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Sample records for monoclonal antibody detects

  1. Monoclonal antibodies that detect live salmonellae.

    PubMed Central

    Torensma, R; Visser, M J; Aarsman, C J; Poppelier, M J; van Beurden, R; Fluit, A C; Verhoef, J

    1992-01-01

    Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor. Images PMID:1476430

  2. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  3. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  4. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    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.

  5. Detection of enterovirus 70 with monoclonal antibodies.

    PubMed

    Anderson, L J; Hatch, M H; Flemister, M R; Marchetti, G E

    1984-09-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70. PMID:6092426

  6. Detection of enterovirus 70 with monoclonal antibodies.

    PubMed Central

    Anderson, L J; Hatch, M H; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70. PMID:6092426

  7. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies

    PubMed Central

    Hutchinson, Alistair P.; Nicklin, Stephen

    2015-01-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  8. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    PubMed

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  9. 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)

  10. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

    Baldwin, R.W.; Byers, V.S. )

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

  11. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    NASA Astrophysics Data System (ADS)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

  12. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  13. 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.

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

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

    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.

  15. Polymorphism of normal factor IX detected by mouse monoclonal antibodies.

    PubMed Central

    Wallmark, A; Ljung, R; Nilsson, I M; Holmberg, L; Hedner, U; Lindvall, M; Sjögren, H O

    1985-01-01

    Hemophilia B is an X-chromosomal recessive disease due to deficiency of coagulation factor IX. Three monoclonal antibodies against factor IX were prepared and used to develop immunoradiometric assays (IRMAs) of factor IX antigen (IX-Ag). IX-Ag was measured in 65 normal individuals with one IRMA based on polyclonal anti-IX antibodies and two IRMAs based on three monoclonal anti-IX antibodies. One of the monoclonal antibodies differed in specificity since it neutralized less than 50% of the clotting activity of factor IX (IX-C), whereas the other two monoclonal antibodies neutralized 80-95%. When the former antibody was used as the solid phase in IRMA, two groups of normal individuals were distinguished: group A with measurable IX-Ag, and group B without demonstrable IX-Ag. There were no differences between the groups either in IX-C or in IX-Ag measured with polyclonal antibodies. A subgroup comprising only women could be distinguished in group A, in whom intermediate IX-Ag concentrations were found. Family studies showed the group B variant of normal factor IX to be transmitted according to the pattern of X-linked recessive inheritance. The allelic frequency of group A was 0.66, and that of group B was 0.34. PMID:3873655

  16. Monoclonal Antibodies for Specific Detection of Encephalitozoon cuniculi

    PubMed Central

    Mo, Lan; Drancourt, Michel

    2004-01-01

    Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia. PMID:15539506

  17. Monoclonal antibodies.

    PubMed

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  18. 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

  19. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    PubMed

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  20. New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, most antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. Methods and Findings Four monoclonal antibodie...

  1. 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.

  2. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    SciTech Connect

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  3. 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.

  4. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

    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. Detection and quantification of circulating antigen in schistosomiasis by a monoclonal antibody. I. Specificity analysis of a monoclonal antibody with immunodiagnostic capacity.

    PubMed Central

    Nogueira-Queiroz, J A; Lutsch, C; Capron, M; Dessaint, J P; Capron, A

    1986-01-01

    Monoclonal antibodies were obtained after immunization of mice with Schistosoma mansoni excretory/secretory antigen, previously shown to contain the circulating cathodic (M) antigen. Among these, the 40:B1 monoclonal antibody proved to be specific for the schistosome genus and to detect only adult worm-derived antigens as shown both by immunoprecipitation and with a two-site immunoradiometric assay using the monoclonal as both the solid-phase and the labelled antibody. The two-site immunoradiometric assay allows a sensitive measurement (detection limit: 5 ng) of circulating schistosome antigen in blood and in urine from patients with schistosomiasis. The amount of circulating schistosome M antigen is correlated with schistosome egg excretion in stool. Images Fig. 2 PMID:3098474

  6. Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

    PubMed Central

    Camargo, Z P; Gesztesi, J L; Saraiva, E C; Taborda, C P; Vicentini, A P; Lopes, J D

    1994-01-01

    Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM. Images PMID:7814469

  7. 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…

  8. 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...

  9. Monoclonal antibody "gold rush".

    PubMed

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush. PMID:17691940

  10. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    PubMed

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh

    2012-12-01

    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

  11. Detection and quantitation of low abundance oligosaccharides in recombinant monoclonal antibodies.

    PubMed

    Ponniah, Gomathinayagam; Nowak, Christine; Gonzalez, Nidia; Miano, Dino; Liu, Hongcheng

    2015-03-01

    Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics. PMID:25647617

  12. In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.

    PubMed

    Iwamoto, Taka-aki; Kobayashi, Nobuhiko; Imoto, Kyoko; Yamamoto, Aya; Nakamura, Yu; Yamauchi, Yukika; Okumura, Hiromi; Tanaka, Akiko; Hanaoka, Fumio; Shibutani, Shinya; Miyagawa, Sachiko; Mori, Toshio

    2004-11-01

    The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells. PMID:15380103

  13. Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

    PubMed Central

    McClelland, R G; Pinder, A C

    1994-01-01

    Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells. Images PMID:7811064

  14. Analysis of monoclonal antibody heterogeneity by post-capillary affinity detection for capillary electrophoresis

    SciTech Connect

    Kelly, J.A.; Lee, Cheng S.

    1997-12-31

    Production of monoclonal antibodies seldom results in a single uniform product. Although the use of hybridomas yields antibodies with a homogeneous amino acid sequence, differences exist in degree of glycosylation. Oligosaccharide variation is known to vary with culture conditions as well as proliferation state. Glycosylation is significant biologically, particularly agalactosyl glycoforms of IgG which can be pathogenic. This suggests a need for rapid analysis of antibody heterogeneity, including glycosylation, during production to optimize quality and yield. Post-capillary affinity detection for capillary electrophoresis is a novel bioanalytical tool which analyzes protein microheterogeneity without interference from complex sample matrices. Mouse monoclonal antibody samples from cell culture media are selectively analyzed by post-capillary affinity detection. Separation of IgG variants is accomplished by capillary zone electrophoresis (CZE) prior to on-line affinity detection with fragment B of Protein A lagged with fluorescein (BF). IgG isoforms are observed while serum proteins and cell culture media are discriminated against.

  15. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

    PubMed Central

    Stanker, Larry H.; Scotcher, Miles C.; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert

    2013-01-01

    Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. PMID:24253240

  16. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

    PubMed Central

    Cheng, Luisa W.; Henderson, Thomas D.; Lam, Tina I.; Stanker, Larry H.

    2015-01-01

    Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  17. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-12-01

    Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  18. Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients.

    PubMed Central

    Chaicumpa, W; Thin-Inta, W; Khusmith, S; Tapchaisri, P; Echeverria, P; Kalambaheti, T; Chongsa-Nguan, M

    1988-01-01

    Monoclonal antibodies were raised against Barber antigen (Ba) of Salmonella typhi 0901. Antibodies produced to antigen 9 of group D salmonellae were used in double- and triple-sandwich antibody enzyme-linked immunosorbent assays (ELISAs) for detecting antigen 9 in urine and plasma specimens from three groups of patients and 49 controls. The triple-antibody ELISA detected the antigen in urine samples from 11 of 18 (65%) patients with hemoculture-proven typhoid (group 1) and 12 of 39 (31%) patients with clinical features compatible with typhoid but whose hemocultures were negative (group 2). This ELISA was negative in three patients from whom Salmonella paratyphi A, Escherichia coli, and Klebsiella pneumoniae (group 3) were isolated by hemoculture and in all healthy controls. The double-antibody sandwich ELISA was positive in 41 and 15% of urine samples from patients in groups 1 and 2, respectively, and was negative with samples from two patients from group 3 and all controls. The sensitivity and specificity compared with those for healthy controls were 65 and 100%, respectively, for the triple-antibody ELISA. Although as little as 7.8 ng of homologous lipopolysaccharide could be detected, background in clinical specimens prevented accurate interpretation of the detection of this antigen in serum. Results were best with urine specimens. Images PMID:3183027

  19. Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody.

    PubMed Central

    Levasseur, S; Husson, M O; Leitz, R; Merlin, F; Laurent, F; Peladan, F; Drocourt, J L; Leclerc, H; Van Hoegaerden, M

    1992-01-01

    Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria. Images PMID:1622220

  20. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  1. Rapid detection of specific polyclonal and monoclonal antibodies against bovine leukemia virus.

    PubMed

    Llames, L; Goyache, J; Domenech, A; de Avila, A; Suarez, G; Gomez-Lucia, E

    1999-10-01

    ELISA and Western blot have been used for detecting specific antibodies or antigens for routine diagnostic laboratory tests and experimental protocols, as well as for screening hybridomas secreting antibodies. Although these techniques are sensitive, some slow growing hybridomas are identified as positive only when they are grown slowly long time. We standardized the dot-ELISA, a more sensitive technique, for the detection of antibodies against BLV. The main advantages of the dot-ELISA described in this study are (a) its sensitivity, detecting hybridomas which would otherwise be considered negative and discarded from the results of indirect ELISA and/or Western blot; and (b) the possibility of economizing reagents using as little as 1 microl of the antigen and 0.5 microl of antibody and conjugate. Different BLV-antigen preparations were bound to nitrocellulose membranes (NC), including cells lysed chemically (LYS) or by sonication (SOC), semi-purified virus (PV), and supernatant from infected cultures, either without treatment (SUP) or sonicated (SOS). The antigen preparations most adequate for detecting monoclonal antibodies against BLV and polyclonal antibodies in cattle sera were undiluted cell lysates (LYS) and semi-purified BLV (PV). When testing bovine sera, the supernatant (SUP) and sonicated supernatant (SOS) antigens gave a high background due to the presence of FCS which reacted with the anti-bovine labeled antibodies. In this study, 59 BLV specific antibody secreting hybridomas were identified using the dot-ELISA, compared to only 20 detected using iELISA, and doubtful reactions due to nonspecific binding to fetal calf serum (FCS) and cellular components were measured. The results of the improved dot-ELISA described may be stored at room temperature for future reference. Results were consistently reproducible in coated nitrocellulose membranes kept at different storage temperatures (-20 degrees C, 4 degrees C, and 25-30 degrees C) 48 h, 1 week and 5

  2. Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

    PubMed

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2014-01-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin. PMID:24830148

  3. Detection of foot-and-mouth disease serotype O by ELISA using a monoclonal antibody.

    PubMed

    Chen, Hao-Tai; Peng, Yun-Hua; Zhang, Yong-Guang; Liu, Xiang-Tao

    2013-02-01

    An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, and A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Furthermore, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations. PMID:23600506

  4. Detection of foot-and-mouth disease serotype O by ELISA using a monoclonal antibody.

    PubMed

    Chen, Hao-tai; Peng, Yun-hua; Zhang, Yong-guang; Liu, Xiang-tao

    2012-12-01

    An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Further, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations. PMID:23244327

  5. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

    PubMed Central

    Gustafsson, B; Rosén, A; Holme, T

    1982-01-01

    A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios. Images PMID:6183214

  6. Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

    PubMed Central

    Quinlan, J J; Foegeding, P M

    1997-01-01

    Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores. PMID:9023926

  7. Monoclonal antibodies to gonadotropin subunits

    SciTech Connect

    Ehrlich, P.H.; Moyle, W.R.; Canfield, R.E.

    1985-01-01

    The production of monoclonal antibodies to peptide hormones, with their unifocal binding sites, can provide tools for understanding hormone structure and function. The paper focuses on techniques that are important for the study of monoclonal antibodies to chorionic gonadotropin (hCG), including hybridoma production, methods of screening for desired clones, properties of the monoclonal antibodies, effect of antibodies on hormone-receptor interaction, inhibition of binding of radiolabeled hCG, inhibition of hCG induced steroidogenesis, determination of relative orientation of epitopes, and synergistic actions of monoclonal antibodies to hCG.

  8. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    PubMed Central

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-01-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  9. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

    PubMed

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-04-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  10. Colorectal carcinoma metastases: Detection with In-111-labeled monoclonal antibody CCR 086

    SciTech Connect

    Abdel-Nabi, H.H.; Levine, G.; Lamki, L.M.; Murray, J.L.; Tauxe, W.N.; Shah, A.N.; Patt, Y.Z.; Doerr, R.J.; Klein, H.A.; Gona, J. )

    1990-07-01

    A phase I/II clinical trial with indium-111-labeled antimucin murine monoclonal antibody (MoAb) CCR 086 was conducted. Seventeen patients with histologically proved colorectal carcinoma and known metastatic disease underwent external scintigraphy after administration of 5.5 mCi (203.5 MBq) of In-111 CCR 086 at doses of 5 and 20 mg. Of 25 known lesions, 17 were detected (sensitivity, 68%). The smallest detected lesion in the lung was 1 cm and in the liver was 1.5 cm. The serum half-life of In-111-labeled CCR 086 MoAb was approximately 64 hours. The formation of human antimouse antibody (HAMA) was detected in the serum of four of five patients who received 20 mg of MoAb. No HAMAs were detected in four patients receiving 5 mg of MoAb. No side effects were encountered. Because of effective detection of liver and lung metastases with lower doses (5-20 mg) of CCR 086 conjugated with In-111, further investigations are warranted to assess clinical and therapeutic potentials of CCR 086 in the management of colorectal cancer.

  11. Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen.

    PubMed Central

    Peterson, R R; Deeb, B J; DiGiacomo, R F

    1997-01-01

    As infection with Pasteurella multocida is common in rabbits, an enzyme immunoassay (EIA) was developed for its detection. A murine immunoglobulin G monoclonal antibody was used to capture a 37-kDa polypeptide of P. multocida serotype A:12 in an EIA to detect antibodies to P. multocida. The 37-kDa antigen was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits. The sensitivity of the P37 EIA, determined with sera from 56 rabbits infected with P. multocida, was 98%. Specificity, evaluated with sera from 62 rabbits from colonies free of P. multocida, was 92%. Titration curves of sera from rabbits immunized with P. multocida serotype A:3 or A:12 coincided, indicating that the P37 EIA was equally efficient in detecting antibodies to the two major serotypes of the organism. Comparison of the P37 EIA with the current serodiagnostic test, a bacterial lysate EIA, revealed relatively good correlation (r = 0.68). However, specificity was greatly improved, as 34% of uninfected rabbits were falsely positive by the lysate EIA whereas only 3% of uninfected rabbits were falsely positive by the P37 EIA. The coefficient of variation for same-day tests was 10%, and that for interday tests was 15%, indicating good reproducibility. The greater sensitivity and specificity of the P37 EIA should significantly enhance diagnostic capability to identify rabbits infected with P. multocida. PMID:8968909

  12. Production of monoclonal antibodies against avidin.

    PubMed

    Ashorn, R; Ashorn, P; Kulomaa, M; Tuohimaa, P; Krohn, K

    1985-01-01

    Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin. PMID:4053566

  13. Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

    PubMed Central

    Fukuma, Aiko; Fukushi, Shuetsu; Yoshikawa, Tomoki; Tani, Hideki; Taniguchi, Satoshi; Kurosu, Takeshi; Egawa, Kazutaka; Suda, Yuto; Singh, Harpal; Nomachi, Taro; Gokuden, Mutsuyo; Ando, Katsuyuki; Kida, Kouji; Kan, Miki; Kato, Nobuyuki; Yoshikawa, Akira; Kitamoto, Hiroaki; Sato, Yuko; Suzuki, Tadaki; Hasegawa, Hideki; Morikawa, Shigeru; Shimojima, Masayuki; Saijo, Masayuki

    2016-01-01

    Background Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. Methodology/Principal Findings We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. Conclusions The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. PMID:27045364

  14. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies. PMID:20954327

  15. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    SciTech Connect

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Pastan, Ira

    2008-01-25

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2{gamma}C, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2{gamma}C and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

  16. Laser-induced breakdown detection of temperature-ramp generated aggregates of therapeutic monoclonal antibody.

    PubMed

    Menzen, Tim; Friess, Wolfgang; Niessner, Reinhard; Haisch, Christoph

    2015-08-01

    The detection and characterization of protein aggregation is essential during development and quality control of therapeutic proteins, as aggregates are typically inactive and may trigger anti-drug-antibody formation in patients. Especially large multi-domain molecules, such as the important class of therapeutic monoclonal antibodies (mAbs), can form various aggregates that differ in size and morphology. Although particle analysis advanced over the recent years, new techniques and orthogonal methods are highly valued. To our knowledge, the physical principle of laser-induced breakdown detection (LIBD) was not yet applied to sense aggregates in therapeutic protein formulations. We established a LIBD setup to monitor the temperature-induced aggregation of a mAb. The obtained temperature of aggregation was in good agreement with the results from previously published temperature-ramped turbidity and dynamic light scattering measurements. This study demonstrates the promising applicability of LIBD to investigate aggregates from therapeutic proteins. The technique is also adaptive to online detection and size determination, and offers interesting opportunities for morphologic characterization of protein particles and impurities, which will be part of future studies. PMID:26158409

  17. Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

    PubMed Central

    Grenfell, Rafaella Fortini Queiroz; Coelho, Paulo Marcos Zech; Taboada, Diana; de Mattos, Ana Carolina Alves; Davis, Ruth; Harn, Donald A.

    2014-01-01

    Background Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity. Methodology/Principal Findings We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure. Conclusions/Significance The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden. PMID:24498191

  18. A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27.

    PubMed

    Hughes, Laura J; Goldstein, Jason; Pohl, Jan; Hooper, Jay W; Lee Pitts, R; Townsend, Michael B; Bagarozzi, Dennis; Damon, Inger K; Karem, Kevin L

    2014-09-01

    The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5 kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology. PMID:25108113

  19. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens.

    PubMed Central

    Gómez, B L; Figueroa, J I; Hamilton, A J; Ortiz, B; Robledo, M A; Hay, R J; Restrepo, A

    1997-01-01

    The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses. PMID:9399534

  20. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens.

    PubMed

    Gómez, B L; Figueroa, J I; Hamilton, A J; Ortiz, B; Robledo, M A; Hay, R J; Restrepo, A

    1997-12-01

    The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses. PMID:9399534

  1. Production of monoclonal antibodies.

    PubMed

    Freysd'ottir, J

    2000-01-01

    The discovery of monoclonal antibodies (mAbs) produced by "hybridoma technology" by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recognized. It took around 10 years to appreciate the importance of using these mAbs in various fields of science other than immunology, such as cell biology, biochemistry, microbiology, virology, para-sitology, physiology, genetics, and molecular biology; and also in areas of clinical medicine, such as pathology, hematology, oncology, and infectious disease. The contribution of mAbs to science and clinical medicine was recognized in 1984 by the award of the Nobel Prize for Medicine to Köhler and Milstein. PMID:21337095

  2. Monoclonal antibodies in myeloma.

    PubMed

    Sondergeld, Pia; van de Donk, Niels W C J; Richardson, Paul G; Plesner, Torben

    2015-09-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting addition to the therapeutic armamentarium. The incorporation of mAbs into current treatment strategies is hoped to enable more effective and targeted treatment, resulting in improved outcomes for patients. A number of targets have been identified, including molecules on the surface of the myeloma cell and components of the bone marrow microenvironment. Our review focuses on a small number of promising mAbs directed against molecules on the surface of myeloma cells, including CS1 (elotuzumab), CD38 (daratumumab, SAR650984, MOR03087), CD56 (lorvotuzumab mertansine), and CD138/syndecan-1 (BT062/indatuximab ravtansine). PMID:26452191

  3. Advantages of detecting monoclonal antibody binding to tissue sections with biotin and avidin reagents in Coplin jars.

    PubMed

    Bindl, J M; Warnke, R A

    1986-04-01

    We describe a method of biotin/avidin-peroxidase detection using second and third stage reagents in Coplin jars. This method allows a large quantity of sections to be stained simultaneously with a minimal amount of technical time involved. A wide range of mouse monoclonal antibodies of varying specificities and isotypes were used to stain both frozen and paraffin-embedded sections of various normal and neoplastic tissues. Three different biotinylated anti-mouse antibodies were tested, including F(ab')2 antibody fragments of one, followed by horseradish peroxidase conjugated avidin. All monoclonal antibodies employed gave good staining, using incubation times of 30-50 minutes. The staining was done during a mean period of 25 to 27 days with an average staining load of 500 sections per Coplin jar. PMID:2420169

  4. Production of a novel monoclonal antibody, JT-95, which can detect antigen of thyroid carcinoma.

    PubMed

    Takeyama, H; Hosoya, T; Sakurai, K; Mori, Y; Watanabe, M; Kisaki, H; Ohno, T

    1996-04-15

    Monoclonal antibody (MAb) JT-95 was produced by immunization of mice with membrane fractions of a human thyroid carcinoma. Immuno-histochemical staining has demonstrated that the antigen recognized by JT-95 is strongly expressed in 95 (95%) of 100 cases of papillary carcinomas and in 3 (75%) of 4 cases of follicular carcinomas. In benign diseases of the thyroid gland, MAb JT-95 reacted with 0 (0%) of 39 adenomas, 1 (4%) of 21 adenomatous goiters, 0 (0%) of 8 hyperthyroidism specimens, and 3 (38%) of 8 chronic thyroiditis specimens. The antigen detected by MAb JT-95 has an apparent Mr 250,000 in thyroid carcinomas. Moreover, circulating antigen in thyroid carcinoma patients was detected by MAb JT-95 in an ELISA and in Western blotting. The circulating antigen has a Mr 105,000. MAb JT-95 conjugated with (131) I was administrated to nude mice bearing a human thyroid carcinoma. JT-95 131I accumulation at the transplanted tumor was visualized by autoradiography with 2.68-14.75-fold higher levels detected at the xenograft compared to that for normal organs. Based on these data, MAb JT-95 may be useful in the diagnosis detection and therapy of thyroid carcinoma. PMID:8620498

  5. Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

    PubMed

    Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

    2016-05-11

    Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing. PMID:27087556

  6. Monoclonal antibody-tagged receptor-targeted contrast agents for detection of cancers

    NASA Astrophysics Data System (ADS)

    Soukos, N. S.; Hamblin, Michael R.; Deutsch, Thomas F.; Hasan, Tayyaba

    2001-07-01

    Oral cancer and precancer overexpress the epidermal growth factor receptor (EGFR) and monoclonal antibodies against EGFR coupled to photoactive dyes may have a potential both as a diagnostic and treatment modalities for oral premalignancy. We asked whether an anti-EGFR mab (C225) conjugated with the fluorescence dye indocyanine Cy5.5 could detect dysplastic changes in the hamster cheek pouch carcinogenesis model. Secondly, we tested whether the same antibody conjugated with the photosensitizer chlorin (e6) could be used together with illumination to reduce levels of expression of EGFR as evaluated by the immunophotodetection procedure. Increased fluorescence appeared to correlate with development of premalignancy when the C225-Cy5.5 conjugate was used. Areas with increased fluorescence signal were found in carcinogen-treated but clinically normal cheek pouches, that revealed dysplastsic changes by histology. The immunophotodetection procedure was carried out after photoummunotherapy with the C225-ce6 conjugate, and showed a significant reduction in fluorescence in the illuminated compared to the non-illuminated areas in the carcinogen- treated but not the normal cheek pouch. The results demonstrate that the use of anti-EGFR Mab targeted photoactive dyes may serve as a feedback controlled optical diagnosis and therapy procedure for oral premalignant lesions.

