and in vitro. While there is evidence in vivo of marked polyclonal B cell activation, primary and secondary antibody responses are impaired. Similarly these cells exhibit spontaneous immunoglobulin secretion upon in vitro culture but do not readily respond to B cell mitogens and recall antigens including HIV. Furthermore, certain of these defects can be reproduced in normal B cells in vitro by incubation with HIV or HIV coded peptides. Individuals infected with HIV develop antibodies to HIV structural proteins (e.g. p17, p24, gp41 and gp120) and regulatory proteins (e.g. vif, nef, RT). Autoantibodies against a number of immunologically important molecules are also frequently observed. The anti-HIV antibodies are predominantly of the IgG1 isotype and exhibit a variety of effects on the virus in vitro. To date, using conventional immortalization strategies, an appreciable number of human monoclonals to HIV have been developed. These have been specific for gp41, gp120 and gag with antibodies of the former specificity predominating. The majority of these antibodies have been of the IgG1 isotype. Only a small number of the antibodies neutralize virus in vitro and most of these react with gp120. The neutralizing antibodies recognize conformational and carbohydrate epitopes or epitopes in amino acid positions 306-322. The predominant epitopes recognized by the anti-gp41 antibodies were in amino acid positions 579-620 and 644-662. A high percentage (congruent to 25%) of these antibodies enhance viral growth in vitro. The problems relating to the production of human monoclonals to HIV are discussed together with strategies that could be used in the future. PMID:1572084

  2. Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups

    Microsoft Academic Search

    S. Bolin; V. Moennig; N. E. Kelso Gourley; J. Ridpath

    1988-01-01

    Summary Isolates of bovine viral diarrhea (BVD) virus were differentiated by monoclonal antibodies (MoAbs) reactive with the 56kD viral polypeptide. Patterns of neutralizing activity of the MoAbs indicate that multiple epitopes are involved in virus neutralization.

  3. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells

    Microsoft Academic Search

    LUC PARDANAUD; CURTIS ALTMANN; PAUL KITOS; FRANCOISE DIETERLEN-LIEVRE; CLAYTON A. BUCK

    1987-01-01

    Summary QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially

  4. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, Larry H. (Livermore, CA); Vanderlaan, Martin (Danville, CA); Watkins, Bruce E. (Livermore, CA); Van Emon, Jeanette M. (Henderson, NV); Bigbee, Carolyn L. (Livermore, CA)

    1992-01-01

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

  5. Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies.

    PubMed Central

    Andersen, A A

    1991-01-01

    Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars. Images PMID:1848867

  6. Production and characterization of monoclonal antibodies to two new mosquito Aedes aegypti salivary proteins

    Microsoft Academic Search

    Zhikang Peng; Jian Yang; Hongsheng Wang; F. Estelle R Simons

    1999-01-01

    Mosquito salivary proteins, which are fundamental to the process of blood feeding, also facilitate disease transmission and cause allergic reactions. The identification and characterisation of these proteins have been hampered by the difficulty of obtaining them in purified form. In this report, we describe the production of mouse monoclonal antibodies (mAbs) against mosquito salivary proteins. BALB\\/c mice were immunised with

  7. Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

  8. Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopus japonicus. In this study, four monoclonal antibodies (MAbs) (3C1, 3D9, 2F2, 2A8) against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no ...

  9. Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

    E-print Network

    Paris-Sud XI, Université de

    Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans , Alexander I. Wanderler5 , Marie Paule Kieny9 * 1 WHO Collaborating Centre for Rabies Surveillance for Reference and Research on Rabies, Wistar Institute, Philadelphia, Pennsylvania, United States of America, 4

  10. Synthesis of Haptens and Derivation of Monoclonal Antibodies for Immunoassay of the Phenylurea Herbicide Diuron

    Microsoft Academic Search

    Alexander E. Karu; Marvin H. Goodrow; Douglas J. Schmidt; Bruce D. Hammock; Michael W. Bigelow

    1994-01-01

    Diuron and related phenylurea herbicides and their metabolites are important candidates for sensitive and specific immunodetection. This paper describes a scheme for the synthesis of two different types of phenylurea haptens for immunization and use as detecting conjugates in enzyme immunoassays (EIAs). The haptens were used to develop indirect and direct EIAs and to derive a panel of monoclonal antibodies

  11. Specificity of a Monoclonal Antibody for Alkaline Phosphatase in Escherichia coli and Shigella Species

    Microsoft Academic Search

    P. A. TRINELY; C. MIELCAREK; F. GAVINI; C. CARON; D. IZARD

    The specificity of monoclonal antibody 2E5 for the alkaline phosphatase of Escherichia coli was studied against the alkaline phosphatases of 251 other bacterial strains. The organisms used included members of the six species of the genus Escherichia (E. coli, E. fergusonii, E. hermannii, E. blattae, E. vulneris, E. adecarboxylata), 41 species representing the family Enterobacteriaceae, and, in addition, Pseudomonas aeruginosa,

  12. Production, characterization and reactivity of monoclonal antibodies to porcine reproductive respiratory syndrome virus (PRRSV)

    Microsoft Academic Search

    Trevor W. Drew; Janneke J. M. Meulenberg; Jennifer J. Sands; David J. Paton

    1995-01-01

    This report describes the preparation of six monoclonal antibodies (MAbs) raised against a British isolate of porcine reproductive and respiratory syndrome virus (PRRSV), their characterization in terms of protein specificity and their reactivity with different PRRS viruses from Europe and the USA. Radioimmunoprecipi- tation and Western blotting studies of MAb reactivity with proteins from cell lysates of infected cells and

  13. INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

    EPA Science Inventory

    Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

  14. ANTIGEN DETECTION WITH MONOCLONAL ANTIBODIES FOR THE DIAGNOSIS OF ADENOVIRUS GASTROENTERITIS

    EPA Science Inventory

    The authors have developed a monoclonal antibody-based enzyme immunoassay (EIA) for direct detection of enteric adenoviruses in stool specimens from patients with gastroenteritis. Tests specific for each of the enteric adenoviruses, adenovirus type 40 (Ad40) and type 41 (Ad41) we...

  15. High-dose monoclonal antibodies via the subcutaneous route: challenges and technical solutions, an industry perspective.

    PubMed

    Narasimhan, Chakravarthy; Mach, Henryk; Shameem, Mohammed

    2012-07-01

    This review summarizes the various challenges in product development involved in subcutaneous administration of high-dose monoclonal antibodies and attempts to provide an industry perspective of some of the available technologies and potential avenues to overcome these challenges. PMID:22900469

  16. CT-SPECT FUSION TO CORRELATE RADIOLABELED MONOCLONAL-ANTIBODY UPTAKE WITH ABDOMINAL CT FINDINGS

    Microsoft Academic Search

    E. L. Kramer; M. E. Noz; J. J. Sanger; A. J. Megibow; G Q Maguire Jr.

    1989-01-01

    To enhance the information provided by computed tomography (CT) and single photon emission computed tomography (SPECT) performed with radiolabeled, anti-carcinoembryonic antigen monoclonal antibody (MoAb), the authors performed fusion of these types of images from eight subjects with suspected colorectal adenocarcinoma. Section thickness and pixel size of the two studies were matched, coordinates of corresponding points from each study were identified,

  17. Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections.