  7. Antibodies and Selection of Monoclonal Antibodies.

    PubMed

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology. PMID:27236550

  8. Label-free detection and characterization of the binding of hemagglutinin protein and broadly neutralizing monoclonal antibodies using terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Sun, Yiwen; Zhong, Junlan; Zhang, Cunlin; Zuo, Jian; Pickwell-MacPherson, Emma

    2015-03-01

    Hemagglutinin (HA) is the main surface glycoprotein of the influenza A virus. The H9N2 subtype influenza A virus is recognized as the most possible pandemic strain as it has crossed the species barrier, infecting swine and humans. We use terahertz spectroscopy to study the hydration shell formation around H9 subtype influenza A virus's HA protein (H9 HA) as well as the detection of antigen binding of H9 HA with the broadly neutralizing monoclonal antibody. We observe a remarkable concentration dependent nonlinear response of the H9 HA, which reveals the formation process of the hydration shell around H9 HA molecules. Furthermore, we show that terahertz dielectric properties of the H9 HA are strongly affected by the presence of the monoclonal antibody F10 and that the terahertz dielectric loss tangent can be used to detect the antibody binding at lower concentrations than the standard ELISA test.

  9. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. )

    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.

  10. Development and application of monoclonal antibodies for detection and analysis of aquareoviruses.

    PubMed

    Gao, Xiao-Chan; Chen, Zhong-Yuan; Liu, Jia; Zhang, Qi-Ya

    2016-01-01

    Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses. PMID:26889962

  11. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

    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.

  12. 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.

  13. Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

    PubMed

    Tsoukas, C D; Lambris, J D

    1988-08-01

    The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage. PMID:2970972

  14. The therapeutic monoclonal antibody market

    PubMed Central

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

  15. Natural monoclonal antibodies and cancer.

    PubMed

    Vollmers, Peter H; Brändlein, Stephanie

    2008-06-01

    Immunity is responsible for recognition and elimination of infectious particles and for removal of cellular waste, modified self structures and transformed cells. Innate or natural immunity acts as a first line defense and is also the link to acquired immunity and memory. By using the human hybridoma technology, a series of monoclonal antibodies and several new tumor-specific targets could be identified. A striking phenomenon of immunity against malignant cells is that all so far isolated tumor-specific antibodies were germ-line coded natural IgM antibodies. And neither in animals nor in humans affinity-maturated tumor-specific IgG antibodies have been detected so far. These IgM's preferentially bind to carbohydrate epitopes on post-transcriptionally modified surface receptors, which are recently patented and preferentially remove malignant cells by inducing apoptosis to avoid inflammatory processes. Our "biology-" or "function-driven" method represents a unique yet powerful approach compared to the typical approaches on screening compounds or antibodies against non-validated targets (mostly differentially expressed). Moreover, the approach creates a competitive patenting strategy of creating proprietary antibodies and validated targets at the same time, which has the potential of further streamlining the discovery of new cancer therapies. PMID:18537750

  16. Production of monoclonal antibodies to hepatitis B surface and core antigens, and use in the detection of viral antigens in liver biopsies.

    PubMed Central

    Tedder, R. S.; Guarascio, P.; Yao, J. L.; Lord, R. B.; Eddleston, A. L.

    1983-01-01

    Hybridomas secreting monoclonal antibodies to HBsAg and HBcAg were prepared from immunized mice. An antibody capture radioimmunoassay was used to detect and select appropriate hybrids for propagation and cloning. The advantages of this assay were discussed. The resulting monoclonal antibodies were compared with conventional polyclonal antisera for the detection of virus antigens in liver tissue and found to give excellent results. Images Plate 2 Plate 1 PMID:6337208

  17. Characterization of a Monoclonal Antibody against Syringate Derivatives: Application of Immunochemical Detection of Methyl Syringate in Honey.

    PubMed

    Kato, Yoji; Fujinaka, Rie; Juri, Maki; Yoshiki, Yui; Ishisaka, Akari; Kitamoto, Noritoshi; Nitta, Yoko; Ishikawa, Hirohito

    2016-08-24

    Syringic acid is one of the key skeletal structures of plant-derived chemicals. The derivatives of syringic acid have certain biological functions. In this study, a monoclonal antibody to syringic acid-based phytochemicals was prepared and characterized. The obtained antibody reacted with methyl syringate, syringic acid, and leonurine. Methyl syringate is a characteristic compound found in manuka honey, other honey varieties, and plants. Manuka honey was fractionated using HPLC, and the reactivity of the fractions with the antibody was examined. The antibody reacted with the fraction in which methyl syringate was eluted. The amount of methyl syringate in honeys as estimated by ELISA using the antibody had a good linearity compared with that estimated by HPLC. These results suggest that the antibody is applicable for the immunochemical detection of syringic acid derivatives in plants and foods. PMID:27477590

  18. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp*

    PubMed Central

    Wongtangprasert, Tossapon; Natakuathung, Wirongrong; Pimpitak, Umaporn; Buakeaw, Anumart; Palaga, Tanapat; Komolpis, Kittinan; Khongchareonporn, Nanthika

    2014-01-01

    A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%–118% for an intra-assay and 96%–113% for an inter-assay. The coefficients of variation of the assays were 3.9%–13.9% and 5.5%–14.9%, respectively. PMID:24510709

  19. Monoclonal Antibodies as Probes for the Detection of Porcine Blood-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2016-05-11

    The lack of effective methods to monitor the use of porcine blood-derived food ingredients (PBFIs) is a concern for the billions of individuals who avoid consuming blood. We therefore sought to develop a panel of porcine blood-specific monoclonal antibodies (mAbs) for use as probes in immunoassays for the detection of PBFIs. Ten selected mAbs were identified that react with either a 60 or 90 kDa protein in the plasma fraction or a 12 kDa protein in the red blood cell fraction of porcine blood. Western blot analysis of commercially produced PBFIs revealed that these antigenic proteins are not affected by various manufacturing processes. The utility of these mAbs was demonstrated in a prototype sandwich ELISA developed for this study using mAbs 19C5-E10 and 16F9-C11. The new assay is porcine blood-specific and capable of detecting ≤0.03% (v/v) of PBFIs in cooked (100 °C for 15 min) ground meats or fish. PMID:27135860

  20. Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies.

    PubMed

    Yamamoto, Takayuki; Kato, Masatoshi; Endo, Kiwamu; Kotoura, Satoshi; Takeda, Zenya

    2016-01-01

    A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt). PMID:26166832

  1. Immunotoxicity of monoclonal antibodies

    PubMed Central

    2009-01-01

    Monoclonal antibodies (mAbs) are large molecules intended to bind to specific targets often expressed on the immune system, and to treat various immunopathological conditions. Therefore, mAbs can be considered to have a high potential for immunotoxicity, which is reflected in the clinical experience accumulated on mAbs-induced adverse effects related to immunosuppression, immunostimulation and hypersensitivity (immunogenicity). So far, non clinical immunotoxicity studies have been inadequate to address all safety issues in relation to the possible immunotoxicity of mAbs, because they are fraught with limitations and pitfalls primarily related to the lack of relevant animal species. In addition, clinical studies rarely include validated end-points dedicated to the prediction of immunotoxicity. With the ongoing development of mAbs as novel therapeutic strategies for a wide variety of diseases, efforts should be paid to improve our understanding of mAbs-induced immunotoxic effects and design dedicated strategies to assess their immunological safety, both non clinically and clinically. PMID:20061816

  2. Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus

    PubMed Central

    2010-01-01

    Background Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. Results In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples. Conclusions Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry. PMID:21192824

  3. Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

    PubMed

    Abhiman, P B; Shankar, K M; Patil, Rajreddy; Suresh Babu, P P; Sahoo, A K; Shamasundar, B A

    2014-05-01

    Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes. PMID:24803705

  4. Development of monoclonal antibodies to detect bovine FOXP3 in PBMCs exposed to a staphylococcal superantigen

    PubMed Central

    Seo, K. S.; Davis, W. C.; Hamilton, M. J.; Park, Y. H.; Bohach, G. A.

    2013-01-01

    The role of regulatory T cells (Tregs) is well documented in immune homeostasis and protection against autoimmune disease. Forkhead box protein 3 (FOXP3) has been shown be essential for the development and function of Treg. Due to the lack of tools for FOXP3 detection in certain species, understanding the role of Treg in a variety of ruminant diseases has been hampered. In this study, we developed monoclonal antibodies (mAbs) against bovine FOXP3 using recombinant bovine FOXP3 lacking the forkhead domain as an immunogen. The specificity of the mAbs was confirmed by immunoblot and mass spectrometry. Expression of FOXP3 was induced in bovine PBMCs after 6 d of exposure to staphylococcal enterotoxin type C1 (SEC1) in vitro. Similar to findings in mice and humans, expression of FOXP3 was restricted to CD4+ CD25+ T cells. Transcriptional analysis of bovine TCR variable regions of the β chain (boVβ) showed that transcription of boVβ sequences reactive with SEC1 increased for 6 d, and then boVβ sequences non-reactive with SEC1 rapidly increased in the cultures. This indicates that induction of FOXP3+ CD4+ CD25+ Tregs by SEC1 is not Vβ restricted. The FOXP3 mAbs developed in this study will be useful in the further investigation of the role of Treg in staphylococcal pathogenesis in bovine mastitis and other ruminant diseases. PMID:19054574

  5. Improved immunobinding test using monoclonal antibodies for detection of Mycoplasma bovis in milk

    PubMed Central

    Infante, F.; Infante, F.; Flores-Gutiérrez, G. H.

    2002-01-01

    The aim of this study was to evaluate an improved immunobinding test (IBT) using monoclonal antibodies to identify Mycoplasma bovis in naturally infected milk. The IBT and the improved IBT were highly specific and had an immunologic sensitivity of 5 × 103 colony-forming units per milliliter. The results for the 2 methods agreed in the 130 milk samples tested. However, the IBT required 158 min, whereas the improved IBT required only 110 min. In addition, the improved IBT used smaller quantities of antibodies and conjugates. PMID:12418785

  6. Preparation and identification of monoclonal antibody against Citreoviridin and development of detection by Ic-ELISA.

    PubMed

    Jin, Ni; Ling, Sumei; Yang, Chi; Wang, Shihua

    2014-11-01

    Citreoviridin (CIT), a neurotoxic mycotoxin produced by Penicillium citreonigrum is generally detected in cereal grains and agricultural products worldwide, and has numerous toxicological effects on human and animal health. Therefore, it is necessary to develop a rapid, sensitive, and reliable immunoassay method for CIT. In this study, artificial antigen CIT-KLH and CIT-BSA was successfully prepared via succinic anhydride and carbodiimide two-step method. CIT-KLH conjugates were injected into Balb/c mice, and titer of the antiserum against CIT was determined using CIT-BSA as coating antigen by ELISA method. A hybridoma cell line 8D8 stably secreting monoclonal antibody against CIT was generated by fusing SP2/0 myeloma cells with the splenocytes from the immunized mice. The titer of 8D8 mAb reached 1: 1.28 × 10(5) after purified by caprylic/ammonium sulfate precipitation (CA-AS) method. The 8D8 mAb was identified as IgG1 subtype. The cross-reactivity results indicated that anti-CIT mAb was highly specific to Citreoviridin, and the average affinity 4.57 × 10(8) L/mol. A sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for CIT was established. Under optimal condition, the linear range to detect CIT was 11.02-2370.48 ng/mL with IC50 of 161.66 ng/mL and the limit of detection of the ic-ELISA was 11.86 ng/mL. With the mean coefficient of variation lowing 5%, the mean recovery in intra-assay and inter-assay were (90.06 ± 1.60)% and (89.65 ± 1.69)%, respectively. Therefore, the anti-CIT mAb secreted by 8D8 hybridoma cell line is useful for analysis of food contaminated with CIT. PMID:25157801

  7. 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.

  8. Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts.

    PubMed

    Lee, J; Siemann, D W; Koch, C J; Lord, E M

    1996-07-29

    While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acet amide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry. To verify the validity of this technique for the detection of hypoxic cells, mice bearing KHT sarcomas were treated with various agents to alter tumor oxygenation and hence vary the fraction of radiobiologically hypoxic tumor cells. The percentage of EF5 binding cells was then compared directly with the clonogenic survival of the tumor cells following radiation treatment under the various pretreatment conditions. The results showed that allowing the mice to breathe carbogen (5% CO2/95% O2) prior to irradiation reduced clonogenic cell survival approx. 6-fold and led to an absence of cells binding high levels of EF5. In contrast, pretreating the tumor-bearing animals with either hydralazine, which decreased tumor blood flow, or phenylhydrazine hydrochloride, which made the mice anemic, increased tumor cell survival following irradiation 2- to 4-fold, indicative of an increase in the fraction of hypoxic tumor cells. EF5 measurements made under identical conditions illustrated a shift in the cells in the tumor to high EF5 binding. Our results demonstrate that flow cytometric measurement by fluorescent MAb binding to EF5 adducts may relate directly to radiobiological hypoxia in KHT tumors measured by conventional methods. PMID:8707411

  9. Utilization of monoclonal antibodies for detection of Plasmodium falciparum antigen in cerebrospinal fluid of cerebral malaria patients.

    PubMed

    Khushiramani, Rekha; Shrivastava, Sandeep; Varma, Subhash; Batra, Harsh Vardhan; Dubey, Mohan Lal

    2008-08-01

    A uniform protein profile of bands at 34, 43, and 52 kDa was obtained with all the cerebrospinal fluid (CSF) samples of malaria (10 in number) and non-malaria patients (31 in number) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An immunoreactive band was observed at 43 kDa in CSF samples of cerebral malaria patients but not in non-malaria cases when tested with rabbit anti-Plasmodium falciparum antibodies by Western blot analysis. Eleven reactive monoclonal antibodies against P. falciparum were stabilized and expanded. Nine monoclonal antibodies were reactive to CSF samples of cerebral malaria and non-malaria and P. falciparum antigen by dot-ELISA and a common immunoreactive band at 43 kDa by Western blot. One clone Cl-2 was reactive at 43 kDa with CSF of the cerebral malaria patients and also in P. falciparum antigen but at 66 kDa with non-malarial CSF samples in Western blot. The other two clones (Cl-6 and 14) reacted with 3/31 (90% specific) and 8/31 (74%) CSF samples of non-malaria patients, respectively. The monoclonal antibody based ELISA reported in the present study using clone-6 can therefore offer another possibility for developing rapid, easy-to-perform, low-cost tests for diagnosis of cerebral malaria in CSF samples. Western blot using clone-2 might be useful for the detection of cerebral malaria antigen in CSF. PMID:18707548

  10. A monoclonal antibody-based latex bead agglutination test for the detection of Bordetella avium.

    PubMed

    Suresh, P; Arp, L H

    1993-01-01

    The purpose of this study was to develop a rapid method to distinguish Bordetella avium from closely related Bordetella avium-like and B. bronchiseptica bacteria. A monoclonal antibody of the IgM isotype was produced in Balb/c mice against live B. avium strain 75. The monoclonal antibody, in the form of ascites fluid, was added to a bovine serum albumin-glycine buffer (pH 8.6) and adsorbed to 3.03-microns-diameter latex beads. Optimum concentrations of antibody, beads, and bacteria were determined. The latex bead conjugate was tested against 40 isolates of B. avium, 24 isolates of B. avium-like bacteria, 17 isolates of B. bronchiseptica, two isolates of Alcaligenes faecalis, and several other common genera. Strong agglutination occurred with all B. avium isolates and the two isolates of A. faecalis. Weak agglutination occurred with Staphylococcus aureus and two isolates of B. bronchiseptica. There was no agglutination with any of the B. avium-like isolates. The latex bead agglutination test may be useful as an aid in the identification of B. avium when used in conjunction with other criteria. PMID:8257369

  11. Development of a monoclonal antibody detection assay for species-specific identification of abalone.

    PubMed

    Lopata, Andreas L; Luijx, Thomas; Fenemore, Bartha; Sweijd, Neville A; Cook, Peter A

    2002-10-01

    Species identification based on biochemical and molecular techniques has a broad range of applications. These include compliance enforcement, the management and conservation of marine organisms, and commercial quality control. Abalone poaching worldwide and illegal trade in abalone products have increased mainly because of the attractive prices obtained and caused a sharp decline in stocks. Alleged poachers have been acquitted because of lack of evidence to correctly identify species. Therefore, a robust method is required that would identify tissue of abalone origin to species level. The aim of this study was to develop immunologic techniques, using monoclonal and polyclonal antibodies, to identify 10 different abalone species and subspecies from South Africa, the United States, Australia, and Japan. The combination of 3 developed monoclonal antibodies to South African abalone (Haliotis midae) enabled differentiation between most of the 10 species including the subspecies H. diversicolor supertexta and H. diversicolor diversicolor. In a novel approach, using antibodies of patients with allergy to abalone, the differentiation of additional subspecies, H. discus discus and H. discus hannai, was possible. A field-based immunoassay was developed to identify confiscated tissue of abalone origin. PMID:14961238

  12. Production of Monoclonal Antibody against Human Nestin.

    PubMed

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  13. Production of Monoclonal Antibody against Human Nestin

    PubMed Central

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  14. Trichinella spiralis: monoclonal antibody against the muscular larvae for the detection of circulating and fecal antigens in experimentally infected rats.

    PubMed

    Zumaquero-Ríos, José-Lino; García-Juarez, Jazmín; de-la-Rosa-Arana, Jorge-Luis; Marcet, Ricardo; Sarracent-Pérez, Jorge

    2012-12-01

    In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were infected with 15 ML per gram of body weight and blood samples were collected weekly for 10 weeks. Antibodies were studied using an indirect ELISA. For detection of circulating antigens and coproantigens, a sandwich ELISA was developed with the use of polyclonal rabbit antibodies obtained against the total extract of ML and an IgM monoclonal antibody (Mab) against ES antigens of ML. No reactivity was observed between Mab and the total worm antigens of Angiostrongylus cantonensis, Ascaris suum, Echinococcus granulosus, Fasciola hepatica, Strongyloides stercoralis, Taenia solium, Toxocara canis and Trichuris trichiura. The IgM Mab recognized antigens of 45, 49, and 55 kDa in ES antigens and was unable to bind ES antigens deglycosylated with trifluoromethanesulphonic acid (TFMS) indicating that a glycan structure is present in the epitope recognized by this Mab. The sensitivity of sandwich ELISA was 1 ng/mL. Circulating antigens were detected in all infected rats between 3 and 8 weeks post infection and coproantigens were found during the first two days post infection. Antibodies were detected since the third week post infection through the end of experiment. These results suggested that antigen detection by our sandwich ELISA could be a useful complementary laboratory test for antibody detection. PMID:23026455

  15. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo.

    PubMed

    Dai, Hua; Xu, Zheng-Zhong; Wang, Meiling; Chen, Jun-Hua; Chen, Xiang; Pan, Zhi-Ming; Jiao, Xin-An

    2016-04-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  16. VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections.

    PubMed Central

    Turley, H; Jones, M; Erber, W; Mayne, K; de Waele, M; Gatter, K

    1994-01-01

    AIMS--To characterise a new mouse monoclonal antibody, VS38, which recognises an intracytoplasmic antigen of 64 kilodaltons present in normal and neoplastic plasma cells; and to establish its value as a diagnostic reagent for routine pathological practice. METHODS--A range of normal and neoplastic tissue sections, both frozen and routinely fixed, were immunostained, using the microwave method of antigen retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range of human cell lines. The molecular weight of the antigen recognised by the antibody was obtained by western blot analysis. FACS analysis was used to demonstrate the cellular location of the antigen and its presence on tonsil cell suspensions and myeloma cases. RESULTS--VS38 recognised normal and neoplastic plasma cells in all of the tissues, including all routinely fixed plasma cell neoplasms tested. The antibody also weakly stained epithelial elements within the tissue but was absent from haemopoietic cells of other lineages. CONCLUSION--Antibody VS38 is of potential value in identifying myeloma or plasmacytoma in bone marrow or other tissues. It differentiates lymphoplasmacytoid lymphoma from lymphocytic and follicular lymphoma. It also subdivides large cell lymphomas into two groups which may be a more reliable method of separating these tumours than morphology alone. Images PMID:7517959

  17. 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.

  18. 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.

  19. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    PubMed

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  20. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

    PubMed

    Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

    2016-01-01

    This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. PMID:27529243

  1. [Monoclonal antibody for cancer treatment].

    PubMed

    Achiwa, Hiroyuki; Sato, Shigeki; Ueda, Ryuzo

    2002-04-01

    Antibodies have for many decades been viewed as ideal molecules for cancer therapy. Although promising from the start, it has taken much of more than two decades to reach the level of clinical application. Genetic engineering of antibodies; that is novel technologies for chimeric or humanizing monoclonal antibodies, has greatly advanced their utility in molecular targeting therapies, and in the past four years some therapeutic monoclonal antibodies for hematologic malignancies and solid tumors, such as Rituximab for B-cell lymphoma and Trastuzumab for metastatic breast cancer, have provided sufficient efficacy and safety to support regulatory approval from the U.S. Food and Drug Administration. They were subsequently approved by the Japanese Ministry of Health, Labour and Welfare in 2001. Many molecular biological and immunological studies have revealed the targeting properties of the host immune system and the biological mechanism of cancer cells for a more specific anticancer effect. Many clinical trials of monoclonal antibodies as a single agent, or in combination protocol with current standard chemotherapy or immunoconjugates have shown promise in the treatment of specific diseases. Furthermore, novel antibody designs and improved understanding of the mode of action of current antibodies lend great hope to the future of this therapeutic approach. The accumulating results from many basic, clinical and translational studies may lead to more individualized therapeutic strategies using these agent directed at specific genetic and immunologic targets. PMID:11977531

  2. Monoclonal Antibodies for Cancer Immunotherapy

    PubMed Central

    Weiner, Louis M.; Dhodapkar, Madhav V.; Ferrone, Soldano

    2008-01-01

    Monoclonal antibodies have emerged as effective therapeutic agents for many human malignancies. However, the ability of antibodies to initiate tumor antigen-specific immune responses has not received as much attention as other mechanisms of antibody action. Here we describe the rationale and evidence for developing anti-cancer antibodies that can stimulate host tumor antigen-specific immune responses. This may be accomplished by inducing antibody-dependent cellular cytotoxicity, by promoting antibody-targeted cross-presentation of tumor antigens or by triggering the idiotypic network. Future treatment modifications or combinations should be able to prolong, amplify and shape these immune responses to increase the clinical benefits of antibody therapy of human cancer. PMID:19304016

  3. 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...

  4. Developing a Noninvasive Procedure Using Labeled Monoclonal Antibody Anti-VEGF (Bevacizumab) for Detection of Endometriosis

    PubMed Central

    Machado, Daniel Escorsim; Perini, Jamila Alessandra; Orlando, Margarida Maria Camoes; Santos-Oliveira, Ralph

    2015-01-01

    The off-label use of bevacizumab labeled with 99mTc as a new radiopharmaceutical for imaging of endometriosis is a promising noninvasive, new clinical procedure. The bevacizumab in monoclonal antibodies targeted at vascular endothelial growth factor (VEGF) is superexpressed in cases of endometriosis. In this study we evaluate the imaging of endometriosis lesion in rats (induced to endometriosis) using bevacizumab-99mTc. The results showed that bevacizumab-99mTc imaged the lesion and support his use for Nuclear Medicine applied to gynecology. Also the results appointed that this radiopharmaceutical has a hepatobiliary excretion. It is important to notice that the dose used was almost 0,01% of the usual dose for the bevacizumab. PMID:26240826

  5. Developing a Noninvasive Procedure Using Labeled Monoclonal Antibody Anti-VEGF (Bevacizumab) for Detection of Endometriosis.

    PubMed

    Machado, Daniel Escorsim; Perini, Jamila Alessandra; Orlando, Margarida Maria Camoes; Santos-Oliveira, Ralph

    2015-01-01

    The off-label use of bevacizumab labeled with 99mTc as a new radiopharmaceutical for imaging of endometriosis is a promising noninvasive, new clinical procedure. The bevacizumab in monoclonal antibodies targeted at vascular endothelial growth factor (VEGF) is superexpressed in cases of endometriosis. In this study we evaluate the imaging of endometriosis lesion in rats (induced to endometriosis) using bevacizumab-99mTc. The results showed that bevacizumab-99mTc imaged the lesion and support his use for Nuclear Medicine applied to gynecology. Also the results appointed that this radiopharmaceutical has a hepatobiliary excretion. It is important to notice that the dose used was almost 0,01% of the usual dose for the bevacizumab. PMID:26240826

  6. Detection of histidine oxidation in a monoclonal immunoglobulin gamma (IgG) 1 antibody.

    PubMed

    Amano, Masato; Kobayashi, Naoki; Yabuta, Masayuki; Uchiyama, Susumu; Fukui, Kiichi

    2014-08-01

    Although oxidation of methionine and tryptophan are known as popular chemical modifications that occur in monoclonal antibody (mAb) molecules, oxidation of other amino acids in mAbs has not been reported to date. In this study, oxidation of the histidine residue in a human immunoglobulin gamma (IgG) 1 molecule was discovered for the first time by mass spectrometry. The oxidation of a specific histidine located at the CH2 domain of IgG1 occurred under light stress, but it was not observed under heat stress. With the forced degradation study using several reactive oxygen species, the singlet oxygen was attributed to a reactive source of the histidine oxidation. The reaction mechanism of the histidine oxidation was proposed on the basis of the mass spectrometric analysis of IgG1 oxidized in deuterium oxide and hydrogen heavy oxide. PMID:24940720

  7. A monoclonal antibody against leptin.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  8. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies.

    PubMed Central

    Moll, R.; Löwe, A.; Laufer, J.; Franke, W. W.

    1992-01-01

    The authors have recently identified a new cytokeratin (CK) polypeptide, CK 20, whose expression is almost entirely confined to the gastric and intestinal epithelium, urothelium, and Merkel cells. Seven monoclonal antibodies (MAbs) specific for CK 20 were raised and characterized by applying immunoblotting and immunocytochemical screening. All of them reacted on frozen tissue sections. A further MAb, IT-Ks20.8, recognized CK 20 in sections of formalin-fixed, paraffin-embedded tissue samples. A total of 711 cases of primary and metastatic cancer, mostly carcinomas, were analyzed immunohistochemically for CK-20 expression, using CK-20 specific guinea-pig antibodies and MAbs. The expression spectrum of CK 20 in carcinomas resembled that seen in the corresponding normal epithelia of origin. CK-20 positivity was seen in the vast majority of adenocarcinomas of the colon (89/93 cases), mucinous ovarian tumors, transitional-cell and Merkel-cell carcinomas and frequently also in adenocarcinomas of the stomach, bile system, and pancreas. Most squamous cell carcinomas in general and most adenocarcinomas from other sites (breast, lung, endometrium), nonmucinous tumors of the ovary, and small-cell lung carcinomas were essentially or completely negative. The authors propose to use CK 20 as a diagnostic marker valuable in distinguishing different types of carcinomas, notably when presenting as metastases. Images Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 Figure 9 Figure 10 PMID:1371204

  9. Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.

    PubMed

    Trap, Catherine; Nato, Farida; Chalbot, Sonia; Kim, Sae-Bom; Lafaye, Pierre; Morfin, Robert

    2005-02-01

    The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues. PMID:15652401

  10. Advances in monoclonal antibody application in myocarditis*

    PubMed Central

    Han, Li-na; He, Shuang; Wang, Yu-tang; Yang, Li-ming; Liu, Si-yu; Zhang, Ting

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories. Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases, inflammatory diseases, cancer, and other immune-associated diseases. This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis, an inflammatory disease of the heart, could be a novel approach in the future. In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis, we, through a significant amount of literature research both domestic and abroad, developed a systematic elaboration of monoclonal antibodies, pathogenesis of myocarditis, and application of monoclonal antibodies in myocarditis. This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future. Under conventional therapy, myocarditis is typically associated with congestive heart failure as a progressive outcome, indicating the need for alternative therapeutic strategies to improve long-term results. Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis, we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above. However, several issues remain. The technology on how to make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues. If we are to

  11. Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

    PubMed

    Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

    2016-07-01

    Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. PMID:27018063

  12. Clinicopathological significance of lymphangiogenesis detected by immunohistochemistry using D2-40 monoclonal antibody in breast cancer.

    PubMed

    Abe, Noriko; Ohtake, Tohru; Saito, Katsuharu; Kumamoto, Kensuke; Sugino, Takashi; Takenoshita, Seiichi

    2016-06-01

    To elucidate the association between the lymphangiogenesis and clinicopathological factors including the survival in breast cancer, 91 Japanese patients with breast cancer were investigated. The lymphangiogenesis was evaluated by the count of lymph vessel density (LVD) with immunohistochemical method using D2-40 monoclonal antibody, a specific marker for lymphatic endothelial cells.D2-40-positive lymph vessels were detected in 87 of 91 cases, and were mainly distributed in the peritumoral lesions or around the tumor edge. There was a significant difference in disease-free survival (DFS) and overall survival (OS) between patients with high LVD and with low LVD (p=0.02, 0.01, respectively, log-rank test). In addition, LVD significantly correlated with the following clinicopathological factors: menopausal status (p<0.01), tumor size (p<0.01), lymph-node status (p=0.01) lymphatic vessel invasion (LVI) (p<0.01), blood vessel invasion (BVI) (p=0.03) and estrogen receptor status (ER) (p=0.02).Those data suggest that D2-40 monoclonal antibody is a useful marker for evaluating the LVD and its evaluation is helpful to predict the survival in breast cancer. PMID:27210308

  13. Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli.

    PubMed

    Zeng, Haijuan; Guo, Wenbo; Liang, Beibei; Li, Jianwu; Zhai, Xuzhao; Song, Chunmei; Zhao, Wenjun; Fan, Enguo; Liu, Qing

    2016-09-01

    We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip. PMID:27370686

  14. Rapid detection of rotavirus in faeces using a dipstick system with monoclonal antibodies and colloidal gold as marker.

    PubMed

    Fernández, D; Valle, I; Llamos, R; Guerra, M; Sorell, L; Gavilondo, J

    1994-07-01

    Rotavirus (RV) is known to be the most common cause of severe diarrhoea in infants and young children, each year leading to an estimated 800,000-900,000 deaths. RV also infects bovines and other species, with high morbidity and mortality. A rapid and simple 'naked-eye' dipstick system was developed to detect human RV in faeces, using nitrocellulose as solid phase, two monoclonal antibodies, and colloidal gold as marker. The system detects 10(4) viral particles (1-2 ng)/g of faeces. For human RV the specificity and sensitivity were 100% when compared with a commercial latex system, and 99% and 98%, respectively, when correlated with traditional RNA-PAGE, and 100% and 98% compared to an ELISA system. PMID:7989446

  15. Use of a monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens.

    PubMed

    Ungar, B L; Yolken, R H; Quinn, T C

    1985-05-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool. PMID:2860814

  16. Liquid chromatography-fluorescence and liquid chromatography-mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody.

    PubMed

    Nowak, Christine; Ponniah, Gomathinayagam; Cheng, Guilong; Kita, Adriana; Neill, Alyssa; Kori, Yekaterina; Liu, Hongcheng

    2016-03-01

    Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography-mass spectrometry (LC-MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease. PMID:26717898

  17. Use of Monoclonal Antibodies in an ELISA for Detecting an Invasive Pest Insect, Liriomyza trifolii (Diptera: Agromyzidae).

    PubMed

    Zhao, W C; Shang, H W; Guo, W; Xu, D; Huang, T Y; Zhu, L X

    2015-04-01

    A monoclonal antibody was prepared by the hybridoma technology. It reacted only with the protein of Liriomyza trifolii (Burgess) and not with that of Chromatomyia horticola Goureau or Liriomyza sativae Blanchard in indirect enzyme-linked immunosorbent assay. It was effective even after being diluted more than 8.192×10(6)-fold. The detection sensitivity of the antibody was 31.3 µg/ml under controlled conditions. Positive reaction was achieved with all laboratory-reared L. trifolii samples, including larvae, pupae, and adults. An indirect enzyme-linked immunosorbent assay system was successfully established to detect L. trifolii in the field. This antibody was successfully used to determine the L. trifolii collected in different locations, from different host plants, and in different seasons. More than 50% of leafminers collected on Brassica chinensis var chinensis, Apium graveolens (Miller) Persoon, Vigna unguiculata (L.) Walpers, Phaseolus vulgaris L., Lactuca sativa L., and Chrysanthemum coronarium (L.) Cassini ex Spach were L. trifolii, indicating that those six plant species might be the preference host plants of L. trifolii. Population of L. trifolii peaked in September, October, or November in Hangzhou, Zhejiang. These results suggest a great potential of using this McAb for precisely identifying L. trifolii and monitoring the population dynamics of L. trifolii in the field. PMID:26470159

  18. Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

    PubMed

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-09-01

    Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:27312522

  19. Monoclonal antibody purification with hydroxyapatite.

    PubMed

    Gagnon, Pete

    2009-06-01

    Hydroxyapatite (HA) has been used for IgG purification since its introduction in the 1950s. Applications expanded to include IgA and IgM in the 1980s, along with elucidation of its primary binding mechanisms and the development of ceramic HA media. With the advent of recombinant monoclonal antibodies, HA was demonstrated to be effective for removal of antibody aggregates, as well as host cell proteins and leached protein A. HA's inherent abilities have been enhanced by the development of elution strategies that permit differential control of its primary binding mechanisms: calcium metal affinity and phosphoryl cation exchange. These strategies support reduction of antibody aggregate content from greater than 60% to less than 0.1%, in conjunction with enhanced removal of DNA, endotoxin, and virus. HA also has a history of discriminating various immunological constructs on the basis of differences in their variable regions, or discriminating Fab fragments from Fc contaminants in papain digests of purified monoclonal IgG. Continuing development of novel elution strategies, alternative forms of HA, and application of robotic high throughput screening systems promise to expand HA's utility in the field. PMID:19491046

  20. Production of thymine glycols in DNA by N-hydroxy-2-naphthylamine as detected by a monoclonal antibody.

    PubMed

    Kaneko, M; Leadon, S A

    1986-01-01

    We have quantitated the production of thymine glycols in DNA following treatment of cultured human fibroblasts or DNA in solution with the carcinogen N-hydroxy-2-naphthylamine. Thymine glycols, detected by using a monoclonal antibody specific to this base damage, were produced in DNA in a dose dependent manner both in vitro and in vivo. Exposure of DNA to N-hydroxy-2-naphthylamine in the presence of catalase and superoxide dismutase, which break down hydrogen peroxide and superoxide anions, respectively, inhibited the production of this base damage. Thymine glycols were efficiently removed from DNA in both normal human fibroblasts and in cells from a patient with xeroderma pigmentosum complementation group A, which are deficient in nucleotide excision repair. PMID:3940211

  1. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  2. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  3. 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.

  4. 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.

  5. Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates.

    PubMed

    Kolberg, J; Aase, A; Michaelsen, T E; Rødal, G

    2001-10-01

    Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein. PMID:11720812

  6. Diagnostic potential of monoclonal antibodies against the capsid protein of chikungunya virus for detection of recent infection.

    PubMed

    Damle, R G; Jayaram, N; Kulkarni, S M; Nigade, K; Khutwad, K; Gosavi, S; Parashar, D

    2016-06-01

    Chikungunya fever is self-limiting. However, neurological and hemorrhagic complications have been seen in recent outbreaks. The clinical manifestations of this disease are similar to those of dengue virus infection, indicating the need for differential diagnosis in areas such as India, which are endemic for both viruses. The aim of the present study was to develop monoclonal antibodies (MAbs) against Chikungunya virus (CHIKV) and assess their use in MAb-based IgM capture ELISA (MAC ELISA). The ELISA detects CHIKV-specific IgM antibodies, a marker of recent infection, in a patient's serum. One IgG1 and two IgM isotype hybrids were obtained. All of the subclones derived from the IgG1 hybrid recognized the C protein of CHIKV. The anti-C MAb ClVE4/D9 was the most promising as a detector antibody in MAC ELISA (C-MAb ELISA) yielding higher positive-to-negative (P/N) ratios. When compared with the CHIKV MAC ELISA kit developed by the National Institute of Virology (NIV), Pune (NIV MAC ELISA), the sensitivity of the test was 87.01 % with 100 % specificity. The positive and negative predictive values (PPV and NPV) were 100 % and 94.47 %, respectively. In precision testing, standard deviation (SD) and coefficient of variation (% CV) values of the C-MAb ELISA were within acceptable limits. The C-MAb ELISA detected anti-CHIKV IgM in serum of patients up to five months after the onset of infection, indicating that anti-C MAbs have strong potential for use in MAC ELISA to detect recent CHIKV infection. PMID:27016930

  7. Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine

    SciTech Connect

    Kvedar, J.C.; Pion, I.A.; Bilodeau, E.B.; Baden, H.P.; Greco, M.A. )

    1992-01-14

    A method providing more sensitive detection of transglutaminase substrates were developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when ({sup 14}C)putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r{sup 2} = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.

  8. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

    PubMed

    Patra, Kailash P; Saito, Mayuko; Atluri, Vidya L; Rolán, Hortensia G; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N; Gotuzzo, Eduardo; Gilman, Robert H; Tsolis, Renee M; Vinetz, Joseph M

    2014-06-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  9. Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

    NASA Astrophysics Data System (ADS)

    Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

    2012-07-01

    The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

  10. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to CPV40 capsid protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAb) were prepared against the 40 kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. By immunoblotting analysis, one MAb, designated MAbCPV40-1, bound to a 40 kDa protein in extracts of C. parvum oocysts, which...

  11. A novel monoclonal antibody specific for cocaine.

    PubMed

    Nakayama, Hiroshi; Kenjyou, Noriko; Shigetoh, Nobuyuki

    2013-08-01

    Detection systems for the illegal drug cocaine need to have a high sensitivity and specificity for cocaine and to be relatively easy to use. In the current study, a monoclonal antibody (MAb) with a high specificity for cocaine was produced. Enzyme-linked immunosorbent assay and fluorescence quenching immunoassay were used to screen the hybridomas. The MAb S27Y (IgG1) was shown to be sensitive and specific for cocaine and quenched fluorescence. Thus, S27Y has the potential to be used in screening assays for the rapid and sensitive detection of cocaine. PMID:23909419

  12. Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies

    SciTech Connect

    Schlimok, G.; Funke, I.; Holzmann, B.; Goettlinger, G.; Schmidt, G.; Haeser, H.; Swierkot, S.; Warnecke, H.H.; Schneider, B.; Koprowski, H.; Riethmueller, G.

    1987-12-01

    The detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-1A epithelial antigen the authors identified immunocytochemically tumor cells in bone marrow of patients with breast cancer and colorectal cancer at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients. Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology and histology. In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. They found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy they demonstrated in 3 patients that infused monoclonal antibody 17-1A can label single tumor cells in bone marrow in vivo. They then used this single approach to follow up on 7 patients undergoing 17-1A therapy in an adjuvant clinical trial.

  13. Development of a multiplex lateral flow strip test for foot-and-mouth disease virus detection using monoclonal antibodies.

    PubMed

    Yang, Ming; Caterer, Nigel R; Xu, Wanhong; Goolia, Melissa

    2015-09-01

    Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses. The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1. The newly developed multiplex-LFI strip test specifically identified serotype O (n=46), A (n=45) and Asia 1 (n=17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively. The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites. PMID:25977185

  14. Generation of monoclonal antibodies against MGA and comparison of their application in breast cancer detection by immunohistochemistry.

    PubMed

    Duan, Cuimi; Yang, Xiqin; Zhang, Xuhui; Feng, Jiannan; Liu, Zhiqiang; Que, Haiping; Johnson, Heather; Zhao, Yanfeng; Fan, Yawen; Lu, Yinglin; Zhang, Heqiu; Huang, Yan; Xiu, Bingshui; Feng, Xiaoyan

    2015-01-01

    Mammaglobin A (MGA) is an organ specific molecular biomarker for metastatic breast cancer diagnosis. However, there are still needs to develop optimal monoclonal antibodies (mAbs) to detect MGA expression in breast carcinoma by immunohistochemistry. In this study, we first generated mAbs against MGA. Then, we used epitope prediction and computer-assisted structural analysis to screen five dominant epitopes and identified mAbs against five epitopes. Further immunohistochemical analysis on 42 breast carcinoma specimens showed that MHG1152 and MGD785 had intensive staining mainly in membrane, while CHH11617, CHH995 and MJF656 had more intensive staining within the cytoplasm. MGA scoring results showed that MJF656 had the highest rate (92.8%) of positive staining among five mAbs, including higher staining intensity when compared with that of MHG1152 (p < 0.01) and CHH995 (p < 0.05) and the highest the mean percentage of cells stained among mAbs. Furthermore, we analyzed the relationship of positive staining rate by mAbs with patient clinical characteristics. The results suggest that MJF656 was able to detect MGA expression, especially in early clinical stage, low grade and lymph node metastasis-negative breast carcinoma. In conclusion, our study generated five mAbs against MGA and identified the best candidate for detection of MGA expression in breast cancer tissues. PMID:26272389

  15. A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies

    PubMed Central

    Stein, H; Bob, R; Dürkop, H; Erck, C; Kämpfe, D; Kvasnicka, H-M; Martens, H; Roth, A; Streubel, A

    2016-01-01

    Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method. PMID:26202929

  16. Monoclonal Antibodies for the Treatment of Cancer

    PubMed Central

    Shuptrine, Casey; Surana, Rishi; Weiner, Louis M.