    PubMed

    Ying, Tianlei; Li, Haoyang; Lu, Lu; Dimitrov, Dimiter S; Jiang, Shibo

    2015-02-01

    The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections. PMID:25456101

  18. Brugia pahangi: production of a monoclonal antibody reactive with the surface of infective larvae.

    PubMed

    Oikawa, Y; Ikeda, T; Horii, Y; Fujita, K; Tsukidate, S

    1992-08-01

    Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection. PMID:1639160

  19. HIV monoclonal antibodies: a new opportunity to further reduce mother-to-child HIV transmission.

    PubMed

    Voronin, Yegor; Mofenson, Lynne M; Cunningham, Coleen K; Fowler, Mary G; Kaleebu, Pontiano; McFarland, Elizabeth J; Safrit, Jeffrey T; Graham, Barney S; Snow, William

    2014-04-01

    Yegor Voronin and colleagues explore how monoclonal antibodies against HIV could provide a new opportunity to further reduce mother-to-child transmission of HIV and propose that new interventions should consider issues related to implementation, feasibility, and access. Please see later in the article for the Editors' Summary. PMID:24714363

  20. Production and characterization of monoclonal antibodies against different epitopes of Ebola virus antigens

    Microsoft Academic Search

    Soraya Shahhosseini; Dipankar Das; Xiangguo Qiu; Heinz Feldmann; Steven M. Jones; Mavanur R Suresh

    2007-01-01

    Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP(1) subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral

  1. Protective Efficacy of Neutralizing Monoclonal Antibodies in a Nonhuman Primate Model of Ebola Hemorrhagic Fever

    Microsoft Academic Search

    Andrea Marzi; Reiko Yoshida; Hiroko Miyamoto; Mari Ishijima; Yasuhiko Suzuki; Megumi Higuchi; Yukie Matsuyama; Manabu Igarashi; Eri Nakayama; Makoto Kuroda; Masayuki Saijo; Friederike Feldmann; Douglas Brining; Heinz Feldmann; Ayato Takada

    2012-01-01

    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed

  2. Development, characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

    Microsoft Academic Search

    Andreas Lucht; Roland Grunow; Peggy Möller; Heinz Feldmann; Stephan Becker

    2003-01-01

    Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the

  3. Ebola GP-Specific Monoclonal Antibodies Protect Mice and Guinea Pigs from Lethal Ebola Virus Infection

    Microsoft Academic Search

    Xiangguo Qiu; Lisa Fernando; P. Leno Melito; Jonathan Audet; Heinz Feldmann; Gary Kobinger; Judie B. Alimonti; Steven M. Jones

    2012-01-01

    Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as

  4. Anti-neurofilament monoclonal antibodies: Reagents for the evaluation of human neoplasms

    Microsoft Academic Search

    J. Q. Trojanowski; V. M.-Y. Lee

    1983-01-01

    Sixty human nervous system neoplasms were examined by immunohistochemistry using a monoclonal antibody against neurofilament triplet proteins. Only those of neuronal origin had tumor cells with intracytoplasmic, immunoreactive neurofilament triplet proteins. However, not all such neoplasms contained labeled tumor cells. Benign or differentiated neuronal tumors more often contained labeled cells than malignant, less differentiated neoplasms of the neuron series. We

  5. Characterization of anti-channel catfish MHC class II monoclonal antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of appro...

  6. Preclinical safety testing of monoclonal antibodies: the significance of species relevance

    Microsoft Academic Search

    Nick Pullen; Mark Graham; Ian Ragan; Kathryn Chapman

    2007-01-01

    Selecting a pharmacologically relevant animal species for testing the safety and toxicity of novel monoclonal antibody (mAb) therapies to support clinical testing can be challenging. Frequently, the species of choice is the primate. With the increased number of mAbs in the pharmaceutical pipeline, this has significant implications for primate use, and so raises several important scientific, ethical and economic issues.

  7. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

    1992-04-28

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

  8. Monoclonal Antibodies in the Lymphatics: Toward the Diagnosis and Therapy of Tumor Metastases

    NASA Astrophysics Data System (ADS)

    Weinstein, John N.; Parker, Robert J.; Keenan, Andrew M.; Dower, Steven K.; Morse, Herbert C.; Sieber, Susan M.

    1982-12-01

    Monoclonal antibodies subcutaneously injected into mice track to regional lymph nodes and specifically label target cells there. The lymphatic route of administration can be expected to provide much higher sensitivity, higher target-to-background ratio, faster localization, and lower toxicity than the intravenous route when the aim is to diagnose or treat tumor metastases or lymphoma in the lymph nodes.

  9. High prevalence of myocardial monoclonal antimyosin antibody uptake in patients with chronic idiopathic dilated cardiomyopathy

    Microsoft Academic Search

    Damià Obrador; Manel Ballester; Ignasi Carrio; Lluis Berna; Guillem Pons-Llado

    1989-01-01

    Monoclonal antimyosin antibody studies were undertaken to assess the presence of myocardial uptake in patients with chronic idiopathic dilated cardiomyopathy. Three groups were studied: 17 patients with chronic (greater than 12 months) idiopathic dilated cardiomyopathy, 12 patients with a large, poorly contracting left ventricle not due to dilated cardiomyopathy (control patients) and 8 normal individuals. The patients in the cardiomyopathy

  10. Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge. (Reannouncement with new availability information)

    SciTech Connect

    Lemley, P.V.; Wright, D.C.

    1992-12-31

    Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approx. 0.5 micrograms/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricin B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titers were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.

  11. Development and Characterization of Monoclonal Antibodies to Detect Klotho

    PubMed Central

    Maltare, Astha; Nietz, Angela K.; Laszczyk, Ann M.; Dunn, Taylor S.; Ballestas, Mary E.; Accavitti-Loper, Mary Ann

    2014-01-01

    Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates. PMID:25513981

  12. Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

    PubMed Central

    Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

    2014-01-01

    The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

  13. Idiotypic analysis of monoclonal antibodies to poly(Glu60Ala30Tyr10).

    PubMed Central

    Ju, S T; Pierres, M; Waltenbaugh, C; Germain, R N; Benacerraf, B; Dorf, M E

    1979-01-01

    Fifteen hybridoma anti-poly(Glu60Ala30Tyr10) (anti-GAT) antibodies were analyzed for the presence of a common set of idiotypic specificities associated with murine anti-GAT antibodies, termed CGAT idiotype, which are present on the anti-GAT antibodies of all mouse strains. Thirteen of these monoclonal anti-GAT antibodies expressed a major fraction of CGAT idiotypic specificities. However, the remaining fraction of CGAT idiotypic specificities were not detected in individual or pooled hybridoma anti-GAT antibodies. Anti-idiotypic antisera made against each of the 15 hybridoma anti-GAT antibodies preferentially bound homologous ligand and showed minimal binding activity to specifically purified serum anti-GAT antibodies. Furthermore, the diversity of the hybridoma anti-GAT antibodies was demonstrated by the presence of individual idiotypic specificities on each of the hybridoma anti-GAT antibodies. However, relatedness among some of the hybridoma antibodies was also apparent since idiotypic analysis revealed that some hybridoma anti-GAT antibodies shared cross-reactive idiotypic specificities not associated with CGAT idiotype. The genetic mechanisms which could account for the generation of such antibody diversity are discussed. PMID:288079

  14. Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies.