    2012-01-01

    Over the past decade, the clinical utility of monoclonal antibodies has been realized and antibodies are now a mainstay for the treatment of cancer. Antibodies have the unique capacity to target and kill tumor cells while simultaneously activating immune effectors to kill tumor cells through the complement cascade or antibody-dependent cellular cytotoxicity (ADCC). This multifaceted mechanism of action combined with target specificity underlies the capacity of antibodies to elicit anti-tumor responses while minimizing the frequency and magnitude of adverse events. This review will focus on mechanisms of action, clinical applications and putative mechanisms of resistance to monoclonal antibody therapy in the context of cancer. PMID:22245472

  17. Production of monoclonal antibodies against Nosema bombycis and their utility for detection of pebrine infection in Bombyx mori L.

    PubMed

    Shamim, M; Ghosh, D; Baig, M; Nataraju, B; Datta, R K; Gupta, S K

    1997-11-01

    Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 micrograms each per ml of 0.4% latex beads) could detect 1 x 10(5) Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1 x 10(4) spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination. PMID:9358341

  18. Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously.

    PubMed

    Sithigorngul, Paisarn; Hajimasalaeh, Warunee; Longyant, Siwaporn; Sridulyakul, Pattarin; Rukpratanporn, Sombat; Chaivisuthangkura, Parin

    2009-12-01

    Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei. PMID:19654023

  19. Monoclonal antibodies to snakehead, Channa striata immunoglobulins: detection and quantification of immunoglobulin-positive cells in blood and lymphoid organs.

    PubMed

    Sood, Neeraj; Chaudhary, Dharmendra K; Rathore, Gaurav; Singh, Akhilesh; Lakra, W S

    2011-02-01

    Snakehead Channa striata is an important freshwater food fish in many Southeast Asian countries. Three monoclonal antibodies (C9, C10 and D10) were developed against purified serum immunoglobulins of Channa striata (Cs-Ig) and characterized. C9 and D10 MAbs were specific to heavy chain, while C10 MAb detected only unreduced Cs-Ig in western blotting. In competitive ELISA, C9 and C10 MAbs were specific to C. striata Ig and showed no cross reactivity with serum Ig of other fish species i.e. Channa punctatus, Channa marulius, Clarias batrachus and Labeo rohita. D10 MAb showed reactivity to serum Ig of C. striata and C. marulius. In FACS analysis of gated lymphocytes, the percentage of Ig+ cells detected by C9 MAb was 18.2%, 27.7% and 10.3% in blood, spleen and kidney, respectively (n=3, body weight 500-600 g). However, only a few cells (0.5%) were found to be Ig+ in thymus (n=5). C9 MAb was also successfully employed to demonstrate Ig+ cells in blood smears and formalin fixed sections of spleen and kidney. These findings suggest that the spleen plays an important role in humoral immunity as compared to head kidney. Further, these MAbs can be useful immunological tool in monitoring health status of cultured C. striata. PMID:21167285

  20. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of bifenthrin in a chemical soil barrier.

    PubMed

    Hua, Xiude; Liu, Xiaofeng; Yin, Wei; Xia, Yazhong; Zhou, Qiujun; Lu, Yiwen; Li, Wei; Shi, Haiyan; Liu, Fengquan; Wang, Minghua

    2015-01-01

    A sensitive monoclonal antibody-based enzyme monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed to detect bifenthrin in a chemical soil barrier for termite control. LBc ((2-methyl[1,1-biphenyl]-3-methoxy) carbonyl propionic acid) was conjugated to bovine serum albumin (BSA) for producing monoclonal antibody. After optimization, the IC50 and limit of detection (LOD, IC10) were 0.05 mg L(-1) and 0.004 mg L(-1), respectively. The sensitivity was improved 40-fold compared to polyclonal antibody-based ELISA reported earlier. No cross-reactivity was measured for the other analogues such as cyhalothrin, cyhalothric acid, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, ethofenprox and tetramethrin except 2-methyl-3-biphenylmethyanol. Spiked recoveries were between 83.5% and 104.7% for the detection of bifenthrin in loess, red soil and black soil. All the relative standard deviations (RSDs) were less than or equal to 15.0%. Moreover, the ELISA for authentic samples showed reliability and high correlation with gas chromatography. The developed ELISA is an alternative tool for simple, sensitive and accurate monitoring of bifenthrin in chemical soil barriers. PMID:25261814

  1. Improved performance and quantitative detection of copro-antigens by a monoclonal antibody based ELISA to diagnose human opisthorchiasis.

    PubMed

    Watwiengkam, Nattaya; Sithithaworn, Jiraporn; Duenngai, Kunyarat; Sripa, Banchob; Laha, Thewarach; Johansen, Maria Vang; Sithithaworn, Paiboon

    2013-12-01

    Copro-antigen detection has been advocated as a promising method for diagnosis of opisthorchiasis, particularly in people that harbored light infection or have had recent drug treatment. This study aimed to improve performance of a monoclonal antibody-based enzyme-linked immunosorbent assay (Mab-ELISA) for detection of Opisthorchis viverrini copro-antigen and assess the correlation between copro-antigen and intensity of infection. Four different treatment methods of 71 samples from the Lawa endemic area, Khon Kaen province were assessed for copro-antigen detection, namely (1) phosphate buffer saline (PBS), (2) heating (70°C 30min), (3) alkaline (pH 9.6 in carbonate buffer), and (4) trichloroacetic acid (TCA) treatment. Comparison of these protocols showed that the TCA method gave the best performance in discriminating O. viverrini positive and negative samples with high sensitivity (97.9%) and moderate specificity (54.2%) compared with other methods. Application of TCA-based Mab-ELISA method for antigen detection in fecal samples obtained from an endemic area of opisthorchiasis revealed that 86 of 141 samples (61.0%) were positive compared with 68 (48.2%) by PBS-based protocol, while the formalin ethyl-acetate concentration technique (FECT) yielded a positive proportion of 71.6%. Among 40 egg-negative samples confirmed by a gold standard parasitological method (FECT) from the same endemic area, 19 (47.5%) were positive by the TCA-based while only 6 (15%) were positive by PBS-based Mab-ELISA protocol. In addition, levels of antigen detection significantly correlated with intensity of infection (egg per gram feces). The results show that the improved Mab-ELISA method has high sensitivity and also quantifiable diagnosis of opisthorchiasis. PMID:24055716

  2. Production of thymine glycols in DNA by radiation and chemical carcinogens as detected by a monoclonal antibody.

    PubMed

    Leadon, S A

    1987-06-01

    In order to understand the role in carcinogenesis of damage indirectly induced by chemical carcinogens, it is important to identify the primary DNA lesions. We have measured the formation and repair of one type of DNA modification, 5,6-dihydroxydihydrothymine (thymine glycol), following exposure of cultured human cells to the carcinogens N-hydroxy-2-naphthylamine or benzo(a)pyrene. The efficiency of production of thymine glycols in DNA by these carcinogens was compared to that by ionizing radiation and ultraviolet light. Thymine glycols were detected using a monoclonal antibody against this product in a sensitive immunoassay. We found that thymine glycols were produced in DNA in a dose dependent manner after exposure to the carcinogens and that their production was reduced if either catalase or superoxide dismutase or both were present at the time of treatment. The efficiency of thymine glycol production following exposure to the chemical carcinogens was greater than that following equi-toxic doses of radiation. Thymine glycols were efficiently removed from the DNA of human cells following treatment with either the chemical carcinogens, ionizing radiation or ultraviolet light. PMID:3477281

  3. Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads

    PubMed Central

    Luciani, Mirella; Di Febo, Tiziana; Zilli, Katiuscia; Di Giannatale, Elisabetta; Armillotta, Gisella; Manna, Laura; Minelli, Fabio; Tittarelli, Manuela; Caprioli, Alfredo

    2016-01-01

    Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 103 E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 102 E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 101 E. coli O104:H4 initial load). The specificity was 100%. PMID:27379071

  4. Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads.

    PubMed

    Luciani, Mirella; Di Febo, Tiziana; Zilli, Katiuscia; Di Giannatale, Elisabetta; Armillotta, Gisella; Manna, Laura; Minelli, Fabio; Tittarelli, Manuela; Caprioli, Alfredo

    2016-01-01

    Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%. PMID:27379071

  5. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

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

    SciTech Connect

    DeLand, F.H. )

    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. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    NASA Astrophysics Data System (ADS)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  8. Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions.

    PubMed Central

    Saliki, J T; House, J A; Mebus, C A; Dubovi, E J

    1994-01-01

    A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation. PMID:8051266

  9. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  10. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  11. Mouse monoclonal antibodies against estrogen receptor.

    PubMed

    De Rosa, Caterina; Rossi, Valentina; Abbondanza, Ciro

    2014-01-01

    The production of monoclonal antibodies, by cloning hybridoma derived from the fusion of myeloma cells and spleen lymphocytes, has allowed to obtain great advances in many fields of biological knowledge. The use of specific antibodies to the estrogen receptor, in fact, has been an invaluable method to bring out its mechanisms of action and its effects, both genomic and extra-genomic. Here we describe, step by step, the production of monoclonal antibodies, starting from protocol for antigen preparation to the selection of antibody-secreting hybridoma. PMID:25182770

  12. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment.

    PubMed

    Krause, B J; Baum, R P; Staib-Sebler, E; Lorenz, M; Niesen, A; Hör, G

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. PMID:9044881

  13. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic. PMID:24037837

  14. Integrated microfluidic capillary electrophoresis-electrospray ionization devices with online MS detection for the separation and characterization of intact monoclonal antibody variants.

    PubMed

    Redman, Erin A; Batz, Nicholas G; Mellors, J Scott; Ramsey, J Michael

    2015-02-17

    Here, we demonstrate an integrated microfluidic capillary electrophoresis-electrospray ionization (CE-ESI) device for the separation of intact monoclonal antibody charge variants with online mass spectrometric (MS) identification. The need for dynamic coating and zwitterionic background electrolyte (BGE) additives has been eliminated by utilizing surface chemistry within the device channels to control analyte adsorption and electroosmotic flow (EOF) while maintaining separation efficiency. The effectiveness of this strategy was illustrated with the separation of charge variants of Infliximab. Three major species corresponding to C-terminal lysine variants were separated with an average resolution of 0.80 and identified by mass difference. In addition to the lysine variants, masses were determined for minor acidic and basic species. The separation of these variants prior to MS analysis facilitated the identification of glycosylation patterns for each of the variants. The general applicability of this method was demonstrated by analyzing two additional monoclonal antibody species: an IgG2 antibody and an IgG1 antibody conjugate. The IgG2 proved to have similar modifications to Infliximab with lower relative abundances of the lysine variants. Analysis of the IgG1 drug conjugate further exemplified the advantages of MS detection; differences in the extent of antibody conjugation were detectable despite limited CE resolution. The CE-ESI-MS methodology described here is a rapid and generic strategy for the separation of intact mAb charge variants and facilitates the identification of variants through MS detection. PMID:25569459

  15. Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

    PubMed

    Shankaran, Dhesingh Ravi; Kawaguchi, Toshikazu; Kim, Sook Jin; Matsumoto, Kiyoshi; Toko, Kiyoshi; Miura, Norio

    2006-11-01

    Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs. PMID:16900380

  16. Trends in Malignant Glioma Monoclonal Antibody Therapy

    PubMed Central

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  17. Kinetics of intralymphatically delivered monoclonal antibodies

    SciTech Connect

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

    1985-05-01

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

  18. Development of a highly sensitive monoclonal antibody based ELISA for detection of benzo[a]pyrene in potable water.

    PubMed

    Matschulat, Diana; Deng, Anping; Niessner, Reinhard; Knopp, Dietmar

    2005-07-01

    In Europe, a limit value of 10 ng L(-1) was set by the European Commission for benzo[a]pyrene (B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore, sensitive and reliable methods are needed to evaluate its presence. We report here on the development of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potable water. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. The immunoassay with the least interference and the best sensitivity was optimized and characterized. As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]P solubilization for use with the developed ELISA. With the purified antibody (clone 22F12) the average IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was 65 ng L(-1) and 24 ng L(-1), respectively. From the 16 EPA-designated PAHs, only chrysene, indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than 20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthracene and benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electric conductivity 1 microS cm(-1)-2.5 mS cm(-1)), and inorganic ions (sodium, copper, iron, aluminium, manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to the Council Directive) on both signal and sensitivity of the ELISA was studied. No significant influence of these parameters on the ELISA competition curve was found. We suggest that the optimized ELISA can be used to monitor potable water samples without previous extraction from the samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levels fall close to the limit value of the new drinking water directive. PMID:15965533

  19. 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, ΔGu, 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 ΔGu versus temperature data from fluorescence gave a ΔCp 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. ΔGu 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

  20. Use of monoclonal antibodies in the sensitive detection and neutralization of botulinum neurotoxin serotype B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed sensitive electrochemiluminescent (ECL) i...

  1. Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.

    PubMed

    Gutay-Tóth, Zsuzsanna; Fenyvesi, Ferenc; Bársony, Orsolya; Szente, Lajos; Goda, Katalin; Szabó, Gábor; Bacsó, Zsolt

    2016-03-01

    The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb. PMID:26704667

  2. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  3. Monoclonal antibodies in acute lymphoblastic leukemia

    PubMed Central

    O’Brien, Susan; Ravandi, Farhad; Kantarjian, Hagop

    2015-01-01

    With modern intensive combination polychemotherapy, the complete response (CR) rate in adults with acute lymphoblastic leukemia (ALL) is 80% to 90%, and the cure rate is 40% to 50%. Hence, there is a need to develop effective salvage therapies and combine novel agents with standard effective chemotherapy. ALL leukemic cells express several surface antigens amenable to target therapies, including CD20, CD22, and CD19. Monoclonal antibodies target these leukemic surface antigens selectively and minimize off-target toxicity. When added to frontline chemotherapy, rituximab, an antibody directed against CD20, increases cure rates of adults with Burkitt leukemia from 40% to 80% and those with pre-B ALL from 35% to 50%. Inotuzumab ozogamicin, a CD22 monoclonal antibody bound to calicheamicin, has resulted in marrow CR rates of 55% and a median survival of 6 to 7 months when given to patients with refractory-relapsed ALL. Blinatumomab, a biallelic T cell engaging the CD3-CD19 monoclonal antibody, also resulted in overall response rates of 40% to 50% and a median survival of 6.5 months in a similar refractory-relapsed population. Other promising monoclonal antibodies targeting CD20 (ofatumumab and obinutuzumab) or CD19 or CD20 and bound to different cytotoxins or immunotoxins are under development. Combined modalities of chemotherapy and the novel monoclonal antibodies are under investigation. PMID:25999456

  4. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition.

    PubMed

    Green, M R; Couchman, J R

    1985-09-01

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed, including the possibility that latent EGF receptors, unable to bind [125I]EGF, may be present in some differentiating epithelial compartments. PMID:2411822

  5. Rapid Detection and Identification of Streptococcus Iniae Using a Monoclonal Antibody-Based Indirect Fluorescent Antibody Technique

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcus iniae is among the major pathogens of a large number of fish species cultured in fresh and marine recirculating and net pen production systems . The traditional plate culture technique to detect and identify S. iniae is time consuming and may be problematic due to phenotypic variations...

  6. A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader.

    PubMed

    Sadler, A M; Krausa, P; Marsh, S G; Heyes, J M; Bodmer, J G

    1992-04-27

    A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies. PMID:1583310

  7. Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection.

    PubMed

    Ma, Yi; Ni, Chao; Dzakah, Emmanuel E; Wang, Haiying; Kang, Keren; Tang, Shixing; Wang, Jihua; Wang, Jufang

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection. PMID:27069923

  8. Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection

    PubMed Central

    Ma, Yi; Ni, Chao; Dzakah, Emmanuel E.; Wang, Haiying; Kang, Keren; Tang, Shixing; Wang, Jihua; Wang, Jufang

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection. PMID:27069923

  9. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for luteoloside detection in Flos Lonicerae Japonicae.

    PubMed

    Zhang, Bo; Nan, Tiegui; Zhan, Zhilai; Kang, Liping; Yang, Jian; Yuan, Yuan; Wang, Baomin; Huang, Luqi

    2016-09-01

    Flos Lonicerae Japonicae (FLJ), the flower bud of Lonicera japonica Thunb. (Caprifoliaceae), is a widely used traditional Chinese medicine with various pharmacological activities. Luteoloside is a major active compound and a quality control marker of FLJ. Luteolin-7-O-glucuronide (LG), an analog of luteoloside, was conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) to create the immunogen and coating antigen, respectively. A sensitive and specific monoclonal antibody (mAb), designated as mAb3A4, was generated with LG-BSA. To screen the authenticity and quality of FLJ, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The concentration of luteoloside producing 50 % inhibition and the working range of the icELISA were 42.3 and 9.1-258.1 μg L(-1), respectively. The icELISA showed cross-reactivity values of 2414, 402, 230, and <1 % for LG, baicalin, scutellarin, and other analogs of luteoloside, respectively. The average recovery of luteoloside in the FLJ samples as determined by icELISA ranged from 83.0 to 112.5 %. The luteoloside content was determined for different Lonicera herbal samples with icELISA, and the results were confirmed by high-performance liquid chromatography analysis. Thus, this icELISA is suitable for the quality assurance of FLJ samples. Graphical abstract Specific monoclonal antibody-based enzyme-linked immunosorbent assay for luteoloside. PMID:26892641

  10. Detection of soluble tumor-associated antigens in sera and effusions using novel monoclonal antibodies, KL-3 and KL-6, against lung adenocarcinoma.

    PubMed

    Kohno, N; Akiyama, M; Kyoizumi, S; Hakoda, M; Kobuke, K; Yamakido, M

    1988-09-01

    Two novel monoclonal antibodies, KL-3 (IgM) and KL-6 (IgG1), which can detect soluble antigens in sera and effusions (molecular weights greater than 1,000 K) were produced against human pulmonary adenocarcinoma VMRC-LCR cells. KL-3 and KL-6 antibodies reacted with asialo- and sialo-carbohydrate antigenic determinants, respectively. Both carbohydrate epitopes appear, from competitive inhibition studies, to be different from Lex, Ley, sialyl Lea and sialyl Lexi which were recognized with FH2, AH6, NS19-9 and FH6 antibodies, respectively. Using an enzyme linked immunosorbent assay, elevated KL-6 antigen levels were frequently observed in the sera of patients with lung adenocarcinoma [52% (17/33)], pancreatic cancer [44% (4/9)] and breast cancer [40% (8/20)], but infrequently in the sera of patients with lung squamous cell carcinoma [18% (4/22)], lung small cell carcinoma [8% (1/13)], gastric cancer [0% (0/19)], colorectal cancer [0% (0/8)] and hepatocellular cancer [13% (1/8)]. The levels and positive rates of serum KL-6 antigen increased with the progression of clinical stage of lung adenocarcinoma. In pleural effusions, the prevalences of lung adenocarcinoma cases with elevated levels of KL-3 and KL-6 antigens were 76% (13/17) and 82% (14/17), respectively. These monoclonal antibodies can define novel soluble antigens in sera and effusions which could be useful in tumor diagnoses and for monitoring tumor progression. PMID:3411786

  11. 5th Annual Monoclonal Antibodies Conference

    PubMed Central

    2009-01-01

    The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca). PMID:20073132

  12. [Current situations and the future prospect of monoclonal antibody products].

    PubMed

    Yamaguchi, Teruhide

    2014-01-01

    Monoclonal antibody products and monoclonal antibody-based biopharmaceuticals have shown considerable effectiveness in the treatment for variety of diseases; cancer, auto-immune/auto-inflammation diseases and so on. Significant advance in monoclonal antibody products for cancer treatments was made with antibody-drug conjugates (ADC), and antibodies for blockade of immune checkpoints. Already 3 ADCs and 2 anti-immune-checkpoint antibodies products have been approved, and these monoclonal antibody-related product pipelines reach over 30. On the other hand, EU approved first monoclonal-antibody biosimilar, RemsimaTM (infliximab), suggesting that other monoclonal-antibody biosmilars will follow to the market. In this paper, several new issues about monoclonal antibody products will be discussed. PMID:25707201

  13. Development of a monoclonal antibody against the left wing of ciguatoxin CTX1B: thiol strategy and detection using a sandwich ELISA.

    PubMed

    Tsumuraya, Takeshi; Takeuchi, Katsutoshi; Yamashita, Shuji; Fujii, Ikuo; Hirama, Masahiro

    2012-09-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL. PMID:22575284

  14. Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

    PubMed

    Schroeijers, A B; Scheffer, G L; Reurs, A W; Pijnenborg, A C; Abbondanza, C; Wiemer, E A; Scheper, R J

    2001-11-01

    The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR. PMID:11668191

  15. 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.

  16. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    PubMed

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-01

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples. PMID:15381269

  17. 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...

  18. Monoclonal antibodies against chicken interleukin-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs that were produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mA...

  19. Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers.

    PubMed

    Gorris, M T; Alarcon, B; Lopez, M M; Cambra, M

    1994-06-01

    Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants. PMID:16349293

  20. Generation of a monoclonal antibody against Mycoplasma spp. following accidental contamination during production of a monoclonal antibody against Lawsonia intracellularis.

    PubMed

    Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

    2012-03-01

    This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis. PMID:22247145

  1. 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.

  2. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  3. Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA.

    PubMed

    Thornton, Christopher R; Slaughter, David C; Davis, R Michael

    2010-10-15

    Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes. PMID:20850192

  4. Comparison of gallium-67 versus indium-111 monoclonal antibody (96. 5, ZME-018) in detection of human melanoma in athymic mice

    SciTech Connect

    Chan, S.M.; Hoffer, P.B.; Maric, N.; Zoghbi, S.S.; Kirkwood, J.M.; Ernstoff, M.S.; Duray, P.H.; Gerich, B.