    PubMed

    Weber, Ekkehard; Barbulescu, Elena; Medek, Rita; Reinheckel, Thomas; Sameni, Mansoureh; Anbalagan, Arulselvi; Moin, Kamiar; Sloane, Bonnie F

    2015-03-01

    Abstract Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B. PMID:25205719

  15. [Monoclonal antitumor antibodies--its application to diagnosis and treatment of tumors].

    PubMed

    Taniguchi, M; Wakabayashi, S

    1983-05-01

    The two types of monoclonal anti-melanoma antibodies (M562 and M2590) were obtained by the fusion of P3U1 myeloma cells and spleen cells of C57BL/6 mice hyperimmunized with mitomycin C-treated syngeneic B16 melanoma cells. The M2590 antibody recognized the cross-species melanoma determinant commonly shared among at least mouse, hamster and human melanoma cells, while the M562 antibody reacted with the mouse (B16) specific melanoma antigenic determinant. The M2590 antibody reacted specifically in radioimmunoassay with NP40 tumor cell extracts obtained from melanoma patients but not from other tumor patients. Thus this antibody would provide a powerful tool for immunodiagnostic evaluation of patients with malignant melanoma. Moreover, these antibodies were found to suppress the growth of melanoma cells in vivo. PMID:6876435

  16. Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies

    Microsoft Academic Search

    Gregory Lee; Bixia Ge

    2010-01-01

    RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed\\u000a immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab\\u000a and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and\\u000a the corresponding rat anti-id Mabs were generated and characterized. Following

  17. CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation.

    PubMed

    Grainger, Rhian K; James, David C

    2013-11-01

    Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that ?1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²? alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface ?1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is possible. Moreover, real-time, or near real-time control can be enabled by facile, rapid measurement of cell surface biomarkers of cellular biosynthetic capability. PMID:23737295

  18. Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

    PubMed Central

    Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

    2013-01-01

    Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

  19. Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells.

    PubMed

    Lin, J J

    1981-04-01

    Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components. PMID:7017730

  20. Human monoclonal antibodies against a recombinant HIV envelope antigen produced by primary in vitro immunization. Characterization and epitope mapping.

    PubMed Central

    Ohlin, M; Broliden, P A; Danielsson, L; Wahren, B; Rosen, J; Jondal, M; Borrebaeck, C A

    1989-01-01

    Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of mu isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 micrograms x (24 hr x 10(6) cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632-646, 677-681 and 687-691. This specificity is very rarely found in immune sera from seropositive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced. PMID:2480328

  1. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, Martin (Danville, CA); Watkins, Bruce E. (Livermore, CA); Stanker, Larry H. (Livermore, CA)

    1991-01-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

  2. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

    1991-10-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

  3. Development of AAV serotype-specific ELISAs using novel monoclonal antibodies.

    PubMed

    Kuck, Dirk; Kern, Andrea; Kleinschmidt, Jürgen A

    2007-03-01

    Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy. More recently, due to the advantages they offer for gene transfer, several AAV serotypes have gained increasing interest. However, monoclonal antibodies for the characterization and quantitation of vectors derived from different serotypes are at present not available. Serotype-specific monoclonal antibodies (mAbs) against the capsids of the serotypes 1/6, 4 and 5 are described. These antibodies, designated as ADK1a and b, ADK4 or ADK5a and b, reacted specifically with the indicated serotype capsids in cell lysates. ADK 1a and b cross-reacted with its highly related AAV6 serotype, but not with the other serotypes tested. The new antibodies recognized exclusively assembled capsids and neither free nor denatured capsid proteins as shown by fractionation experiments. In immunofluorescence experiments, the mAbs stained only distinct intranuclear foci in cells expressing the capsid protein. The development of capture ELISAs for quantitation of AAV1 and 6, AAV4 or AAV5 capsids illustrates that these novel monoclonal antibodies provide valuable tools for characterization of vector stocks. PMID:17126418

  4. Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A.

    PubMed

    Kuhn, H M; Brade, L; Appelmelk, B J; Kusumoto, S; Rietschel, E T; Brade, H

    1992-06-01

    A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes. PMID:1375194

  5. Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

    PubMed Central

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

    2014-01-01

    CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

  6. Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

    PubMed

    Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

    2013-05-01

    B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. PMID:23084371

  7. Radiolabeled anti platelet monoclonal antibody for imaging in-vivo thrombi

    SciTech Connect

    Srivastava, S.C.; Coller, B.S.; Meinken, G.E.

    1993-07-06

    A radiolabeled monoclonal antibody radiolabeled with a halogen or metallic radionuclide selected from the group consisting of [sup 123]I, [sup 125]I, [sup 131]I, [sup 111]In-DTPA, [sup 99]mTc and [sup 97]Ru, wherein the monoclonal antibody is of the class IgG, produced by a hybridoma formed by fusion of cells from a BALB/C mouse myeloma line, X63.Ag8.653 and spleen cells from a BALB/c mouse previously immunized with human blood platelets, which antibody: (a) reacts readily with normal human platelets and with dog platelets; (b) fails to react with thrombasthenic human platelets or human platelets whose GPIIb/IIIa complex is dissociated with EDTA and (c) reacts slowly with inactivated platelets and substantially twice as rapidly with ADP activated platelets; and (d) completely blocks the interaction of fibrinogen with platelets induced by ADP.

  8. Radioimmunotherapy of transplanted small cell lung cancer with 131I-labelled monoclonal antibody.

    PubMed

    Yoneda, S; Fujisawa, M; Watanabe, J; Okabe, T; Takaku, F; Homma, T; Yoshida, K

    1988-09-01

    Monoclonal antibody TFS-4 has previously been shown to react selectively with human small cell lung cancer (SCLC). We evaluated the use of 131I-labelled TFS-4 for the treatment of established human SCLC transplanted in nude mice. The specific accumulation of the antibody in the transplanted tumour was recorded by both scintigraphic and biodistribution studies. Administration of 200 microCi 131I-labelled TFS-4 inhibited tumour growth when compared with the same radiation dose of the control monoclonal antibody. The therapeutic effect was dose-dependent and complete disappearance of the tumour was observed transiently in one out of the three animals following the administration of 500 microCi 131I-labelled TFS-4. PMID:2846023

  9. Purification of bovine thyroid-stimulating hormone by a monoclonal antibody

    SciTech Connect

    Lock, A.J.; van Denderen, J.; Aarden, L.A.

    1988-01-01

    A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by (/sup 3/H)thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.

  10. Photoregulation process of sorghum leaf phosphoenolpyruvate carboxylase: study with monoclonal antibodies

    SciTech Connect

    Thomas, M.; Cretin, C.; Keryer, E.; Vidal, J.; Gadal, P.; Bidart, J.M.; Buhuon, C.

    1987-02-27

    Monoclonal antibodies were produced against the G isozyme subunit of PEP carboxylase (PEPC) from Sorghum leaves by the hybridoma technique. More than 400 antibodies-producing hybridomas to PEPC were produced from the fusion of spleen cells from immunized mice with NS1 myeloma cells. By using an ELISA, three hybridomas (91-G, 83-G, 49-EG) were selected. Monoclonal antibodies were subsequently characterized in a Western experiment; Mabs 83-G and 91-G were found to be highly specific to the G isozyme whereas Mab 49-EG recognized both forms (E and G isozymes) of the enzyme. Addition of Mabs to the enzyme preparation did not modify its catalytic activity nor its activation by glycine. Use of these probes provided direct and definite evidence of the specific enhancing effect of light on the G form and on its corresponding mRNA.

  11. Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies.

    PubMed

    Mould, Diane R; Dubinsky, Marla C

    2015-03-01

    Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. PMID:25707964

  12. A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.

    PubMed

    Flieger, D; Kufer, P; Beier, I; Sauerbruch, T; Schmidt-Wolf, I G

    2000-10-01

    Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. PMID:11043851

  13. Production and Use of Monoclonal Antibodies for Identification of Strains of Rhizobium trifolii

    PubMed Central

    Wright, Sara F.; Foster, Joyce G.; Bennett, Orus L.

    1986-01-01

    We produced a monoclonal antibody against Rhizobium trifolii 162×95. This antibody in cell culture supernatant was used in an indirect enzyme-linked immunosorbent assay to differentiate strain 162×95 from naturalized strains in the Appalachian region. Nodules crushed in 0.1 to 0.2 ml of phosphate-buffered saline and used to charge enzyme-linked immunosorbent assay plates gave strong absorbance readings. Heat-inactivated and noninactivated portions of 162×95 cultures were strongly reactive, indicating that the antigen is probably a carbohydrate. Of 10 strains from California, where 162×95 was isolated, 6 strongly cross-reacted with the antibody. The cellular protein patterns in a sodium dodecyl sulfate-polyacrylamide gradient gel of cross-reactive strains were essentially identical. A Western blot analysis indicated that the antibody was against a 19.8-kilodalton band. The Western blot analysis also revealed that the polyvalent antiserum contained other strongly reacting antibodies with molecular weights of approximately 20,000, indicating the possibility that other monoclonal antibodies to detect strain 162×95 may be produced. However, the available antibody has been shown to be useful for short-term experiments. Based upon protein profiles and immunological reactions, there are 4 or 5 California strains rather than 10. Images PMID:16347098

  14. Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance

    NASA Astrophysics Data System (ADS)

    Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.

    1989-10-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

  15. Biokinetics of radiolabeled monoclonal antibodies in heterotransplanted nude rats: Evaluation of corrected specific tissue uptake

    SciTech Connect

    Ingvar, C.; Norrgren, K.; Strand, S.E.; Brodin, T.; Joensson, P.E.S.; Sjoegren, H.O. (Lund Univ. (Sweden))

    1989-07-01

    A tumor model is presented to study the biokinetics and localization of radiolabeled monoclonal antibodies (MAb) in the nude rat (Rowett RNu/RNu) heterotransplanted with human melanoma metastases. The nude rat is larger, less sensitive, and lives longer than the nude mouse. It is, therefore, well suited for in vivo studies of tumor localization with radiolabeled monoclonal antibodies. The tumor-to-host weight ratio was closer to the human situation for the nude rat than for the mouse, and quantitative imaging could be performed with a parallel hole collimator. We followed the antibody biokinetics for as long as 8 days, with repeated blood sampling and imaging. Specific uptake of MAb was higher in tumor tissue than in all other tissues except blood. Initial high uptake was also recorded in the bone marrow. The lymph glands showed a slow uptake of specific and control antibody. A simple in vitro correction procedure is described to calculate the corrected specific tissue uptake (STUcorr) that takes the blood activity into account. Thus it was shown that 80% of the tissue uptake in the dissected liver at 30 hr was due to labeled antibodies circulating in the blood. The specific tissue uptake ratio of antibodies 96.5 and OKT3 (nonspecific control) was unity for all other organs except for tumor tissue, where the ratio was greater than two and even higher when correction for blood content of labeled antibody was made.

  16. Cell surface antigens of human melanoma identified by monoclonal antibody.

    PubMed Central

    Yeh, M Y; Hellström, I; Brown, J P; Warner, G A; Hansen, J A; Hellström, K E

    1979-01-01

    Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10 lymphoblastoid cell lines and peripheral blood lymphocytes from 68 normal donors and 12 chronic lymphocytic leukemia patients. This suggests that we have identified one or more determinants of a melanoma-associated antigen(s), whose expression is limited to a small proportion of melanomas. PMID:288077

  17. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

    SciTech Connect

    Eisenberg, R.J. (Univ. of Pennsylvania, Philadelphia, PA); 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.

  18. A technetium-labeled monoclonal antibody for imaging metastatic melanoma

    SciTech Connect

    Frytak, S.; Creagan, E.T.; Brown, M.L.; Salk, D.; Nelp, W. (Mayo Clinic, Rochester, MN (USA))

    1991-04-01

    Twenty patients with histologically proven metastatic melanoma were scanned with a 99mtechnetium ({sup 99}mTc)-labeled melanoma antibody to determine the detection rate of known malignant lesions and to evaluate the antibody's ability to discover occult metastases. Isotope localization in different organs was as follows: liver 100%, bone 100%, subcutaneous lesions 80%, lymph nodes 54%, and lung 33%. Four unsuspected bone lesions and 16 occult subcutaneous lesions were found. False positive lesions were noted in two instances--one benign thyroid adenoma, and one arthritic bone lesion. One patient developed an atypical serum sickness reaction with a rash and arthralgias that responded rapidly to treatment. The {sup 99}mTc antimelanoma antibody is a safe and effective method to detect metastatic melanoma. It has potential use for screening newly diagnosed melanomas that carry an increased risk of recurrence.

  19. Reagent Target Request for Monoclonal Antibody Production and Characterization

    Cancer.gov

    Requests will be reviewed and considered for merit based on their justification and contribution to existing NCI-funded projects. Priority will be given to projects applying the antibodies to proteomic research. Requests should be submitted electronically by completing the accompanying form.