    1987-09-01

    We compared the biodistribution of two radiolabeled, whole, tumor selective monoclonal antibodies (( /sup 111/In)96.5, (/sup 111/In)ZME-018) to /sup 67/Ga in nude mice bearing a human melanoma known to express p97 antigen. Localization of gallium was determined 48 hr following i.v. injection. Localization of the radiolabeled antibodies was determined at 3 days and 7 days following i.v. injection. All agents showed more or less similar absolute tumor uptake which varied between 22% and 36% of the injected dose per gram of tumor. Only the tumor uptake of (/sup 111/In)96.5 antibody at 7 days was significantly lower than the /sup 67/Ga uptake at 48 hr. However, uptake in normal tissues was generally higher for both antibodies at 3 and 7 days than for /sup 67/Ga uptake at 48 hr. Therefore, the tumor-to-blood ratio for /sup 67/Ga was tenfold higher than that for either antibody, the tumor-to-muscle ratio was twofold higher. Bone was the only organ in which the tumor-to-organ ratio was consistently higher with radiolabeled antibody than with /sup 67/Ga. The tumor-to-liver and tumor-to-intestine ratios were comparable. Localization of the two tumor selective antibodies was greater than a nonspecific control antibody (( /sup 111/In)CEA) and change in specific activity from 0.17 mCi/mg to 3.3 mCi/mg did not influence localization. From these animal data it may be anticipated that tumor imaging with (/sup 111/In)96.5 or (/sup 111/In)ZME-018 will not be superior to imaging with 67Ga for detection of melanoma.

  5. Application of monoclonal antibodies in a rapid sandwich dot-enzyme linked immunosorbent assay for identification and antigen detection of Leptospira serovars.

    PubMed

    Sharma, Rashmi; Tuteja, Urmil; Khushiramani, Rekha; Shukla, Jyoti; Batra, H V

    2008-04-01

    Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars--namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes. PMID:18642676

  6. Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection.

    PubMed

    Sengupta, K; Das, P; Johnson, T M; Chaudhuri, P P; Das, D; Nair, G B

    1993-01-01

    Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool. PMID:8292992

  7. Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

    PubMed

    Jeoung, Mee Hyun; Kim, Taek-Keun; Shim, Hyunbo; Lee, Sukmook

    2016-09-01

    It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera. PMID:27288967

  8. Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

    PubMed

    Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M

    2016-10-01

    changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7-12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology. PMID:27260838

  9. 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.

  10. Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

    PubMed

    Giddens, John P; Wang, Lai-Xi

    2015-01-01

    Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications. PMID:26082235

  11. Development and characterization of monoclonal antibodies against avian reovirus sigma C protein and their application in detection of avian reovirus isolates.

    PubMed

    Hsu, Chien J; Wang, Chi Y; Lee, Long H; Shih, Wen L; Chang, Chi I; Cheng, Hsueh L; Chulu, Julius L C; Ji, Wen T; Liu, Hung J

    2006-08-01

    Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates. PMID:16854646

  12. Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein

    PubMed Central

    2014-01-01

    Background Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen documented to cause respiratory disease, mastitis, and arthritis in cattle throughout China since 2008. Here, we report the development of a direct competitive enzyme-linked immunosorbent assay (Dc-ELISA) to detect M. bovis antibody. Results We used a recombinant P48 protein and monoclonal antibody (mAb) 10E. MAb 10E, prepared against the recombinant P48 protein of M. bovis, identified all M. bovis strains with no cross-reactivity with other related pathogens. Coating micro plates with P48 protein instead of whole M. bovis cells as well as the use of mAb 10E produced a specific and sensitive Dc-ELISA for M. bovis antibody detection with a cut-off percent inhibition (PI) value of 32%. Compared with two commercial indirect ELISA (i-ELISA) kits, our Dc-ELISA offered a higher positive detection rate in 165 clinical bovine serum samples. Conclusions A rapid, sensitive, and reliable serological diagnosis method was developed for M. bovis, which can facilitate M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology of M. bovis. PMID:24533468

  13. Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin

    PubMed Central

    2013-01-01

    Background PAM4, an antibody that has high specificity for pancreatic ductal adenocarcinoma (PDAC), compared to normal pancreas, benign lesions of the pancreas, and cancers originating from other tissues, is being investigated as a biomarker for early detection, as well as antibody-targeted imaging and therapy. Therefore, the identity of the antigen bound by this monoclonal antibody (MAb) can provide information leading to improved use of the antibody. Prior results suggested the antigen is a mucin-type glycoprotein rich in cysteine disulfide bridges that provide stable conformation for the PAM4-epitope. Methods Indirect and sandwich enzyme immunoassays (EIA) were performed to compare and contrast the reactivity of PAM4 with several anti-mucin antibodies having known reactivity to specific mucin species (e.g., MUC1, MUC4, MUC5AC, etc.). Studies designed to block reactivity of PAM4 with its specific antigen also were performed. Results We demonstrate that MAbs 2-11 M1 and 45 M1, each reactive with MUC5AC, are able to provide signal in a heterologous sandwich immunoassay where PAM4 is the capture antibody. Further, we identify MAbs 21 M1, 62 M1, and 463 M1, each reactive with MUC5AC, as inhibiting the reaction of PAM4 with its specific epitope. MAbs directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6 are not reactive with PAM4-captured antigen, nor are they able to block the reaction of PAM4 with its antigen. Conclusions These data implicate MUC5AC as a specific mucin species to which PAM4 is reactive. Furthermore, this realization may allow for the improvement of the current PAM4 serum-based immunoassay for detection of early-stage PDAC by the application of anti-MUC5AC MAbs as probes in this sandwich EIA. PMID:24257318

  14. Monoclonal antibodies: the promise and the reality.

    PubMed

    Coons, T

    1995-01-01

    Monoclonal antibodies, or "MoAbs," have revolutionized clinical approaches to diagnostic imaging and therapy of many diseases. The use of MoAbs for diagnosing and treating cancer has been especially promising. However, the full potential of these "magic bullets" has yet to be realized. This article examines the current and potential uses of MoAbs, describes problems with the technology and looks at potential solutions. Along with descriptions of how MoAbs are made and prepared for use in the clinic, the article provides examples of the ways in which MoAbs can be used to complement and expand the information obtained from standard diagnostic imaging modalities. Specific examples of the use of monoclonal antibodies for treating cancer and other diseases also are provided. PMID:7491408

  15. 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

  16. Innovative Monoclonal Antibody Therapies in Multiple Sclerosis

    PubMed Central

    Kieseier, Bernd C.

    2008-01-01

    The recent years have witnessed great efforts in establishing new therapeutic options for multiple sclerosis (MS), especially for relapsing–remitting disease courses. In particular, the application of monoclonal antibodies provide innovative approaches allowing for blocking or depleting specific molecular targets, which are of interest in the pathogenesis of MS. While natalizumab received approval by the US Food and Drug Administration and the European Medicines Agency in 2006 as the first monoclonal antibody in MS therapy, rituximab, alemtuzumab, and daclizumab were successfully tested for relapsing-remitting MS in small cohorts in the meantime. Here, we review the data available from these recent phase II trials and at the same time critically discuss possible pitfalls which may be relevant for clinical practice. The results of these studies may not only broaden our therapeutic options in the near future, but also provide new insights into disease pathogenesis. PMID:21180564

  17. 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.

  18. Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor

    PubMed Central

    Aamelfot, Maria; Weli, Simon C; Dale, Ole B; Koppang, Erling O; Falk, Knut

    2013-01-01

    Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon. PMID:23439106

  19. Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

    PubMed

    Wongwairot, Sirima; Kueakhai, Pornanan; Changklungmoa, Narin; Jaikua, Wipaphorn; Sansri, Veerawat; Meemon, Krai; Songkoomkrong, Sineenart; Riengrojpitak, Suda; Sobhon, Prasert

    2015-01-01

    Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans. PMID:25324133

  20. Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection.

    PubMed

    Lesur, Antoine; Varesio, Emmanuel; Hopfgartner, Gérard

    2010-01-01

    Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 degrees C and 37 degrees C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 degrees C with a 750rpm mixing applied for at least 30min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 degrees C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version ((13)C(4), (15)N(1)) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation. PMID:19939394

  1. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    PubMed Central

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  2. Monoclonal IgA Antibodies for Aflatoxin Immunoassays.

    PubMed

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2-50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  3. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    PubMed

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. PMID:26416205

  4. Selection of monoclonal antibodies for the identification of D variants: ability to detect weak D and to split epD2, epD5 and epD6/7.

    PubMed

    Jones, J; Filbey, D

    1996-01-01

    Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and RoHar cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 micrograms/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (Du) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with RoHarr cells, while only one IgG antibody was positive. PMID:8740011

  5. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  6. A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences.

    PubMed

    Wernike, Kerstin; Brocchi, Emiliana; Cordioli, Paolo; Sénéchal, Yann; Schelp, Christian; Wegelt, Anne; Aebischer, Andrea; Roman-Sosa, Gleyder; Reimann, Ilona; Beer, Martin

    2015-01-01

    A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics. PMID:25889366

  7. 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.

  8. Use of a Monoclonal Antibody-Based Immunoassay for the Detection and Quantification of Heliscus lugdunensis Colonizing Alder Leaves and Roots.

    PubMed

    Bermingham, S.; Dewey, F.M.; Fisher, P.J.; Maltby, L.

    2001-12-01

    A genus-specific monoclonal antibody, NG-CF10, raised in a previous study to the fungal pathogen Nectria galligena, was found to recognize the aquatic hyphomycete Heliscus lugdunensis (anamorph) and its teleomorph Nectria lugdunensis. Using this MAb in a plate trapped antigen- ELISA we could detect and determine the biomass of Heliscus lugdunensis in mixed assemblages in both naturally occurring and artificially inoculated leaves and roots of Alnus glutinosa trees. Initial studies indicate that the biomass associated with naturally occurring leaf material is significantly lower than that recorded with laboratory inoculated leaves, suggesting that biomass production is limited in the natural environment. Significantly lower biomass was associated with roots when compared with leaf material, which supports the proposition that rather than a major substrate for the growth of aquatic hyphomycetes, roots act as a refugium for fungal growth. PMID:12024233

  9. Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay.

    PubMed Central

    Burns, J W; Welch, S K; Nakata, S; Estes, M K

    1989-01-01

    Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus. Images PMID:2536755

  10. Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment.

    PubMed

    Zhen, Le; Ford, Nicole; Gale, Debra K; Roesijadi, Guritno; Rorrer, Gregory L

    2016-05-15

    A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene). PMID:26774089

  11. Comparison of an acid-fast stain and a monoclonal antibody-based immunofluorescence reagent for the detection of Cryptosporidium oocysts in faecal specimens from cattle and pigs.

    PubMed

    Quílez, J; Sánchez-Acedo, C; Clavel, A; del Cacho, E; López-Bernad, F

    1996-12-01

    A commercially available direct immunofluorescence (IF) assay with monoclonal antibodies (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) was compared with the modified Ziehl-Neelsen (MZN) acid-fast technique for the detection of Cryptosporidium oocysts in faecal samples from cattle and pigs. Stool specimens individually collected from 108 bovines and 90 pigs were examined in a blind test. The results of the two procedures corresponded (both positive or negative) in 102 (94.4%) cattle samples and 80 (88.9%) pig faecal samples. However, the remaining six (5.5%) cattle specimens and 10 (11.1%) pig stool samples, all of them harboring few oocysts (0-1 oocysts per 20 x field), were negative by MZN and positive by IF. False-negative results of the acid-fast stain occurred in suckling (17.2% of discrepant results) and weaned calves (2.9%) as well as weaned piglets (43.7%) and fattening pigs (10%). Stool specimens from the remaining age groups were negative by both techniques. The MacNemar's chi-square test showed that differences between both methods were statistically significant (P < 0.05). Compared with immunofluorescence procedure, the sensitivity of MZN technique in samples from cattle and pigs was 79.3% and 67.7% and the negative predictive value was 92.9% and 85.5% respectively. The specificity and positive predictive values of the acid-fast stain were 100% in both animal species. It is concluded that the monoclonal antibody-based immunofluorescence reagent evaluated is more efficient that the MZN technique, especially for detecting a low number of Cryptosporidium oocysts, in faecal specimens from both cattle and pigs. PMID:9011016

  12. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    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.

  13. Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

    PubMed

    Brindle, R J; Bryant, T N; Draper, P W

    1989-03-01

    A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1. PMID:2654183

  14. Proficiency monitoring of monoclonal antibody cocktail-based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs.

    PubMed

    Lee, Kenneth W; Blankenship, Karen; McKinney, Brennan; Kern, Gerhard; Buch, Jesse; Greenwood, Janice; Brazis, Pilar; Drouet, Laurent; Tambone, Cecilia; Faas, Rebecca; Weaver, Gareth

    2015-07-01

    The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA. PMID:26069227

  15. Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.

    PubMed

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  16. Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

    PubMed Central

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  17. 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.

  18. The Role of Monoclonal Antibodies in the Management of Leukemia

    PubMed Central

    Al-Ameri, Ali; Cherry, Mohamad; Al-Kali, Aref; Ferrajoli, Alessandra

    2010-01-01

    This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML). As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  19. Development of a bispecific monoclonal antibody for use in molecular hybridisation.

    PubMed

    Auriol, J; Guesdon, J L; Mazié, J C; Nato, F

    1994-02-28

    A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise. PMID:8133070

  20. Generation of anti-trenbolone monoclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay for detection of trenbolone in animal tissues, feed and urine.

    PubMed

    Zhang, Yuanyang; He, Fangyang; Wan, Yuping; Meng, Meng; Xu, Jing; Yi, Jian; Wang, Yabin; Feng, Caiwei; Wang, Shanliang; Xi, Rimo

    2011-01-15

    Trenbolone (TRE) is a steroid used by veterinarians on livestock to increase appetite and body weight. The use of TRE has been restricted because of its harmful side effect for consumers. To effectively control TRE residue in food and food product, a rapid and convenient immunoassay was developed by preparing an anti-TRE monoclonal antibody. The immunogen and coating antigen were prepared by coupling TRE hapten with carrier proteins via 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method. The optimized method gave an average IC(50) value of 0.323 ng mL(-1) towards TRE and an average detection limit (LOD) of 0.06 ng mL(-1), which is much lower than the maximum residue levels (2.0 ng g(-1)) accepted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The specificity of the antibody was evaluated by measuring cross-reactivity of six structurally related compounds, including 19-nortestosterone (9.7%), testosterone (0.13%), methyltestosterone (<0.01%), methandrostenolone (<0.01%), (+)-dehydroisoandrosterone (<0.001%) and β-estradiol (<0.001%). The recovery rates of the test in detection of TRE-fortified animal tissue, urine and animal feed samples were in the range of 81.3-89.4%, while the intra- and inter-assay coefficients of variation were less than 12.0%. PMID:21147313

  1. Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

    PubMed

    Wang, Z-Y; Cai, J-P; Qiu, L-W; Hao, W; Pan, Y-X; Tung, E T K; Lau, C C Y; Woo, P C Y; Lau, S K P; Yuen, K-Y; Che, X-Y

    2012-11-01

    Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis. PMID:22669560

  2. Current status of tumor imaging with monoclonal antibodies.

    PubMed

    Blend, M J

    1991-12-01

    Although the full potential of MoAb imaging has yet to be realized, technologic advances continue with great intensity at a number of academic and industrial research institutions. Continuous production of MoAbs will eventually yield a variety of highly specific antibodies and novel approaches for improving cancer detection. As new diagnostic and therapeutic methods continue to be developed, MoAbs will begin to play a major role as targeted carriers, provided adequate funding from industry and government can be readily obtained. At present, the future of monoclonal antibodies in diagnosis and therapy for cancer patients appears promising. PMID:1790666

  3. Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

    PubMed Central

    Cercenado, E; Edelstein, P H; Gosting, L H; Sturge, J C

    1987-01-01

    Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes. PMID:3320084

  4. Anaphylaxis to chemotherapy and monoclonal antibodies.

    PubMed

    Castells, Mariana C

    2015-05-01

    Hypersensitivity reactions are increasingly prevalent, although underrecognized and underreported. Platins induce immunoglobulin E-mediated sensitization; taxenes and some monoclonal antibodies can induce reactions at first exposure. Severe hypersensitivity can preclude first-line therapy. Tryptase level at the time of a reaction is a useful diagnostic tool. Skin testing provides a specific diagnosis. Newer tests are promising diagnostic tools to help identify patients at risk before first exposure. Safe management includes rapid drug desensitization. This review provides information regarding the scope of hypersensitivity and anaphylactic reactions induced by chemotherapy and biological drugs, as well as diagnosis, management, and treatment options. PMID:25841555

  5. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    PubMed

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  6. 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 IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  7. Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

    PubMed

    Hoare, R; Thompson, K D; Herath, T; Collet, B; Bron, J E; Adams, A

    2016-01-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen. PMID:27434377

  8. Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

    PubMed Central

    Hoare, R.; Thompson, K. D.; Herath, T.; Collet, B.; Bron, J. E.; Adams, A.

    2016-01-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen. PMID:27434377

  9. Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.

    PubMed Central

    Sokol, P A; Woods, D E

    1986-01-01

    Hybridomas secreting specific monoclonal antibodies against the Pseudomonas aeruginosa ferripyochelin-binding protein (FBP) were isolated. These monoclonal antibodies reacted with FBP in immunoblots of outer membrane preparations from all serotypes of P. aeruginosa. Two of the monoclonal antibodies also reacted with FBP in strains of P. putida, P. fluorescens, and P. stutzeri. These antibodies did not react with outer membranes of P. cepacia, "P. multivorans," P. maltophilia, or other gram-negative organisms. The monoclonal antibodies were opsonophagocytic and blocked the binding of [59Fe]ferripyochelin to isolated outer membranes of strain PAO. By indirect immunofluorescence techniques, the monoclonal antibodies were used to demonstrate that FBP is present on the cell surface of P. aeruginosa cells grown in low-iron but not high-iron medium. These observations were confirmed by using 125I in surface-labeling techniques. Images PMID:3091506

  10. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    PubMed Central

    Hamm, Melissa; Wang, Yang; Rustandi, Richard R.

    2013-01-01

    The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis. PMID:24276024

  11. A monoclonal antibody-based immunoperoxidase monolayer (micro-isolation) assay for detection of type 1 and type 2 bovine viral diarrhea viruses.

    PubMed Central

    Deregt, D; Prins, S

    1998-01-01

    A monoclonal antibody (mAb)-based immunoperoxidase monolayer assay (IPMA) for detection of bovine viral diarrhea virus (BVDV) was developed and compared with an existing bovine polyclonal antibody (pAb)-based IPMA. A pool of 5 mAbs, 4 mAbs produced to a type 1 BVDV and 1 mAb produced to a type 2 BVDV, was utilized in the mAb-IPMA. The mAbs were chosen for inclusion in the pool because of their broad cross-reactivities with type 1 and/or type 2 BVDV, their apparent avidities for antigen, their reactivity to different BVDV proteins, and their lack of competition for binding sites or their binding to unusual BVDV isolates. The mAb-IPMA outperformed the pAb-IPMA in staining, ease of reading test results, and relative sensitivity with a panel of known BVDV positive and negative sera. The relative sensitivities of the mAb-IPMA and pAb-IPMA were 100% and 93.5%, respectively, for 62 positive samples including several that were known to contain type 2 BVDV. With retesting, the pAb-IPMA gave a similar level of sensitivity as that of the mAb-IPMA. Both tests gave a specificity of 100% for 40 negative serum samples obtained from a BVDV-free herd. PMID:9553717

  12. Production of monoclonal antibodies against canine leukocytes.

    PubMed

    Aguiar, Paulo Henrique Palis; Borges dos Santos, Roberto Robson; Lima, Carla Andrade; Rios de Sousa Gomes, Hilton; Larangeira, Daniela Farias; Santos, Patrícia Meira; Barrouin-Melo, Stella Maria; Conrado dos-Santos, Washington Luis; Pontes-de-Carvalho, Lain

    2004-04-01

    A panel of anti-canine leukocyte monoclonal antibodies (MAbs) was produced by immunizing BALB/c mice with canine peripheral blood mononuclear cells (PBMC), either resting or stimulated with concanavalin A (ConA). Three out of 28 clones-IH1, AB6, and HG6-screened by ELISA and producing antibody with the highest specificity for canine cell immunostaining, were subjected to three subsequent subcloning steps by limiting dilution, and selected for further characterization. These MAbs belonged to IgG1 (HG6 and IH1) and IgG2a (AB6) isotypes. The distribution of cell populations expressing the antigen recognized by the antibodies was identified by indirect immunoflorescence on canine PBMC and on tissue sections of lymph node, spleen, liver and skin. The possible crossreactivity with human PBMC was also examined in immunocytochemistry. One of the antibodies specifically recognized macrophages. The MAbs presented here can be foreseen as possible valuable diagnostic and research tools to study immune functions in dogs. PMID:15165486

  13. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    PubMed Central

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  14. Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production.

    PubMed

    Lin, Jisheng; Smith, Mark A; Benjamin, William H; Kaminski, Robert W; Wenzel, Heather; Nahm, Moon H

    2016-08-01

    There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency. PMID:27280622

  15. A Monoclonal Antibody Toolkit for C. elegans

    PubMed Central

    Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

    2010-01-01

    Background Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. Methodology/Principal Findings We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working

  16. Rapid diagnosis of whooping cough using monoclonal antibody.

    PubMed Central

    Boreland, P C; Gillespie, S H; Ashworth, L A

    1988-01-01

    A counterimmunoelectrophoresis (CIE) method for antigen detection using monoclonal antibody was assessed for its ability to aid in the rapid diagnosis of Bordetella pertussis in 59 patients. A positive diagnosis from a combination of results from tests of serum and urine was obtained in 51 (87%) of cases. For sera, CIE had a sensitivity of 85% and a specificity of 94%; for urine samples the sensitivity was 81% and a specificity of 100%. Antigen detection by CIE is simple to perform and yields results on the same day, thus allowing treatment to begin at an early stage. PMID:2898488

  17. Characterization of Tritrichomonas foetus antigens by use of monoclonal antibodies.

    PubMed Central

    Hodgson, J L; Jones, D W; Widders, P R; Corbeil, L B

    1990-01-01

    The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at approximately 155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complement-dependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity. Images PMID:2201645

  18. An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs.

    PubMed

    Lin, Jianhan; Wang, Ronghui; Jiao, Peirong; Li, Yuntao; Li, Yanbin; Liao, Min; Yu, Yude; Wang, Maohua

    2015-05-15

    Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation. PMID:25263315

  19. A new tool for monoclonal antibody analysis

    PubMed Central

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

    2014-01-01

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

  20. Monoclonal antibodies based on hybridoma technology.

    PubMed

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs. PMID:24237029

  1. Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

    PubMed

    Guo, Yancheng; Ngom, Babacar; Le, Tao; Jin, Xiue; Wang, Liping; Shi, Deshi; Wang, Xiliang; Bi, Dingren

    2010-09-15

    A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods. PMID:20726505

  2. Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

    PubMed Central

    van Tiel, F H; Boere, W A; Vinjé, J; Harmsen, T; Benaissa-Trouw, B J; Kraaijeveld, C A; Snippe, H

    1984-01-01

    Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes. PMID:6386855

  3. Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1

    PubMed Central

    Liu, Xing; Kato, Yukinari; Kaneko, Mika Kato; Sugawara, Masato; Ogasawara, Satoshi; Tsujimoto, Yuta; Naganuma, Yasushi; Yamakawa, Mitsunori; Tsuchiya, Takashi; Takagi, Michiaki

    2013-01-01

    Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 occur in gliomas, acute myeloid leukemia, and cartilaginous tumors. Somatic mosaic IDH1/2 mutations are also reported in Ollier disease and Maffucci syndrome, which are characterized by multiple central cartilaginous tumors. Although IDH1/2 mutation analysis against osteosarcoma has been performed in several studies, no IDH1/2 mutation has been reported. Herein, we newly report the IDH2-R172S mutation in three of 12 (25%) osteosarcoma patients, which was detected by direct DNA sequencing. No monoclonal antibody (mAb) has been reported against IDH2-R172S mutation. However, we demonstrate that the IDH2-R172S peptide was recognized by our established multi-specific anti-mutated IDH1/2 mAb, MsMab-1, in enzyme-linked immunosorbent assay. Western blot analysis revealed that MsMab-1 reacts with PA tag combined recombinant proteins of IDH2-R172S. Furthermore, MsMab-1 stained IDH2-R172S-expressing osteosarcoma tissues in immunohistochemistry. The MsMab-1 stained nine of 32 (28.1%) osteosarcomas in a tissue microarray. This report is the first describing IDH2 mutations in osteosarcoma, which can be detected by MsMab-1 mAb. Taken together, these results show that MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing osteosarcoma. PMID:24403254

  4. 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.

  5. The birth pangs of monoclonal antibody therapeutics

    PubMed Central

    2012-01-01

    This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor's development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development. PMID:22531443

  6. Monitoring therapeutic monoclonal antibodies in brain tumor

    PubMed Central

    Ait-Belkacem, Rima; Berenguer, Caroline; Villard, Claude; Ouafik, L’Houcine; Figarella-Branger, Dominique; Beck, Alain; Chinot, Olivier; Lafitte, Daniel

    2014-01-01

    Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling. PMID:25484065

  7. A humanized monoclonal antibody targeting Staphylococcus aureus.

    PubMed

    Patti, Joseph M

    2004-12-01

    This current presentation describes the in vitro and in vivo characterization of Aurexis (tefibazumab), a humanized monoclonal antibody that exhibits a high affinity and specificity and for the Staphylococcus aureus MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) protein ClfA. Aurexis inhibited ClfA binding to human fibrinogen, and enhanced the opsonophagocytic uptake of ClfA-coated beads. Preclinical in vivo testing revealed that a single administration of Aurexis significantly protected against an IV challenge with a methicillin resistant S. aureus (MRSA) strain in murine septicemia and rabbit infective endocarditis (IE) models. Safety and pharmacokinetic data from a 19-patient phase I study support continued evaluation of Aurexis in phase II studies. PMID:15576200

  8. 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

  9. Building better monoclonal antibody-based therapeutics

    PubMed Central

    Weiner, George J.

    2015-01-01

    For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, yet there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anti-cancer mAbs function via a variety of mechanisms including directly targeting the malignant cells, modifying the host response to the malignant cells, delivering cytotoxic moieties to the malignant cells or retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This article reviews the various approaches to using mAb-based therapeutics to treat cancer, the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. PMID:25998715

  10. Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies.

    PubMed

    Scharinger, Eva J; Dietrich, Richard; Kleinsteuber, Ina; Märtlbauer, Erwin; Schauer, Kristina

    2016-04-01

    Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method. PMID:26850303

  11. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    PubMed

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  12. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  13. Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

    PubMed Central

    Fu, Xiaoxiang; Wang, Ali; Wang, Xiaohan; Lin, Fengke; He, Lishan; Lai, Daowan; Liu, Yang; Li, Qing X.; Zhou, Ligang; Wang, Baoming

    2015-01-01

    Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples. PMID:26343725

  14. Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It.

    PubMed

    Wei, Julie Y; Bou-Assaf, George M; Houde, Damian; Weiskopf, Andrew

    2015-11-01

    Protein self-association or aggregation is a property of significant concern for biopharmaceutical products due to the potential ability of aggregates to cause adverse toxicological and immunological effects. Thus, during the development of a protein biopharmaceutical, it is important to detect and quantify the level and nature of aggregate species as early as possible in order to make well-informed decisions and to mitigate and control potential risks. Although a deeper understanding of the mechanism of aggregation (i.e., protein-protein interactions) is desirable, such detailed assessment is not always necessary from a biopharmaceutical process development point of view. In fact, the scope of characterization efforts is often focused on achieving a well-controlled process, which generates a product that reliably meets established acceptance criteria for safety and efficacy. In this brief note, we evaluated the utility of size-exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation in their simplest forms, to effectively reveal and confirm the presence of concentration-dependent reversible self-association (RSA) in a monoclonal antibody in the early stages of formulation development. Using these techniques, we also initiated preliminary work aimed at reducing the occurrence of this RSA behavior by varying the pH of the formulation buffer. PMID:26308556

  15. Drug Development of Therapeutic Monoclonal Antibodies.

    PubMed

    Mould, Diane R; Meibohm, Bernd

    2016-08-01

    Monoclonal antibodies (MAbs) have become a substantial part of many pharmaceutical company portfolios. However, the development process of MAbs for clinical use is quite different than for small-molecule drugs. MAb development programs require careful interdisciplinary evaluations to ensure the pharmacology of both the MAb and the target antigen are well-understood. Selection of appropriate preclinical species must be carefully considered and the potential development of anti-drug antibodies (ADA) during these early studies can limit the value and complicate the performance and possible duration of preclinical studies. In human studies, many of the typical pharmacology studies such as renal or hepatic impairment evaluations may not be needed but the pharmacokinetics and pharmacodynamics of these agents is complex, often necessitating more comprehensive evaluation of clinical data and more complex bioanalytical assays than might be used for small molecules. This paper outlines concerns and strategies for development of MAbs from the early in vitro assessments needed through preclinical and clinical development. This review focuses on how to develop, submit, and comply with regulatory requirements for MAb therapeutics. PMID:27342605

  16. Monoclonal antibody therapy for Junin virus infection.

    PubMed

    Zeitlin, Larry; Geisbert, Joan B; Deer, Daniel J; Fenton, Karla A; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N; Kuehne, Ana I; Aman, M Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W

    2016-04-19

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  17. Monoclonal antibody therapy for Junin virus infection

    PubMed Central

    Zeitlin, Larry; Geisbert, Joan B.; Deer, Daniel J.; Fenton, Karla A.; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N.; Kuehne, Ana I.; Aman, M. Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W.

    2016-01-01

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  18. Licensed monoclonal antibodies and associated challenges.

    PubMed

    Khan, Amjad Hayat; Sadroddiny, Esmaeil

    2015-12-23

    Monoclonal antibodies (mAbs) are the leading class of targeted therapeutics and remarkably effective in addressing autoimmune diseases, inflammations, infections, and various types of cancer. Several mAbs approved by US food and drug administration (FDA), are available on the market and a number are pending for approval. Luckily, FDA approved mAbs have played a pivotal role in the treatment and prevention of lethal diseases. However, claiming that licensed mAbs are 100% safe is still debatable, because infections, malignancies, anaphylactoid, and anaphylactic reactions are the more frequently associated adverse events. To evaluate benefit to risk ratio of mAbs, it is important for the clinical research staff or physicians to monitor and follow-up the patients who are receiving mAbs dozes. It is recommended that patients, physicians, biopharmaceutical companies, and researchers should keep in touch to highlight and resolve antibody-based adverse events. In this review we underscore the associated challenges of mAbs, approved by FDA from 2007-2014. PMID:27472864

  19. Immunoenzymatic detection of three kinds of 43,000-molecular-weight antigens by monoclonal antibodies in the insoluble fraction of Toxoplasma gondii.

    PubMed Central

    Ogata, K; Kasahara, T; Shioiri-Nakano, K; Igarashi, I; Suzuki, M

    1984-01-01

    Monoclonal antibodies (TpM 3, TpM 6, and TpM 19) against Toxoplasma gondii insoluble antigens were produced by the hybridization of NS-1, a mouse myeloma cell line, with spleen cells from mice immunized with T. gondii insoluble antigens. TpM 3, TpM 6, and TpM 19 were characterized by the dye test, the latex agglutination test, two types of enzyme-linked immunosorbent assays, using either T. gondii supernatant antigens or T. gondii insoluble antigens, and immunoperoxidase staining. TpM 3, TpM 6, and TpM 19 antigens were analyzed by the immunoblotting method, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer of the antigens to nitrocellulose sheets. TpM 3, TpM 6, and TpM 19 were all negative by the dye test, the latex agglutination test, and the enzyme-linked immunosorbent assay, using T. gondii supernatant antigens but positive by the enzyme-linked immunosorbent assay, using T. gondii insoluble antigens. Each antibody gave unique spot patterns in the cytoplasm of tachyzoites, and each detected antigens of ca. 43,000 molecular weight with different electrophoretic patterns. Purified TpM 3 antigen bound only to TpM 3 but not to TpM 6 or TpM 19. Comparable results were obtained with TpM 6 and TpM 19 antigens. Rabbit anti-T. gondii sera reacted with both TpM 3 and TpM 19 antigens. However, human anti-T. gondii sera reacted only with TpM 3 antigen. These results indicate that T. gondii insoluble antigens contained at least three types of 43,000-molecular-weight antigens that have been revealed for the first time in this paper. Images PMID:6698600

  20. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Cancer.gov

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  1. 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.

  2. Simplified solid-phase membrane immunobead assay (MIA) with monoclonal anti-ciguatoxin antibody (MAb-CTX) for detection of ciguatoxin and related polyether toxins.

    PubMed

    Hokama, Y; Nishimura, K; Takenaka, W; Ebesu, J S

    1998-02-01

    The development of a simplified and modified procedure for the assessment of ciguatoxin (CTX) and related polyethers from ciguateric contaminated fish tissues is presented in this study. The previous method, stick-enzyme immunoassay (S-EIA) used an organic correction fluid-coated bamboo paddle stick for the solid phase; this new procedure, membrane immunobead assay (MIA), uses a plastic stick with a synthetic membrane laminated onto one end. The membrane is hydrophobic and serves as the solid-phase receptor for the binding of methanol-extracted CTX or its related polyether lipids from fish tissues. Detection of the bound polyether toxin(s) on the membrane is carried out with colored polystyrene beads coated with monoclonal antibody to CTX (MAb-CTX). Intensity of the color on the membrane is proportional to the amount of toxic polyethers on the membrane. The MIA is compared with previous procedures developed for CTX detection (S-EIA and solid-phase immunobead assay, SPIA) using State of Hawaii Department of Health (DOH) implicated ciguateric fishes, and reef fishes from Hawaii and Kwajalein. The data presented show good correlation between the three test systems, especially with ciguatera implicated fish and toxic, routinely assessed fishes in the mouse toxicity (MT) bioassay. Variations between MT results and those of the S-EIA, SPIA, and MIA of routine fishes are generally attributable to diverse toxins present in the fish species examined. The MIA is a simple, rapid, sensitive, and specific detection method for CTX and its related polyethers, with no reported false negative results. The test is useful for field and personal use and can be adapted to the laboratory for large-scale screening of potentially ciguateric fishes. PMID:9689599

  3. Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

    PubMed

    Khamehchian, S; Madani, R; Rasaee, M J; Golchinfar, F; Kargar, R

    2007-06-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance. PMID:17668032

  4. Monoclonal antibody specific for a pigmentation associated antigen

    SciTech Connect

    Thomson, T.M.; Mattes, M.J.; Old, L.J.; Lloyd, K.O

    1989-01-17

    Monoclonal antibody TA99, which specifically binds to a pigmentation associated antigen present on melanoma cells is described. Additionally, the hybridoma cell line deposited with the ATCC under Accession Number HB 8704 from which the antibody is derived, as well as methods for using the antibody are described.

  5. 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

  6. Development and application of a monoclonal antibody against Thiothrix spp.

    PubMed Central

    Brigmon, R L; Bitton, G; Zam, S G; O'Brien, B

    1995-01-01

    Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml. PMID:7887596

  7. Anti-CD155 and anti-CD112 monoclonal antibodies conjugated to a fluorescent mesoporous silica nanosensor encapsulating rhodamine 6G and fluorescein for sensitive detection of liver cancer cells.

    PubMed

    Tao, Liang; Song, Chaojun; Huo, Chenyang; Sun, Yuanjie; Zhang, Chunmei; Li, Xiaohua; Yu, Shaojuan; Sun, Mingyu; Jin, Boquan; Zhang, Zhujun; Yang, Kun

    2016-08-01

    A novel method for sensitive detection of liver cancer cells using anti-CD155 and anti-CD112 monoclonal antibodies conjugated to ultrabright fluorescent mesoporous silica nanoparticles (FMSNs) encapsulating Rhodamine 6G and fluorescein was developed. The diameter of the obtained nanoparticles was 90 nm, and the quantum yield was 69%. Because the emission of fluorescein has a high degree of overlap with the excitation of Rhodamine 6G, and these two dyes were sufficiently close to each other on the nanoparticles, fluorescence resonance energy transfer can occur between these two dyes. This transfer not only maintains the original feature of the nanochannels and the skeletal network of the silica weakening the inner filtering of the dye, but also makes the excitation peak of the nanoparticles wider and increases the useful load amount of the dye. Because the wider Stokes shifts weaken the interference of excitation, the detection sensitivity is enhanced at the same time. The NaIO4 oxidation method does not use a cross-linker but rather uses covalent immobilization of the monoclonal antibodies on the FMSNs. This method can maintain the activity of the monoclonal antibodies more easily than the glutaraldehyde method. These advantages ensure that the nanosensor has high sensitivity and specificity for detecting liver cancer SMMC-7721 and HHCC cells. The in vivo imaging experiment also ensured that the biosensor can target tumor tissue in mice. PMID:27301350

  8. Immunoscintigraphic detection of venous thrombosis of the lower extremities by means of human antifibrin monoclonal antibodies labeled with sup 111 In

    SciTech Connect

    Lusiani, L.; Zanco, P.; Visona, A.; Breggion, G.; Pagnan, A.; Ferlin, G. )

    1989-07-01

    A new monoclonal antibody specific for the beta-chain of human fibrin (C22A) and labeled with 111In has been obtained and successfully used in rabbits and dogs for the in vivo detection of venous thrombosis. Studies in humans are currently ongoing. In order to assess the diagnostic value of 111In-antifibrin for the detection of venous thrombosis of the lower extremities, the authors investigated 25 consecutive patients. Ten patients had clinical and instrumental (contrast phlebography and duplex scanning) evidence of acute deep venous thrombosis (DVT), 3 had a long-standing DVT with relapsing episodes of swelling and pain, 5 had superficial venous thrombosis, and the remaining 7 had no signs of thrombosis at all. Twenty patients were being treated with heparin. All patients received 111In-antifibrin at the dose of 74 MBq IV and were scanned with a large field of view gamma camera coupled with a high-energy, parallel-hole collimator at 30 minutes and three, six, and twenty-four hours postinjection. Only the persistence of an abnormal uptake at twenty-four hours confirmed by two observers at visual inspection was considered as positive. A positive result was obtained in 9 of 10 DVT patients (90% sensitivity) and in all SVT patients. The single DVT patient with a negative 111In-antifibrin test had the longest interval between scintigraphy and onset of symptoms (fifty-five days). Thus, the age of thrombi represented a substantial limitation for the test. A false-positive result was obtained in a single SVT patient, in whom also a deep involvement, unconfirmed by phlebography, was suspected (91.6% specificity).

  9. Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection.

    PubMed

    Bai, Xiaofei; Shaozhou, Wulin; Zhang, Qingshan; Li, Chenxi; Qiu, Na; Meng, Runzhe; Liu, Ming; Zhang, Yun

    2015-03-01

    The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds. PMID:25588821

  10. The generation of monoclonal antibodies and their use in rapid diagnostic tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

  11. Monoclonal Antibodies Targeting Tumor Growth | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NCI Nanobiology Program, Protein Interaction Group is seeking parties to license or co-develop, evaluate, or commercialize monoclonal antibodies against the insulin-like growth factor for the treatment of cancer.

  12. Technological progresses in monoclonal antibody production systems.

    PubMed

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-01-01

    Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on-line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices. PMID:20043321

  13. Preparation of Monoclonal Antibodies Against Bovine Haptoglobin

    PubMed Central

    Wang, Caihong; Gu, Cheng; Guo, Donghua; Gao, Jing; Li, Chunqiu; Liu, Na; Geng, Yufei; Su, Mingjun; Wang, Xinyu

    2014-01-01

    Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6×108 and 1:8.2×104, respectively, and that of MAb 6D6 were 1:4.4×105 and 1:1.0×104, respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp. PMID:25358005

  14. Monoclonal Antibody Purification (Nicotiana benthamiana Plants)

    PubMed Central

    Husk, Adam; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2016-01-01

    Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of “deconstructed” virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a mAb per kg of leaf material in 7 days. A representative case for the broadly neutralizing anti-HIV and anti-influenza mAbs, VRC01 and CR6261 respectively, is shown (Hamorsky et al., 2013). Leaf tissue is homogenized and the extract is clarified by filtration and centrifugation. The mAb is purified by fast protein liquid chromatography (FPLC) using Protein A affinity and Phenyl HP hydrophobic interection resins.

  15. [Monoclonal antibodies from neurological and neuropsychological perspective].

    PubMed

    Piusińska-Macoch, Renata

    2013-05-01

    The role of monoclonal antibodies and other proinflammatory cytokines in the regulatory processes of the central and peripheral nervous system is not yet fully understood. Clinical studies show that they are involved in the pathogenesis of Alzheimer's disease, Parkinson's disease or other neurodegenerative disabilities with cognitive impairments. Genetic basis of these disorders is still in research. In the past few years it has been shown that increased levels of TNF-alpha and IL-6 in plasma play role in patients with ischemic stroke in the acute phase as well as transient ischemic episodes. Also the negative impact of TNF-alpha has been demonstrated on neck and coronary vessels, including the composition of plaques in the carotid arteries. A few reports indicate the involvement of tumor necrosis factor in such complex processes such as emotions, behavior or personality. Recent studies point to the important role of proinflammatory cytokines in the pathogenesis of sleep disorders such as narcolepsy, cataplexy and sleep paralysis. TNF-alpha can also activate nociceptive pathways, causing the intensity of neuropathic pain. However discloses asymmetric subtypes share TNF-1, TNF-2 in the induction and the maintenance of pain. The phenomenon of complex neurohormonal control mechanism support the proinflammatory cytokines is not fully understood and needs further empirical verification. PMID:23894773

  16. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    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.

  17. Therapeutic Monoclonal Antibodies and Fragments: Bevacizumab.

    PubMed

    Klein, Ainat; Loewenstein, Anat

    2016-01-01

    Bevacizumab (Avastin) is a recombinant humanized monoclonal immunoglobulin antibody that has two antigen-binding domains and blocks all active forms of vascular endothelial growth factor-A. It was originally designed and is still in use as antitumor agent (for colorectal and non-small cell lung cancers). Besides inhibiting vessel growth and neovascularization, the drug promotes the regression of existing microvessels and induces 'normalization' of surviving mature vasculature, stabilizes vessels and prevents leakage. Its molecular weight is 149 kDa and its estimated terminal half-life is approximately 20 days for both men and women. The effectiveness and safety of bevacizumab was proven in retrospective and prospective controlled clinical trials for the treatment of neovascular age-related macular degeneration, neovascularization in proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusion and retinopathy of prematurity, especially for zone I. Uncontrolled trials have shown its effectiveness in various other conditions as myopic and uveitic choroidal neovascularization and neovascular glaucoma. There are no absolute contraindications to intravitreal injection though it is recommended to withhold treatment in patients who have recently suffered from a cardiovascular or cerebrovascular event and during pregnancy. Ocular complications from intravitreal use are usually mild and transient (corneal abrasion, chemosis, subconjunctival hemorrhage and vitreous hemorrhage). Bacterial endophthalmitis is rare (about 0.1%). New or progressive subretinal hemorrhages, tears of the retinal pigment epithelium and an increased incidence of geographic atrophy have also been reported. PMID:26502311

  18. Monoclonal antibody-directed radioimmunoassay of specific cytochromes P-450

    SciTech Connect

    Song, B.J.; Fujino, T.; Park, S.S.; Friedman, F.K.; Gelboin, H.V.