  20. Monoclonal antibodies against 24?kDa surface antigen of hepatitis B viruses.

    PubMed

    Abolhassani, Mohsen; Nejad-Moghaddam, Amir; Modaresi, Mohammad Hossein

    2013-04-01

    Hepatitis B virus (HBV) infection is one of the major public health problems worldwide. Effective control of HBV transmission in areas of high and intermediate endemicity would not be possible without vaccination of the vulnerable group. The diagnosis of acute and chronic hepatitis B infection is based on the detection of hepatitis B surface antigen (HBsAg). In this study, we prepared nine hybridomas that produced monoclonal antibodies specific for HBsAg. BALB/c mice were immunized with 24?kDa HBsAg and the immune spleen cells were fused with SP2/0 myeloma cell line. We obtained seven IgG1, one IgM, and one IgG2b positive clones. The stable hybrids were sub-cloned and ascitic fluid was prepared in the BALB/c mice. After antibody purification by protein G, the affinity column was prepared and the pure 24?kDa HBsAg from cell extract was eluted from the column. Western blot analysis showed that all monoclonal antibodies are specific for 24?kDa antigen. Since 24?kDa HBsAg is used for vaccination against hepatitis, these monoclonal antibodies are the best candidate for the isolation and purification of recombinant HBsAg from yeast expression vector by affinity column to be used for vaccination. PMID:23607349

  1. SPECT assay of radiolabeled monoclonal antibodies. Third yearly progress report, September 1991--February 1992

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  2. Monoclonal Antibody Therapy Directed Against Human Acute Myeloid Leukemia Stem Cells

    PubMed Central

    Majeti, Ravindra

    2015-01-01

    Accumulating evidence indicates that many human cancers are organized as a cellular hierarchy initiated and maintained by self-renewing cancer stem cells. This cancer stem cell model has been most conclusively established for human acute myeloid leukemia (AML), although controversies still exist regarding the identity of human AML stem cells (LSC). A major implication of this model is that in order to eradicate the cancer and cure the patient, the cancer stem cells must be eliminated. Monoclonal antibodies have emerged as effective targeted therapies for the treatment of a number of human malignancies, and given their target antigen specificity and generally minimal toxicity, are well positioned as cancer stem cell-targeting therapies. One strategy for the development of monoclonal antibodies targeting human AML stem cells, involves first identifying cell surface antigens preferentially expressed on AML LSC compared to normal HSC. In recent years, a number of such antigens have been identified including CD123, CD44, CLL-1, CD96, CD47, CD32 and CD25. Moreover, monoclonal antibodies targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML. PMID:21076471

  3. Specific biodetection of B16 mouse melanoma in vivo by syngeneic monoclonal antibody.

    PubMed

    Yamasaki, T; Wakabayashi, S; Inoue, O; Ando, K; Kusakabe, K; Kawasaki, Y; Okamoto, S; Taniguchi, M

    1987-09-01

    The specific detection of tumors in vivo using a radiolabeled syngeneic monoclonal antibody made by fusion of P3U1 (BALB/c myeloma cells) and C57BL/6 spleen cells primed with syngeneic B16 melanoma cells was investigated by color imaging, autoradiography, and biodistribution. The radiolabeled antimelanoma antibody specifically accumulated only in the tumor lesions, whereas no radioactivity was observed in normal tissues or organs. The distribution patterns of the radioactive antibody in the tumor lesions depended on the sizes of the tumor. Almost the entire region of the small metastatic tumor in lymph nodes was labeled, whereas the radioactive antibody was irregularly localized mainly in the center of the medium-sized tumor. However, only the peripheral region of the large primary tumor was labeled. The highest uptake of radioactivity (tumor:blood ratio) was observed in the small lymph node metastatic tumor lesions rather than in the large primary tumor. Furthermore, high resolution color imaging of B16 melanoma was also obtained by using 125I-labeled monoclonal antibody. Tumor location was specifically visible without subtraction or enhancement methods 3-5 days after injection of the radiolabeled antibody. PMID:3624896

  4. 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

  5. Molecular characterization of monoclonal antibodies against aflatoxins: a possible explanation for the highest sensitivity.

    PubMed

    Li, Xin; Li, Peiwu; Zhang, Qi; Li, Yuanyuan; Zhang, Wen; Ding, Xiaoxia

    2012-06-19

    We screened and established seven hybridoma cell lines that secrete anti-aflatoxin monoclonal antibodies with different sensitivities. Among these antibodies, 1C11 exhibited the highest sensitivity against all four major kinds of aflatoxins (AFB1, AFB2, AFG1, and AFG2) (IC(50) 0.0012-0.018 ng mL(-1) in the enzyme linked immunosorbent assay (ELISA) system, visual limit of detection of 0.03-0.25 ng mL(-1)). To better understand the interactions between these antibodies and aflatoxins, as well as to guide their potential sensitivity improvement in recombinant antibodies, we used multiple sequence alignment and molecular modeling combined with molecular docking to clarify the molecular mechanism of the highest sensitivity of 1C11 against aflatoxins. Our results show that hydrogen bond and hydrophobic interaction formed by Ser-H49 and Phe-H103 in the antibody with the hapten played the most important roles in determining the binding affinity. Further experiments performed on antibody mutants, designed on the basis of the computational models, supported the prediction of the interaction mode between the antibody and the hapten. Although the factors that influence antibody sensitivity are highly interdependent, our experimental and modeling studies clearly demonstrate how structural differences influence the binding properties of antibodies against the target hapten with different sensitivities. PMID:22548609

  6. Lyophilized Silk Fibroin Hydrogels for the Sustained Local Delivery of Therapeutic Monoclonal Antibodies

    PubMed Central

    Guziewicz, Nicholas; Best, Annie; Perez-Ramirez, Bernardo; Kaplan, David L.

    2011-01-01

    The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering ?-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications. PMID:21216004

  7. Optimization of monoclonal antibody delivery via the lymphatics: the dose dependence

    SciTech Connect

    Steller, M.A.; Parker, R.J.; Covell, D.G.; Holton, O.D. 3d.; Keenan, A.M.; Sieber, S.M.; Weinstein, J.N.

    1986-04-01

    After interstitial injection in mice, antibody molecules enter local lymphatic vessels, flow with the lymph to regional lymph nodes, and bind to target antigens there. Compared with i.v. administration, delivery via the lymphatics provides a more efficient means for localizing antibody in lymph nodes. An IgG2a (36-7-5) directed against the murine class I major histocompatibility antigen H-2Kk has proved useful for studying the pharmacology of lymphatic delivery. At very low doses, most of the antibody remains at the injection site in Kk-positive animals. As the dose is progressively increased, most effective labeling occurs first in nodes proximal to the injection site and then in the next group of nodes along the lymphatic chain. At higher doses, antibody overflows the lymphatic system and enters the blood-stream via the thoracic duct and other lymphatic-venous connections. Once in the blood, antibody is rapidly cleared, apparently by binding to Kk-bearing cells. These findings indicate that the single-pass distribution of monoclonal antibodies in the lymphatics can be strongly dose dependent, a principle which may be of clinical significance in the improvement of immunolymphoscintigraphic imaging, especially with antibodies directed against normal and malignant lymphoid cells. Monoclonal antibodies directed against normal cell types in the lymph node may be useful for assessing the integrity of lymphatic chains by immunolymphoscintigraphy or, more speculatively, for altering the status of regional immune function. The results presented here indicate that a low or intermediate antibody dose may optimize the signal:noise ratio for imaging. In Kk-negative animals, the percentage of dose taken up in the major organs was essentially independent of the dose administered; there was no evidence for saturable sites of nonspecific binding.