    1984-02-10

    A rapid solid phase radioimmunoassay (RIA) for cytochromes P-450 has been developed utilizing specific monoclonal antibodies to major forms of rat liver cytochrome P-450 that are induced by 3-methylcholanthrene (MC-P-450) and phenobarbital (PB-P-450). Monoclonal antibodies (MAbs) that were endogenously labeled with (/sup 35/S)methionine were used to detect MAb-specific cytochromes P-450 in liver microsomes from untreated rats and rats pretreated with 3-methylcholanthrene (MC) or phenobarbital. The competitive binding assays are rapid and can detect cytochrome P-450 in less than 100 ng of microsomal protein. Tthe RIA was used to examine the distribution of MAb-specific cytochromes P-450 in extrahepatic tissues of MC-treated rats; an approximately 30- to 50-fold greater amount of MC-P-450 in liver relative to lung and kidney was observed, which corresponds well with aryl hydrocarbon hydroxylase activity in these tissues. The inducibility of MAb-specific cytochromes P-450 were observed in MC-treated rats, guinea pigs, and C57BL/6 mice, all highly inducible for aryl hydrocarbon hydroxylase; little increase was observed for the relatively noninducible DBA/2 mouse strain.

  19. Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

    PubMed Central

    Robertson, S M; Kettman, J R; Miller, J N; Norgard, M V

    1982-01-01

    Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed. PMID:7047388

  20. Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

    PubMed

    Hunt, Ann R; Hall, Roy A; Kerst, Amy J; Nasci, Roger S; Savage, Harry M; Panella, Nicholas A; Gottfried, Kristy L; Burkhalter, Kristen L; Roehrig, John T

    2002-06-01

    An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay. PMID:12037058

  1. Considerations for the development of therapeutic monoclonal antibodies.

    PubMed

    Swann, Patrick G; Tolnay, Mate; Muthukkumar, Subramanian; Shapiro, Marjorie A; Rellahan, Barbara L; Clouse, Kathleen A

    2008-08-01

    An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality. Recent findings highlight the need to more fully characterize these products and their activity. Advances in product characterization tools, immunogenicity assessments, and other bioanalytical assays can be used to better understand product performance and facilitate development. PMID:18586093

  2. Clinical experience with radiolabelled monoclonal antibodies in the detection of colorectal and ovarian carcinoma recurrence and review of the literature.

    PubMed

    Pinkas, L; Robins, P D; Forstrom, L A; Mahoney, D W; Mullan, B P

    1999-08-01

    A retrospective study was carried out to determine the diagnostic value of OncoScint CR/OV immunoscintigraphy in assessing patients with suspected recurrence of carcinoma of the colon and ovary. The scintigraphic results of 31 patients were compared with surgical and histopathological findings, conventional radiological examinations and clinical disease outcome over an average 3-year follow-up. Detected lesions were divided by location into hepatic or extrahepatic and the latter group was classified as local recurrence at the resection site, pelvic or abdominal regional lymph node involvement and distant metastatic disease. The combined sensitivity and accuracy of immunoscintigraphy in the detection of extra-hepatic disease was significantly higher than that of cross-sectional radiological imaging (87% and 83% vs 44% and 53% respectively) with equal specificity of 74%. Scintigraphy identified 14 (36%) of 39 extra-hepatic malignant lesions not diagnosed by conventional radiological techniques and influenced therapeutic planning in 8 (26%) of 31 patients studied. In the liver, conventional imaging had a significantly higher detection rate than immunoscintigraphy (sensitivity 93% vs 28%). In conclusion, these results show that OncoScint scintigraphy is a sensitive method for the detection of local recurrence and extra-hepatic metastases in colorectal and ovarian carcinoma and has an important role in the therapeutic decision-making process. PMID:10451876

  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. )

    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. Development of a monoclonal antibody specific to envelope domain III with broad-spectrum detection of all four dengue virus serotypes.

    PubMed

    Kim, Sae-Hae; Kim, Yu Na; Truong, Thang Thua; Thu Thuy, Nguyen Thi; Mai, Le Quynh; Jang, Yong-Suk

    2016-05-13

    Dengue virus (DENV) is a mosquito-borne pathogen that annually infects more than 390 million people in 100 different countries. Symptoms of the viral infection include a relatively weak dengue fever to severe dengue hemorrhagic fever/dengue shock syndrome, which are mortal infectious diseases. As of yet, there is no commercially available vaccine or therapeutic for DENV. Currently, passive immunotherapy using DENV-specific antibody (Ab) is a considered strategy to treat DENV infection. Here, we developed a monoclonal Ab (mAb), EDIIImAb-61, specific to the DENV domain III of the envelope glycoprotein (EDIII) with broad-spectrum detection ability to all four DENV serotypes (DENV-1∼4) to use as a therapeutic Ab. Although EDIII contains non-immunodominant epitopes compared to domains I and II, domain III plays a critical role in host receptor binding. EDIIImAb-61 exhibited cross-reactive binding affinity to all four DENV serotypes that had been isolated from infected humans. To further characterize EDIIImAb-61 and prepare genes for large-scale production using a heterologous expression system, the sequence of the complementarity determining regions was analyzed after cloning the full-length cDNA genes encoding the heavy and light chain of the mAb. Finally, we produced Ab from CHO-K1 cells transfected with the cloned EDIIImAb-61 heavy and light chain genes and confirmed the binding ability of the Ab. Collectively, we conclude that EDIIImAb-61 itself and the recombinant Ab produced using the cloned heavy and light chain gene of EDIIImAb-61 is a candidate for passive immunotherapy against DENV infection. PMID:27059141

  5. Boronated monoclonal antibody conjugates for neutron capture therapy

    SciTech Connect

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    This paper describes the effectiveness of /sup 10/B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link /sup 10/B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs.

  6. Increased concentration of two different advanced glycation end-products detected by enzyme immunoassays with new monoclonal antibodies in sera of patients with rheumatoid arthritis

    PubMed Central

    2010-01-01

    Background Levels of pentosidine (representative of advanced glycation end-products) in sera of patients with rheumatoid arthritis are increased when compared with sera of other diagnoses or healthy controls. These levels have been reported to correlate with clinical indices of rheumatoid arthritis activity and with laboratory markers of inflammation. The purpose of this study was to find out if these findings pertain to other advanced glycation end-products. Methods We have developed two immunoassays based on new monoclonal antibodies to advanced glycation end-products. Antibody 103-E3 reacts with an unidentified antigen, formed in the reaction of proteins with ribose, while antibody 8-C1 responds to Nε-(carboxyethyl)lysine. We have used these monoclonal antibodies to measure levels of advanced glycation end-products in sera of patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and healthy controls. We calculated the correlations between advanced glycation end-product levels in rheumatoid arthritis sera and the Disease Activity Score 28 (DAS28), age, disease duration, CRP, anti-CCP, rheumatoid factor and treatment with corticosteroids, respectively. Results Levels of both glycation products were significantly higher in sera of patients with rheumatoid arthritis when compared with sera of patients with systemic lupus erythematosus, osteoarthritis, or the healthy controls. Neither the level of Nε-(carboxyethyl)lysine nor the level of the 103-E3 antigen in rheumatoid arthritis sera correlated with the DAS28-scored rheumatoid arthritis activity. The levels of both antigens in rheumatoid arthritis sera did not correlate with age, gender, corticosteroid treatment, or levels of CRP, anti-CCP antibodies, and rheumatoid factor in sera. Conclusions We report highly specific increases in the levels of two advanced glycation end-products in sera of patients with rheumatoid arthritis. This increase could be explained neither by rheumatoid

  7. 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.; Friedman, A.M.; Hines, J.J.

    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.

  8. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

    PubMed Central

    Rosseels, Joëlle; Van den Brande, Jeff; Violet, Marie; Jacobs, Dirk; Grognet, Pierre; Lopez, Juan; Huvent, Isabelle; Caldara, Marina; Swinnen, Erwin; Papegaey, Anthony; Caillierez, Raphaëlle; Buée-Scherrer, Valerie; Engelborghs, Sebastiaan; Lippens, Guy; Colin, Morvane; Buée, Luc; Galas, Marie-Christine; Vanmechelen, Eugeen; Winderickx, Joris

    2015-01-01

    A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential. PMID:25540200

  9. Coupling of the 99mtechnetium-nitrido group to monoclonal antibody and use of the complexes for the detection of tumors in mice

    SciTech Connect

    Kanellos, J.; Pietersz, G.A.; McKenzie, I.F.; Bonnyman, J.; Baldas, J.

    1986-08-01

    The in vivo detection of tumors by immunoscintigraphy using /sup 99m/Tc labelled monoclonal antibodies (MoAb) was explored in this study. A simple method for the labelling of microgram quantities of MoAb with /sup 99m/Tc based on the substitution reaction of MoAb and /sup 99m/TcNCl/sub 4//sup -/ is described. The selective activity of the /sup 99m/technetium-nitrido-MoAb (/sup 99m/TcN-MoAb) complexes was proved in vitro by a binding assay with different target cells. The /sup 99m/TcN-MoAb complexes were shown to bind reactive cells up to 20 times more avidly than nonreactive cells. The specificity of the /sup 99m/TcN-MoAb complexes was shown in vivo. (C57BL/6 X BALB/c)F1 mice bearing palpable tumors (0.3-1.5 cm in diameter) were given an iv injection of 1 of 2 MoAb (one reactive and the other nonreactive) identically labeled with /sup 99m/TcNCl/sub 4//sup -/ and then scanned with a gamma camera, and/or the tissues were removed and the localization of /sup 99m/Tc-nitrido group-labeled MoAb was measured. Tumor localization of the reactive MoAb (1.8-2.2% of the injected dose) was four times greater than that of the nonreactive /sup 99m/TcN-MoAb (0.3-0.4% of the injected dose). The localization of specific /sup 99m/TcN-MoAb to a murine thymoma was observed in the gamma camera image at just 2 hours after injection. At 27 hours, tumors could readily be detected by /sup 99m/TcN-MoAb without the need for background subtraction. Nonreactive /sup 99m/TcN-MoAb did not image the tumors. The use of /sup 99m/TcN-MoAb offers substantial improvement over radioiodinated (/sup 125/I or /sup 131/I) MoAb for the detection of tumors. The use of /sup 99m/TcNCl/sub 4//sup -/ as a labeling agent results in /sup 99m/Tc-labeled MoAb with high specific activity and specificity when compared with the specific activity and specificity of the /sup 99m/Tc-MoAb prepared by using the conventional SnCl/sub 2/ reduction of pertechnetate.

  10. Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

    PubMed

    Khoudi, H; Laberge, S; Ferullo, J M; Bazin, R; Darveau, A; Castonguay, Y; Allard, G; Lemieux, R; Vézina, L P

    1999-07-20

    The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs. PMID:10397849

  11. Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: characterization of stages of the division cycle of centrosomes.

    PubMed

    Schatten, H; Walter, M; Mazia, D; Biessmann, H; Paweletz, N; Coffe, G; Schatten, G

    1987-12-01

    A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle. PMID:3120191

  12. Structural Basis for Broad Detection of Genogroup II Noroviruses by a Monoclonal Antibody That Binds to a Site Occluded in the Viral Particle

    SciTech Connect

    Hansmana, Grant S.; Taylor, David W.; Smith, Thomas J.; McLellan, Jason S.; Georgiev, Ivelin; Tame, Jeremy R.H.; Park, Sam-Yong; Yamazaki, Makoto; Gondaira, Fumio; Miki, Motohiro; Katayama, Kazuhiko; Murata, Kazuyoshi; Kwong, Peter D.

    2012-03-13

    Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-{angstrom} resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at {approx}10-{angstrom} resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised {approx}15 {angstrom} off the shell domain and rotated {approx}40{sup o} relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.

  13. 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)

  14. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies.

    PubMed

    Ferguson, Carly N; Gucinski-Ruth, Ashley C

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics. Graphical Abstract ᅟ. PMID:26988372

  15. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

  16. Complete De Novo Assembly of Monoclonal Antibody Sequences.

    PubMed

    Tran, Ngoc Hieu; Rahman, M Ziaur; He, Lin; Xin, Lei; Shan, Baozhen; Li, Ming

    2016-01-01

    De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216-441 AA, at 100% coverage, and 96.64-100% accuracy. PMID:27562653

  17. Complete De Novo Assembly of Monoclonal Antibody Sequences

    PubMed Central

    Tran, Ngoc Hieu; Rahman, M. Ziaur; He, Lin; Xin, Lei; Shan, Baozhen; Li, Ming

    2016-01-01

    De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216–441 AA, at 100% coverage, and 96.64–100% accuracy. PMID:27562653

  18. 90Y-labeled monoclonal antibodies for cancer therapy.

    PubMed

    Washburn, L C; Hwa Sun, T T; Crook, J E; Byrd, B L; Carlton, J E; Hung, Y W; Steplewski, Z S

    1986-01-01

    Monoclonal antibody 17-1A, which has specificity for colorectal carcinoma, was labeled with 90Y (10-20% radiolabeling yield). Tissue distribution studies in tumor-bearing nude mice were carried out. 90Y-labeled 17-1A showed good uptake in the SW 948 colon carcinoma cell line. However, 90Y-labeled A5C3, a monoclonal antihepatitis virus antibody studied as a control, showed similar uptake in this tumor. Neither antibody was taken up well by a WM-9 melanoma. It is believed that the loss of specificity observed is due to the low specific activity of the 90Y-labeled monoclonal antibody preparations used. This hypothesis is supported by radioimmunoassay data. PMID:3793501

  19. Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.

    PubMed

    Li, Yi; Hewitt, Daniel; Lentz, Yvonne K; Ji, Junyan A; Zhang, Taylor Y; Zhang, Kelly

    2014-05-20

    Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to

  20. Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

    PubMed Central

    Polanco, Juan Carlos; Wang, Bei; Zhou, Qi; Chy, Hun; O'Brien, Carmel; Laslett, Andrew L.

    2013-01-01

    Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30Hi-GCTM-2Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30Neg-GCTM-2Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30Neg-GCTM-2Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30Hi-GCTM-2Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched

  1. 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

  2. Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

    PubMed

    Luo, Quanzhou; Chung, Hyo Helen; Borths, Christopher; Janson, Matthew; Wen, Jie; Joubert, Marisa K; Wypych, Jette

    2016-01-01

    Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody. PMID:26629796

  3. Coarse grained modeling of transport properties in monoclonal antibody solution

    NASA Astrophysics Data System (ADS)

    Swan, James; Wang, Gang

    Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

  4. Improved iodine radiolabels for monoclonal antibody therapy.

    PubMed

    Stein, Rhona; Govindan, Serengulam V; Mattes, M Jules; Chen, Susan; Reed, Linda; Newsome, Guy; McBride, Bill J; Griffiths, Gary L; Hansen, Hans J; Goldenberg, David M

    2003-01-01

    A major disadvantage of (131)iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodotyrosine from target cells after internalization and catabolism of the radioiodinated MAbs. We recently reported that a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had impeded the development of residualizing iodine for clinical use. To determine the factors governing the therapeutic index of the labeled MAb, as well as the factors required for production of radioiodinated MAb in high yield and with high specific activity, variations in the peptide structure of IMP-R1 were evaluated. A series of radioiodinated, diethylenetriaminepentaacetic acid-appended peptide moieties (IMP-R1 through IMP-R8) that differed in overall hydrophilicity and charge were compared. Radioiodinations of the peptides followed by conjugations to disulfide-reduced RS7 (an anti-epithelial glycoprotein-1 MAb) furnished radioimmunoconjugates in good overall incorporations, with immunoreactivities comparable to that of directly radioiodinated RS7. Specific activities of up to 8 mCi/mg and yields > 80% have been achieved. In vitro processing experiments showed marked increases in radioiodine retention with all of the adducts; radioiodine retention at 45 h was up to 86% greater in cells than with directly iodinated RS7. Each of the (125)I-peptide-RS7 conjugates was compared with (131)I-RS7 (labeled by the chloramine-T method) in paired-label biodistribution studies in nude mice bearing human lung tumor xenografts. All of the residualizing substrates exhibited significantly enhanced retention in tumor in comparison to directly radioiodinated RS7, but the nontarget uptakes differed significantly among the residualizing labels. The best labels were IMP-R4 and IMP-R8, showing superior tumor-to-non-tumor ratios

  5. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples.

    PubMed

    Wang, Yuzhen; Wei, Dapeng; Yang, Hong; Yang, Yuan; Xing, Weiwei; Li, Yuan; Deng, Anping

    2009-03-15

    The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ngmL(-1). The values of IC(50) for nine standard curves were in the range of 1.1-2.0 ngmL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N=3) was 0.07-0.14 ngmL(-1). The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC(50) value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n=9). The mAb-based ELISA proven to be a feasible quantitative

  6. 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.

  7. Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas.

    PubMed

    Groves, D J; Morris, B A

    2000-06-01

    The generation of monoclonal antibodies from species other than rats and mice has developed slowly over the last 20 years. The advent of antibody engineering and realization of the advantages of nonmurine antibodies, in terms of their superior affinities and specificities, and their potential as components of human and veterinary therapeutics has increased their relevance recently. There have been significant advances in the development of myeloma and heteromyeloma fusion partners. This is an opportune moment to consolidate experiences of MAb production across the range of species of veterinary interest and place it into context with other developments in the field of monoclonal antibodies. The background to the development of antibodies from species other than the mouse is discussed. The species and antigens used to date are reviewed, as are the methods and results reported. A suggested protocol is provided for first attempts to exploit the huge potential of this aspect of hybridoma technology and suggestions are made for its further expansion. PMID:10952409

  8. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    SciTech Connect

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

  9. Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays.

    PubMed Central

    Smith, K J

    1985-01-01

    Monoclonal antibodies have been used to demonstrate a polymorphism of human plasma coagulation factor IX antigen in double antibody solid-phase immunoradiometric assays. This polymorphism is detected in an assay where a monoclonal antibody (A-1) adsorbed to microtiter wells is used to bind factor IX from diluted plasma samples. Plasma samples with the factor IX polymorphism have less than 0.2 U/ml of apparent antigen when tested with the A-1 antibody, while assays with other monoclonal antibodies and assays with goat antisera to factor IX show normal amounts of factor IX antigen. Factor IX coagulant activity was normal in samples from donors with the polymorphism. The thin-layer polyacrylamide gel isoelectric focusing pattern of factor IX purified from a donor with the factor IX polymorphism (IXp) was identical to that obtained with factor IX prepared from a donor who did not have the polymorphism (IXn). Purified radiolabeled factor IX prepared from a donor with the polymorphism showed a Ka for the A-1 antibody that was threefold less than that measured for IXn. The gene frequency of IXp in male blood donors is 0.25. This polymorphism may be useful as a marker for the X chromosome in genetic studies on plasma samples. Further studies are necessary to determine the explanation for decreased reaction of IXp with the A-1 monoclonal antibody. Images Fig. 1 PMID:9556657

  10. Preparation and identification of anti-rabies virus monoclonal antibodies.

    PubMed

    Wang, Wen-juan; Li, Xiong; Wang, Li-hua; Shan, Hu; Cao, Lei; Yu, Peng-cheng; Tang, Qing; Liang, Guo-dong

    2012-06-01

    To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents. PMID:22684471

  11. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

    PubMed Central

    Ball, H J; Finlay, D; Mackie, D P; Greer, D; Pollock, D; McNair, J

    1991-01-01

    A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs. Images PMID:1761683

  12. 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.

  13. Monoclonal antibodies and Fc fragments for treating solid tumors.

    PubMed

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  14. Monoclonal antibodies and Fc fragments for treating solid tumors

    PubMed Central

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  15. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed Central

    Sato, H; Sato, Y

    1990-01-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23

  16. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed

    Sato, H; Sato, Y

    1990-10-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23

  17. Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

    PubMed

    Fotso Fotso, Aurélien; Mediannikov, Oleg; Nappez, Claude; Azza, Saïd; Raoult, Didier; Drancourt, Michel

    2016-01-01

    Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field. PMID:26598566

  18. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed Central

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-01-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies. Images PMID:3267219

  19. Elicited antibody nature of human monoclonal protein with anti-streptolysin O activity--analysis with monoclonal anti-idiotype antibody.

    PubMed

    Sawada, S; Shida, M; Suenaga, R; Mizuma, H; Karasaki, M; Hashimoto, M; Kawano, K; Amaki, I

    1986-01-01

    Sera from 7 patients with multiple myeloma having antistreptolysin O (ASO) activity in high titers were detected by a streptolysin O (SLO) inhibition assay. However, activity was in low titer when assayed by a passive agglutination assay. The discrepancy between these 2 assays raised some doubts as to whether these monoclonal proteins (M.protein) bond to SLO in the same manner as elicited antibodies. Immunochemical analysis and idiotope analysis using monoclonal antibody to one of these M.proteins strongly suggest that M.protein with ASO activity bind to SLO in a manner similar to elicited antibody. The discrepancy between the 2 assays might be due to differences in the antigenic structure of different forms of the SLO molecule. PMID:2422380

  20. 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...

  1. Targeted therapeutics for severe refractory asthma: monoclonal antibodies.

    PubMed

    Grainge, Christopher L; Maltby, Steven; Gibson, Peter G; Wark, Peter A B; McDonald, Vanessa M

    2016-07-01

    Severe asthma is a complex multifactorial disease that requires specialist multidisciplinary input for optimal clinical outcomes. Following multidimensional assessment for optimisation of current therapy, self-management skills and comorbidities, all patients should be accurately phenotyped. Only after this assessment has been completed should new monoclonal antibody therapies be considered. In this review, we summarise the new antibody approaches targeting identified pathological pathways in severe refractory asthma. PMID:27018798

  2. Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

    PubMed

    Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

    2012-05-01

    Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF. PMID:22808513

  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. Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

    PubMed Central

    Tabll, Ashraf; Abbas, Aymn T; El-Kafrawy, Sherif; Wahid, Ahmed

    2015-01-01

    Hepatitis C virus (HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies (mAb) of mice are predominantly used for the immunodiagnosis of several viral, bacterial, and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mAb are used in the immunotherapy of several blood malignancies, such as lymphoma and leukemia, as well as for autoimmune diseases. In this review article, we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mAb in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV, which is a major health problem throughout the world, particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mAb. Finally, we will discuss available and new trends to produce antibodies, such as egg yolk-based antibodies (IgY), production in transgenic plants, and the synthetic antibody mimics approach. PMID:26464752

  5. Survey of citrus tristeza virus populations in Central California that react with MCA13 monoclonal antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Citrus Pest Detection Program (CPDP) of the Central California Tristeza Eradication Agency monitors Citrus tristeza virus (CTV) in Central California. MCA13 is a severe strain discriminating monoclonal antibody used to screen for potentially virulent CTV isolates. MCA13-reactive CTV isolates are...