  8. Monoclonal antibodies to Gliocladium roseum, a potential biological control fungus of sap-staining fungi in wood

    Microsoft Academic Search

    COLETTE BREUIL; BRIAN T. LUCK; L. Rossignol; JUDY LITTLE; CHRISTOPHE J. ECHEVERRI; SRABANI BANERJEE; DAVID L. BROWN

    1992-01-01

    Immunological probes were developed to discriminate between a potential biological control fungus and sap- staining fungi present in wood. This paper describes the production of monoclonal antibodies to isolated cell wall fragments of the biological control fungus Gliocladium roseum. Two monoclonals, designated 6A5 and 3F12, were characterized. Their specificity was assessed by ELISA, by immunogold silver staining light microscopy, by

  9. COORDINATION OF PHYTOCHROME LEVELS IN PHYB MUTANTS OF ARABIDOPSIS AS REVEALED BY APOPROTEIN-SPECIFIC MONOCLONAL ANTIBODIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accumulating evidence indicates that individual members of the hytochrome family of photoreceptors have differential but interactive roles in controlling plant responses to light. To investigate possible crossregulation of these receptors, we have identified monoclonal antibodies that specifically d...

  10. Evaluating delivery of a monoclonal antibody using a linear Lorentz-force actuated needle-free injector

    E-print Network

    Jin, Tiffany

    2011-01-01

    The medical application of injection of monoclonal antibodies using a controllable auto-loading needle-free jet injector has been evaluated for two potentially limiting factors: viscosity of the formulation and shearing ...

  11. Method for differential diagnosis of T cell leukemias using monoclonal antibodies

    SciTech Connect

    Knowles, R.W.; Dupont, B.; Naito, K.; Morishima, Y.

    1987-02-24

    This patent describes monoclonal antibody 3-3 (HB8369) and 3-40 (HB8368) not capable of immunological reaction with normal, human peripheral T or B blood cell antigens, normal human thymocyte antigens or normal, human bone marrow precursor cell antigens but capable of immunological reaction with separate and distinct T-ALL leukemia antigens (T-ALL) having molecular weights of approximately 35-40,000 KD. The monoclonal antibodies are capable of distinguishing T-ALL leukemia from cutaneous T-cell lymphoma (CTCL), adult T cell leukemia (ATL) and T-cell chronic lymphocytic leukemia (T-CLL) and are further capable of subsetting T-ALL leukemia into E-Rosette positive and E-Rosette negative cells.

  12. Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.

    PubMed Central

    Smith, J S; Reid-Sanden, F L; Roumillat, L F; Trimarchi, C; Clark, K; Baer, G M; Winkler, W G

    1986-01-01

    Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species. PMID:2429983

  13. Production and characterization of monoclonal antibodies against the recombinant nucleoprotein of Araucaria hantavirus.

    PubMed

    Mazzarotto, Giovanny A C A; Raboni, Sonia M; Stella, Vanessa; Carstensen, Suzana; de Noronha, Lucia; Levis, Silvana; Zanluca, Camila; Zanetti, Carlos R; Bordignon, Juliano; Duarte dos Santos, Claudia N

    2009-12-01

    Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). From the first detection of infection in Brazil in 1993 until 2009, 1161 cases of HPS have been reported, with mortality rates of around approximately 40%. Currently, due to the absence of a vaccine or specific antiviral therapy, the only way to reduce mortality by hantavirus infection is a fast and precise diagnosis that allows for supportive clinical health care. To improve the detection of hantavirus infection, we developed monoclonal antibodies (Mabs) against the nucleoprotein (rNDelta85) of the Araucaria hantavirus strain (ARAUV). The specificity of generated Mabs for rNDelta85 was demonstrated by western blot, indirect immunofluorescence and immunohistochemistry. These are the first monoclonal antibodies to be produced and characterized against the South American hantavirus strain, and may be of special interest in the development of diagnostic assays and epidemiological studies. PMID:19654026

  14. Legionella pneumophila serogroup 1 subgrouping by monoclonal antibodies--an epidemiological tool.

    PubMed Central

    Watkins, I. D.; Tobin, J. O.; Dennis, P. J.; Brown, W.; Newnham, R.; Kurtz, J. B.

    1985-01-01

    A panel of 10 monoclonal antibodies was used to subgroup 326 strains of Legionella pneumophila serogroup 1. All but two strains could be classified into three major subgroups named after their representative strains Pontiac 1, Olda and Bellingham 1. Of the 50 isolates from patients, 44 representing 32 separate incidents were of the Pontiac subgroup. This subgroup was also found in 16 of 18 buildings epidemiologically associated with Legionnaires' Disease. In contrast, strains of the Olda subgroup predominated in buildings where no infections had occurred. In 9 of the 11 incidents where isolates were available from at least one patient as well as from the suspected environmental source, the monoclonal antibody reaction patterns of strains from patients were identical to those of one or more of their environmental counterparts. PMID:3905954

  15. Monoclonal antibody against bacterial lipopolysaccharide cross-reacts with DNA-histone.

    PubMed

    Sumazaki, R; Fujita, T; Kabashima, T; Nishikaku, F; Koyama, A; Shibasaki, M; Takita, H

    1986-10-01

    Monoclonal antibodies to bacterial lipopolysaccharide (LPS) were prepared by fusing spleen cells from BALB/c mice immunized with Salmonella Minnesota Re 595 LPS to the mouse myeloma cell line P3U1. One of them, designated RS01, revealed a strong positive antinuclear activity and reacted with DNA-histone. RS01 also bound specifically to Salmonella Minnesota Re 595 LPS and eliminated the biological activity of LPS. The Salmonella completely inhibited the ANA activity of RS01 and DNA-histone blocked the reactivity of RS01 with LPS. Thus, it is clear that an anti-LPS monoclonal antibody, RS01 cross-reacts with DNA-histone. PMID:3542315

  16. Application of a monoclonal antibody to a comparative study of alpha-lactalbumins from various species

    SciTech Connect

    Kaminogawa, S.; Shimoda, M.; Kurisaki, J.; Yamauchi, K.

    1989-05-01

    A monoclonal antibody to bovine alpha-lactalbumin was prepared and purified. The binding ability of alpha-lactalbumin from different species (cow, goat, giraffe, horse, pig, human, monkey, and guinea pig) was examined by a competitive radioimmunoassay. The order in strength of the binding affinity was cow goat, giraffe, horse, cynomolgus monkey and human, pig, and guinea pig. The order of evolutional divergence calculated from the amino acid composition was cow, goat, giraffe, horse, pig, guinea pig and human, and monkey. The orders in both cases were similar. Hence, it is suggested that immunological divergence as deduced by a monoclonal antibody is likely to be close to the evolutional divergence of alpha-lactalbumin.

  17. Monoclonal antibodies against discoidin I and discoidin II of the cellular slime mold, Dictyostelium discoideum.

    PubMed

    Fukuzawa, M; Ochiai, H

    1988-05-01

    The preparation and properties of monoclonal antibodies against carbohydrate-binding proteins (discoidin I and discoidin II) in the cellular slime mold, Dictyostelium discoideum are described. Monoclonal antibody (mAb) ndI,II-1 bound both discoidins I and II specifically. mAb nI-1 and mAb dI-1 bound only discoidin I but their binding specificities were different: nI-1 recognized the native form and dI-1 the denatured form. mAb dII-1 bound only denatured discoidin II. In preliminary work mAbs dII-1 and nI-1 were found to be useful for localizing discoidins I and II immunohistochemically. PMID:2460438

  18. Anti-idiotypic monoclonal antibodies reacting with idiotope on isolated-denatured chains of an anti-CD4 monoclonal antibody.

    PubMed Central

    Perosa, F; Ferrone, S; Dammacco, F

    1991-01-01

    Hybridization of a non-secreting mouse myeloma cell line NSO with splenocytes from a BALB/c mouse hyperimmunized with the anti-CD4 monoclonal antibody (mAb) HP2/6 generated the anti-idiotypic (anti-id) mAb F11-2113, F11-2302 and F11-2444, which recognize idiotope(s) (id) that are the same or spatially close to each other and are located outside the antigen-combining site of the immunizing mAb. Binding and inhibition assay showed that id are not expressed either on other mouse anti-CD4 mAb or polyclonal immunoglobulins (Ig). Western blotting analysis showed that the id defined by anti-id mAb F11-2113, F11-2302 and F11-2444 are similarly expressed on separated heavy and light chains of mAb HP2/6 and suggested they are likely to be 'sequence-dependent' since their expression is conserved following SDS and reducing reagents treatment. This finding is unique inasmuch as 'sequence-dependent' id similarly expressed on heavy and light chains have been described only on a mouse monoclonal auto-antibody with immunoregulatory properties. Images Figure 2 PMID:1783432

  19. Monoclonal Antibodies Detect a Spectrin-Like Protein in Normal and Dystrophic Human Skeletal Muscle

    NASA Astrophysics Data System (ADS)

    Appleyard, S. T.; Dunn, M. J.; Dubowitz, V.; Scott, M. L.; Pittman, S. J.; Shotton, D. M.

    1984-02-01

    Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against ? -spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against ? -spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive ? -chain similarities to erythrocyte spectrin but differs significantly with respect to the ? -subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.

  20. Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein.

    PubMed

    Ferns, R B; Tuke, P W; Sweenie, C H

    1996-11-01

    Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160. PMID:8923286

  1. A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies

    Microsoft Academic Search

    Gelin Xu; Patrick Weber; Qiaoling Hu; Honggang Xue; Laurent Audry; Chengping Li; Jie Wu; Herve Bourhy

    2007-01-01

    Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO

  2. Monoclonal Antibody PR92 with Restricted Specificity for Tumor-associated Antigen of Prostate and Breast Carcinoma

    Microsoft Academic Search

    Yung D. Kim; Deborah Y. Robinson; Joseph T. Tornita

    A unique mouse hybridoma, PR92, was obtained using human prostate adenocarcinoma cell line 1)11-15.The monoclonal antibody produced by the PR92 clone was reactive with DU145, MCF-7 (breast adenocarci noma), and CHACO (lung carcinoma), but not with normal cell lines and 16 other cell lines of cancerous origin. A homologous solid-phase sand wich radioimmunoassay (PR92-RIA) was developed utilizing PR92 monoclonal antibody.

  3. The effect of polyvinylpyrrolidone (PVP) on the heavy chain monoclonal antibody production from plant suspension cultures

    Microsoft Academic Search

    Walter LaCount; Gynheung An; James M. Lee

    1997-01-01

    A heavy chain monoclonal antibody (HC MAb) was produced by the suspension cultures of genetically modified tobacco cells. Though the HC MAb was secreted to the culture medium by the cells, it was unstable in plant cell media. The addition (0.75 g\\/L) of polyvinylpyrrolidone (MW 360,000) to the medium stabilized the extracellular HC MAb and increased its production 35 fold

  4. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

    Microsoft Academic Search

    Jason D Sterling; Jeffrey T Regan; John R Gasdaska; Karen K Frantz; Charles G Peele; Amelia Black; David Passmore; Cristina Moldovan-Loomis; Mohan Srinivasan; Severino Cuison; Pina M Cardarelli; Lynn F Dickey; Kevin M Cox

    2006-01-01

    N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb

  5. Effect of monoclonal antibody on expression of lipid metabolism related genes in porcine adipocytes

    Microsoft Academic Search

    S. M. Zhao; Q. H. Wan; M. L. Cheng; Y. Huang; W. Z. Li; Y. Y. Zhang; S. Z. Gao

    2009-01-01

    In order to study the mechanism of monoclonal antibody (McAb) against a porcine 40-kDa adipocyte-specific plasma membrane protein in reducing fat deposition, porcine primary adipocytes were treated with the McAb during the process of adipocyte differentiation; its effect on expression of lipid metabolism related genes was investigated. Adipocytes were treated with 1-methyl-3-isobutylmethylxanthine (IDX) plus 10?g\\/mL of the McAb or without

  6. Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes

    Microsoft Academic Search

    Marc Mercken; Marc Vandermeeren; Ursula Liibke; Jan Six; Jef Boons; André Voorde; Jean-Jacques Martin; Jan Gheuens

    1992-01-01

    A modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimer's disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity

  7. Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies

    Microsoft Academic Search

    Norbert Kartner; Deanna Evernden-Porelle; Grace Bradley; Victor Ling

    1985-01-01

    One reason for the failure of chemotherapy in the treatment of advanced cancers may be the outgrowth of multidrug-resistant tumour cells1-5. Multidrug resistance has been modelled in numerous mammalian cell lines in which the phenotype is characterized by a pleiotropic cross-resistance to unrelated drugs1-7. In the study reported here, we have produced monoclonal antibodies whose binding to plasma membranes of

  8. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

    Microsoft Academic Search

    Amie L. Ingold; Stanley A. Cohn; Jonathan M. Scholey

    1988-01-01

    Abstract, We have prepared and characterized seven mouse,monoclonal,antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immuno- blots, SUK 3 and SUK 4 cross-reacted with Drosoph- ila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven

  9. REMISSION INDUCTION IN NON-HODGKIN LYMPHOMA WITH RESHAPED HUMAN MONOCLONAL ANTIBODY CAMPATH-1H

    Microsoft Academic Search

    G. Hale; M. J. S. Dyer; M. R. Clark; J. M. Phillips; R. Marcus; L. Riechmann; G. Winter; H. Waldmann

    1988-01-01

    A genetically rehaped human IgG1 monoclonal antibody (CAMPATH-1H) was used to treat two patients with non-Hodgkin lymphoma. Doses of 1-20 mg daily were given intravenously for up to 43 days. In both patients lymphoma cells were cleared from the blood and bone marrow and splenomegaly resolved. One patient had lymphadenopathy which also resolved. These effects were achieved without myelosuppression, and

  10. Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

    Microsoft Academic Search

    Camila Zanluca; Luan Renato dos Passos Aires; Paula Pazzini Mueller; Vanessa Valgas dos Santos; Maria Luiza Carrieri; Aguinaldo Roberto Pinto; Carlos Roberto Zanetti

    2011-01-01

    Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3?, IgG2a?, IgM?, and an IgG2b? isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs

  11. Sindbis Virus Conformational Changes Induced by a Neutralizing Anti-E1 Monoclonal Antibody

    Microsoft Academic Search

    Raquel Hernandez; Angel Paredes; Dennis T. Brown

    2008-01-01

    A rare Sindbis virus anti-E1 neutralizing monoclonal antibody, Sin-33, was investigated to determine the mechanism of in vitro neutralization. A cryoelectron microscopic reconstruction of Sindbis virus (SVHR) neutral- ized with FAb from Sin-33 (FAb-33) revealed conformational changes on the surface of the virion at a resolution of 24 Å. FAb-33 was found to bind E1 in less than 1:1 molar

  12. Establishment and usefulness of an anti-human CD57 IgG1 monoclonal antibody

    Microsoft Academic Search

    Junko Tajima; Yuji Heike; Kazunori Kato; Yoshinori Ikarashi; Rumiko Asada-Mikami; Mitsuji Yoshida; Tadashi Kasahara; Hiro Wakasugi

    2000-01-01

    In the present study we established a new monoclonal antibody, JNK-1, which recognizes all cells recognized by CD57\\/HNK-1 mAb. JNK-1 and CD57 mAbs inhibited the binding of each other, suggesting that the molecules they recognize are either identical or sufficiently close to cause steric hindrance in the binding assay. JNK-1 mAb detected the 110-kDa protein, which is identical to the

  13. Genetic Immunization: A New Monoclonal Antibody for the Detection of BCL6 Protein in Paraffin Sections

    Microsoft Academic Search

    José-Francisco García; Juan-Fernando García; Lorena Maestre; Elena Lucas-Campeño; Lydia Sánchez-Verde; Silvia Romero-Chala; Miguel-Ángel Piris; Giovanna Roncador

    2006-01-01

    Genetic immunization can be combined with hybridoma technology to generate high-affinity monoclonal antibodies (MAbs). A new anti-BCL-6 MAb (GI191E\\/A8) was produced by cloning full-length BCL-6 cDNA into a eukaryotic vector and delivering this into mouse epidermis using a helium gene gun. A comparative study was made of the specificity and the effects of formalin fixation on immunohistochemistry quality of GI191E\\/A8

  14. Antigenic Sites on the CVS Rabies Virus Glycoprotein: Analysis with Monoclonal Antibodies

    Microsoft Academic Search

    MONIQUE LAFON; TADEUSZ J. WIKTOR; RODERICK I. MACFARLAN

    1983-01-01

    SUMMARY Antigenic variation in the glycoprotein of rabies (CVS-11) virus was studied. Neutralization-resistant variant viruses were isolated in vitro at high frequency (10-* to 10 -5) in the presence of anti-glycoprotein monoclonal antibody. Analysis of these variants identified at least three functionally independent antigenic sites, based on the grouping of variants that were no longer neutralized by one or more

  15. Production and Characterization of Monoclonal Antibodies to Peste des Petits Ruminants (PPR) Virus

    Microsoft Academic Search

    R. P. Singh; S. K. Bandyopadhyay; B. P. Sreenivasa; P. Dhar

    2004-01-01

    Peste des petits ruminants (PPR) is an acute, febrile viral disease of small ruminants, caused by a virus of the genus Morbillivirus. PPR and rinderpest viruses are antigenically related and need to be differentiated serologically. In the present study,\\u000a 23 mouse monoclonal antibodies were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma\\u000a cells. Among these, two belong to

  16. A Monoclonal Antibody against a-Smooth Muscle Actin: A New Probe for Smooth Muscle Differentiation

    Microsoft Academic Search

    Omar Skalli; Patricia Ropraz; Arnold Trzeciak; Gilbert Benzonana; Dieter Gillessen; Giulio Gabbiani

    1986-01-01

    A monoclonal antibody (anti-ctsm-1) recog- nizing exclusively a-smooth muscle actin was selected and characterized after immunization of BALB\\/c mice with the NH2-terminal synthetic decapeptide of a-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-ctsm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized

  17. Studies on tissue distribution of M1 sperm antigen using sperm- specific monoclonal antibody

    Microsoft Academic Search

    Mahanem Mat Noor; Alene Tawang; Harry D. M. Moore

    2006-01-01

    It was reported that M1 monoclonal antibody (M1 mab) inhibited hamster sperm-egg fusion in a dose-dependent manner. The antigen was localized to the equatorial segment (ES) of the hamster sperm, and that after the acrosome reaction, it was exposed on the surface of the sperm plasma membrane overlying the ES. The M1 antigen there- fore fulfilled the main requirements of

  18. Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

    Microsoft Academic Search

    A. Hekman; A. Honselaar; W. M. J. Vuist; J. J. Sein; S. Rodenhuis; W. W. ten Bokkel Huinink; R. Somers; Ph. Rümke; C. J. M. Melief

    1991-01-01

    Summary Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15–30 mg. After the mAb infusions the number of circulating tumour cells was temporarily

  19. Methodological approaches to immunohistochemical demonstration of ? hexosaminidase in human placental and renal tissue with monoclonal antibodies

    Microsoft Academic Search

    Mohamed Elhassan Elsafi; A. Isaksson; B. Hultberg; I. Hägerstrand; U. Stenram

    1990-01-01

    Summary  The lysosomal enzyme,?- hexosaminidase (Hex), was studied in full-term human placentas and in renal tissue using monoclonal antibodies raised against\\u000a Hex purified from human placentas. The immunohistochemical reaction for Hex was pronounced in trophoblastic cells and macrophages\\u000a of the basal plate and the smooth chorion, but was faint or negative in the amnion as well as in the syncytiotrophoblast and

  20. Immunophotosensitizer: the preparation and antitumor properties of monoclonal antibody-hematoporphyrin conjugate

    Microsoft Academic Search

    Shan Bai; Cheng-Gui Liu; Zhong-He Guo

    1993-01-01

    The immunophotosensitizer was prepared by conjugating hematoporphyrin (HP) with anti-CEA and anti-colonic cancer monoclonal antibody (McAb), respectively. In vitro, the anti-CEA McAb-HP conjugate showed cytotoxicity for human colonic cancer cell line SW1116 which was CEA positive, but no cytotoxicity for the CEA-negative Hep-2 cell line. The cytotoxicity of immunophotosensitizer was much higher than the McAb alone, the HP alone, or