  6. 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...

  7. Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary.

    PubMed

    Kim, Dae-Jung; Park, Chae-Won; Byambaragchaa, Munkhzaya; Kim, Shin-Kwon; Lee, Bae-Ik; Hwang, Hyung-Kyu; Myeong, Jeong-In; Hong, Sun-Mee; Kang, Myung-Hwa; Min, Kwan-Sik

    2016-09-01

    Monoclonal antibodies were generated against recombinant follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica; rec-FSH was produced in Escherichia coli and purified using Ni-NTA Sepharose column chromatography. In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary. PMID:27331121

  8. Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries*

    PubMed Central

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezső, Balázs; Karger, Barry L.; Takács, László

    2011-01-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  9. Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries.

    PubMed

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezso, Balázs; Karger, Barry L; Takács, László

    2011-12-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  10. Identification of two antigenic determinants in pseudomurein by monoclonal antibodies

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Kandler, O.; Wolin, M.J.

    1982-01-01

    Pseudomurein is a unique peptidoglycan found only in the wall of methanogenic bacteria (MB) of the family Methanobacteriaceae. Although its chemical composition has recently been determined, its immunologic properties have not been elucidated. Methanobacteriaceae elicit antibodies in rabbits and mice. The authors have produced monoclonal antibodies against the bacteria. Antigenic determinants on the MB's surface were resolved with the monoclonal antibodies by means of inhibition-blocking procedures combined with immunoenzymatic assays devised for the structural analysis of bacterial antigens. One monoclonal antibody against Methanobrevibacter arboriphilus DHl recognized a determinant involving the ..gamma..-Glu-Ala end of the pseudomurein peptide. A second antibody did not react with the above determinant but with another involving N-acetylglucosamine. The latter antibody reacted with the immunizing MB, i.e. Methanobacterium thermoautotrophicum ..delta..H and with another strain of this species, GGl, but it did not react with the rest of the pseudomurein-containing bacteria. The data show that pseudomurein possess at least two different determinants, one in the C-terminus of the peptide moiety and the other in the backbone structure and indicate that the spatial arrangement of the peptidoglycan components is distinctive for the species examined and plays a role in antigenicity.

  11. Humanization of a chicken anti-IL-12 monoclonal antibody.

    PubMed

    Tsurushita, Naoya; Park, Minha; Pakabunto, Kanokwan; Ong, Kelly; Avdalovic, Anamarija; Fu, Helen; Jia, Audrey; Vásquez, Max; Kumar, Shankar

    2004-12-01

    Chicken anti-IL-12 monoclonal antibodies were isolated by phage display using spleen cells from a chicken immunized with human and mouse IL-12 as a source for library construction. One of the chicken monoclonal antibodies, DD2, exhibited binding to both human and mouse IL-12 in the single-chain Fv form and also after conversion to chicken-human chimeric IgG1/lambda antibody. The chicken DD2 variable regions were humanized by transferring their CDRs and several framework amino acids onto human acceptor variable regions. In the Vlambda, six chicken framework amino acids were identified to be important for the conformation of the CDR structure by computer modeling and therefore were retained in the humanized form; likewise, five chicken amino acids in the VH framework regions were retained in the humanized VH. The affinities of humanized DD2 IgG1/lambda to human and mouse IL-12 measured by competitive binding were nearly identical to those of chicken-human chimeric DD2 IgG1/lambda. This work demonstrates that humanization of chicken monoclonal antibodies assisted by computer modeling is possible, leading to a new way to generate therapeutic humanized antibodies against antigens to which the rodent immune system may fail to efficiently raise high affinity antibodies. PMID:15627607

  12. Development and evaluation of monoclonal antibodies for paxilline

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

  13. Bacterial surface antigens defined by monoclonal antibodies: the methanogens

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Magarinos, M.C.; Jovell, R.J.; Kandler, O.

    1982-01-01

    The methanogens (MB) are unique microbes of great evolutionary interest with applications in biotechnology-bioengineerings and are important in digestive processes. Their cell-wall composition is distinctively different from that of Eubacteria, e.g. the Methanobacteriaceae possess the peptidoglycan pseudomurein rather than murein. The range of cell-wall compositions among MB and their evolutionary and functional significance is not well known. The authors undertook a systematic study of the MB's surface structure using monoclonal antibodies through the following steps: (1) generation of hybridomas that produce antibody to several MB from 3 of their 4 families; (2) development of immunoenzymatic assays for MB's antigens and antibodies; (3) determination of the fine specificity of monoclonal antibodies by inhibition-blocking tests using cell-wall extracts and compounds of known structure; thus a set of monoclonal probes of predetermined specificity was assembled; and (4) resolution of surface determinants of MB representative of the Methanobacteriaceae using the monoclonal probes. Specific markers of MB strains were characterized. Two epitopes were identified within the pseudomurein molecule.

  14. 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...

  15. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    PubMed

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology. PMID:25894652

  16. Trimerization Dictates Solution Opalescence of a Monoclonal Antibody.

    PubMed

    Yang, Teng-Chieh; Langford, Alex Jacob; Kumar, Sandeep; Ruesch, John Carl; Wang, Wei

    2016-08-01

    Opalescence, sometimes observed in antibody solutions, is thought to be mediated by light scattering of soluble oligomers or insoluble particulates. However, mechanistic features, such as stoichiometry and self-association affinity of oligomeric species related to opalescence, are poorly understood. Here, opalescence behavior of a monoclonal antibody (mAb-1) solution was studied over a wide range of solution conditions including different protein concentrations, pH, and in the presence or absence of salt. Hydrodynamic and thermodynamic properties of mAb-1 solutions were studied by analytical ultracentrifugation and dynamic light scattering. Opalescence in mAb-1 solutions is pH and concentration dependent. The degree of opalescence correlates with reversible monomer-trimer equilibrium detected by analytical ultracentrifugation. Increased trimer formation corresponds to increased opalescence in mAb-1 solutions at higher pH and protein concentrations. Addition of NaCl shifts this equilibrium toward monomer and reduces solution opalescence. This study demonstrates that opalescence in mAb-1 solutions does not arise from the light scattering of monomer or random molecular self-associations but is strongly correlated with a specific self-association stoichiometry and affinity. Importantly, at pH 5.5 (far below isoelectric point of mAb-1), the solution is not opalescent and with nonideal behavior. This study also dissects several parameters to describe the hydrodynamic and thermodynamic nonideality. PMID:27373839

  17. Development of antibody arrays for monoclonal antibody Higher Order Structure analysis

    PubMed Central

    Wang, Xing; Li, Qing; Davies, Michael

    2013-01-01

    Antibody arrays were developed to probe a monoclonal antibody's three-dimensional structure (3-D structure). Peptides with overlapping regions were designed to cover the whole mAb light chain and heavy chain, respectively, and used to generate polyclonal antibodies after the conjugation of the peptides to a carrier protein, KLH. It was shown that good peptide specificity was achieved from the antibodies generated. Using more than 30 different polyclonal antibodies to measure the surface epitope distribution, it was shown that the mAb antibody array can detect epitope exposure as low as 0.1% of defined mAb populations. This ELISA-based analysis of mAb epitope exposure can be considered as a measurement of “conformational impurity” in biologics development, similar to the analysis of other product-related impurities such as different forms of glycosylation, deamidation, and oxidation. This analysis of “conformational impurity” could provide valuable information on the mAb conformational comparability for biosimilar mAbs as well as novel mAbs, especially in the area of protein immunogenicity. Furthermore, stability studies indicated that there are several conformational “hot spots” in many mAbs tested, especially in the hinge region. This antibody array technology can be used for novel mAb Higher Order Structure (HOS) analysis during process and formulation development. Another important area of application is for biosimilar mAb development where the innovator molecule and biosimilar molecule could be compared based on their systemic “fingerprint” from the 30 plus antibodies. PMID:23970865

  18. 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

    ... Human Monoclonal Antibodies Against DR4 AGENCY: National Institutes of Health, Public Health Service... Monoclonal Antibodies Against DR4'' (HHS Ref. No. E-158-2010/0) to Customized Biosciences, Inc., which is... relates to the development of two human monoclonal antibodies (mAbs) that bind to death receptor 4...

  19. 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.

  20. Monoclonal antibodies produced by muscle after plasmid injection and electroporation.

    PubMed

    Tjelle, Torunn Elisabeth; Corthay, Alexandre; Lunde, Elin; Sandlie, Inger; Michaelsen, Terje E; Mathiesen, Iacob; Bogen, Bjarne

    2004-03-01

    Antibodies are useful for the treatment of a variety of diseases. We here demonstrate that mouse muscle produced monoclonal antibodies (mAb) after a single injection of immunoglobulin genes as plasmid DNA. In vivo electroporation of muscle greatly enhanced antibody production. For chimeric antibodies, levels of 50-200 ng mAb/ml serum were obtained but levels declined after 7-14 days due to an immune response against the xenogeneic parts of the antibody. By contrast, fully mouse antibodies persisted in serum for at least 7 months. mAb produced by the muscle had correct structure, specificity, and biological effector functions. The findings were extended to a larger animal, the sheep, in which mAb serum levels of 30-50 ng/ml were obtained. Sustained levels of serum mAb, induced by single injection of Ig genes and electroporation of muscle cells, may offer significant advantages in the treatment of human diseases. PMID:15006599

  1. Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

    PubMed Central

    Darsley, M J; Rees, A R

    1985-01-01

    The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence. PMID:2410256

  2. 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

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

    SciTech Connect

    Marx, A.; Hock, B.

    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).

  4. Development and Evaluation of Monoclonal Antibodies for Paxilline.

    PubMed

    Maragos, Chris M

    2015-10-01

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. PMID:26426046

  5. Development and Evaluation of Monoclonal Antibodies for Paxilline

    PubMed Central

    Maragos, Chris M.

    2015-01-01

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. PMID:26426046

  6. Species- and infective stage-specific monoclonal antibodies to Leishmania major produced by an in vitro immunization method.

    PubMed

    Wu, S J; Rowton, E D; Ma, M; Andre, R G

    1990-12-01

    Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies (Phlebotomus papatasi) 7-10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag.653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes. PMID:2087235

  7. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  8. 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.

  9. Monkey-derived monoclonal antibodies against Plasmodium falciparum.

    PubMed Central

    Stanley, H A; Reese, R T

    1985-01-01

    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 Mr 95,000 antigen. Images PMID:3898084

  10. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

    SciTech Connect

    Elthon, T.E.; Nickels, R.L.; McIntosh, L. )

    1989-04-01

    The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states. AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

  11. 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.

  12. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    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.

  13. Scintigraphic detection of overexpressed c-erbB-2 protooncogene products by a class-switched murine anti-c-erbB-2 protein monoclonal antibody

    SciTech Connect

    Saga, T.; Endo, K.; Akiyama, T.; Sakahara, H.; Koizumi, M.; Watanabe, Y.; Nakai, T.; Hosono, M.; Yamamoto, T.; Toyoshima, K. )

    1991-02-01

    Class-switched monoclonal antibody SV2-61r recognized the extracellular domain of c-erbB-2 protooncogene products separate from the epidermal growth factor receptor. We studied the potential of SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells, which has been reported to have prognostic value in adenocarcinoma patients. Radiolabeled SV2-61r specifically bound to various adenocarcinoma cells in addition to c-erbB-2-transfected NIH-3T3 cells (A4) with the affinity constant of 4.4 x 10(8) M-1. SV2-61r injected i.v. localized well to A4 cells xenografted in nude mice. Tumor uptake and localization index of radioiodinated SV2-61r were lower than those of 111In-labeled SV2-61r, probably due to the internalization and dehalogenation of formed antibody-antigen complexes. Biodistribution and specificity of targeting were assessed by comparison among three cells, A4, lung cancer SBC-3 (c-erbB-2 weakly positive) and B-lymphoblastoid Manca cells (c-erbB-2 negative). Tumor:blood ratios, obtained 48 h after injection, were 5.63, 1.45, and 0.68, respectively, indicating the potential of 111In-labeled SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells. Because of its close relationship with carcinogenesis and the uniform expression, c-erbB-2 protooncogene products seem to be the optimal target of imaging and therapy of adenocarcinoma patients.

  14. 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.

  15. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

    PubMed Central

    Williamson, R A; Burioni, R; Sanna, P P; Partridge, L J; Barbas, C F; Burton, D R

    1993-01-01

    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. Images Fig. 1 Fig. 2 PMID:7683424

  16. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  17. Immunohistochemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoccidioides brasiliensis in biopsy material and partial characterization by using species-specific monoclonal antibodies.

    PubMed Central

    Figueroa, J I; Hamilton, A; Allen, M; Hay, R

    1994-01-01

    Two murine monoclonal antibodies (MAbs) specific to Paracoccidioides brasiliensis (as determined by enzyme-linked immunosorbent assay [ELISA] and Western blot [immunoblot]) were produced by using a modification of standard hybridization protocols, with cyclophosphamide included as an immunomodulator to abolish responses to highly cross-reactive immunodominant epitopes. MAbs PS14 and PS15 are two different clones which exhibit similar characteristics by ELISA and Western blot. They are directed against a 22- to 25-kDa antigen which is present in P. brasiliensis and which could not be identified in other dimorphic fungi by ELISA or Western blot. Partial purification of the antigen was accomplished by isoelectric focusing, and deglycosylation studies suggested that the 22- to 25-kDa antigen is a glycoprotein with a pI of between 4.5 and 5 and that O-linked sugars may be part of the recognized epitope. The MAbs stained the cytoplasm of P. brasiliensis yeast and hyphal cells in cryostat sections of fresh cultures of the fungus. In addition, the MAbs stained the wall of paracoccidioidomycotic granulomas, as well as the cytoplasm of the fungus, as determined by the use of immunofluorescence, immunoperoxidase, and immuno-alkaline phosphatase staining techniques in paraffin-embedded sections of human biopsy material, and they failed to stain granulomas resulting from other clinical conditions. These findings suggest that these MAbs have potential use in the immunohistochemical identification of P. brasiliensis. Images PMID:8077405

  18. 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

    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

  19. Treatment of leukemia with radiolabeled monoclonal antibodies.

    PubMed

    Sgouros, G; Scheinberg, D A

    1993-01-01

    In contrast to radioimmunotherapy of solid disease, wherein the primary obstacle to success is access of radiolabeled antibody to antigen-positive cells, in the treatment of leukemia delivering a lethal absorbed dose to the isolated cell appears to be the primary obstacle. The isolated cell is defined as one that is exposed only to self-irradiation (from internalized or surface-bound radiolabeled antibody) and to irradiation from free antibody in the blood. It is isolated in the sense that the particulate (beta, electron, alpha) emissions from its nearest neighboring antigen-positive cell do not contribute to its absorbed dose. Disease in the bone marrow and other tissues, since it is confined to a smaller volume, is more easily eradicated because the absorbed dose to a given cell nucleus is enhanced by emissions from adjacent cells (a smaller fraction of the emission energy is 'wasted'). The optimization simulations presented above for the M195 antibody suggest that the optimum dose of antibody that should be administered is that required to yield a concentration within the distribution volume of the antibody that is approximately equal to the concentration of antigen sites as determined by the tumor burden. Although not specifically considered in the modeling example presented above, antibody internalization and catabolism may be expected to play an important role in radioimmunotherapy treatment planning of leukemia. Depending upon the kinetics of internalization and catabolism, the absorbed dose to the red marrow and to antigen-positive cells may be reduced considerably, since catabolism, assuming that it is followed by rapid extrusion of the radioactive label, would decrease the cells' exposure time considerably. The recently demonstrated effectiveness of radioimmunotherapy in certain cases of B-cell lymphoma and in reducing tumor burden in acute myelogenous leukemia suggests that radioimmunotherapy is beginning to fulfill the promise held when it was initially

  20. Cysteinylation of a monoclonal antibody leads to its inactivation.

    PubMed

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  1. Molecular specificities of monoclonal antibodies directed against virulent Treponema pallidum.

    PubMed Central

    Marchitto, K S; Selland-Grossling, C K; Norgard, M V

    1986-01-01

    Radioimmunoprecipitation (RIP) and Western blot analyses with specific anti-Treponema pallidum subsp. pallidum monoclonal antibodies were used to identify antigens with apparent masses of 102, 84, 54, 53, 52, 47, 32, 29, and 24 kilodaltons (kDa). Cross-reactivity of these antibodies with T. pallidum subsp. pertenue antigens and lack of cross-reactivity with T. phagedenis biotype Reiter, T. vincentii, T. refringens, T. scoliodontum, and T. denticola were also demonstrated by RIP and Western blot analyses. Reactivities in the T. pallidum immobilization test, along with the RIP of lactoperoxidase-catalyzed iodination products, suggested that the identified antigens were surface associated. The abundance and surface association of the 47- and 84-kDa antigens were supported by reactivity in the microhemagglutination test for T. pallidum and by strong reactivity of monoclonal antibodies upon indirect immunofluorescence assays with rabbit-cultivated T. pallidum subsp. pallidum, respectively, but not with T. phagedenis biotype Reiter. Anti-47-kDa and anti-84-kDa monoclonal antibodies were also reactive in indirect immunofluorescence assays using treponemes found in dark-field-positive smears of human genital ulcers. Images PMID:3510168

  2. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    SciTech Connect

    Miller, M.R.; Heyneman, C.; Walker, S.; Ulrich, R.G.

    1986-03-01

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S/sub 1/ nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by (/sup 3/H)TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.

  3. Human antiglioma monoclonal antibodies from patients with astrocytic tumors.

    PubMed

    Dan, M D; Schlachta, C M; Guy, J; McKenzie, R G; Dorscheid, D R; Sandor, V A; Villemure, J G; Price, G B

    1992-04-01

    The current management of malignant gliomas is unsatisfactory compared to that of other solid tumors; the expected median survival period is less than 1 year with the patient undergoing conventional surgery, radiotherapy, and chemotherapy treatment. Immunological reagents could be a useful adjunct. Human monoclonal antibodies derived from patients with astrocytic tumors might recognize subtle antigenic specificities that would differ from those recognized by xenogeneic (murine) systems. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8, were produced from the fusion of peripheral blood lymphocytes of four patients with astrocytic tumors to the human myeloma-like cell line TM-H2-SP2. This cell line has a 46, XX karyotype and is negative for hypoxanthine guanine phosphoribosyltransferase. All five human monoclonal antibodies produced 2.4 to 44 micrograms/ml of immunoglobulin M, had a similar but not identical pattern of reactivity against a panel of human tumor cell lines, and failed to react with normal human astrocytes. Labeling of four neuroectodermal tumor explant cultures by BT27/2A3 was demonstrated by flow cytometry. Karyotyping of three of the five hybridomas demonstrated that two were pseudodiploid (2-3n) and one hypodiploid (less than 2n). The monoclonality of the hybridomas was evaluated by Southern blot analysis of JH gene rearrangements, revealing two types of rearrangements for each hybridoma, both consistent with monoclonality. Preliminary antigen characterization indicated that at least four of the five human monoclonal antibodies were directed to cell-surface glycolipids. PMID:1545260

  4. Development of highly specific monoclonal antibodies for the diagnosis of Vibrio cholerae 01.

    PubMed

    Castillo, L; Castillo, D; Silva, W; Zapata, L; Reid, M; Ulloa, M T; Seoane, M; Maldonado, A; Valenzuela, M E; Bustos, R

    1995-06-01

    We report here the development of two monoclonal antibodies, termed 5G8 and 5C12, belonging to the IgM and IgG1 class, respectively, suitable for the identification of Vibrio cholerae 01 in clinical and environmental samples. The specificities of the monoclonals were evaluated by ELISA and indirect immunofluorescent microscopy of microorganisms normally present in stool samples and with two bacterial panels. One panel included 72 potentially antigenically related bacterial strains and the second panel included 20 pathogenic bacterial strains involved in diarrhea cases. The results of these extensive analyses indicate that monoclonal antibodies 5G8 and 5C12 are highly specific and suitable for the clinical diagnosis of Vibrio cholerae 01 in human stool samples by indirect immunofluorescent microscopy. Although the antigenic sites recognized by these antibodies were not identified in this study, the observation of Western blot patterns suggested that 5G8 and 5C12 monoclonal antibodies bind to LPS epitopes, a good structural marker for the detection of V. cholerae 01 because it is present in all bacterial cell walls. PMID:7590791

  5. Monoclonal Antibody Drugs for Systemic Lupus Erythematosus.

    PubMed

    Kamenarska, Zornitsa G; Hristova, Maria H; Vinkov, Anton I; Dourmishev, Lyubomir A

    2015-01-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease which engages most of the immune cells in its development. Various studies concerning the application of antibodies against TNF-α, BlyS, CD20, CD22, IL-6R and complement factors in treatment of SLE have been recently conducted and in spite of the good results reported by some of them, no definite conclusion on their risk-benefit profile can be drawn. The current review summarizes the results obtained in the field and reveals the perspectives for the development of new and more effective strategies for SLE treatment in combination with other immunomodulating drugs. PMID:26933777

  6. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: Application for cell surface antigen detection

    PubMed Central

    Boeggeman, Elizabeth; Ramakrishnan, Boopathy; Pasek, Marta; Manzoni, Maria; Puri, Anu; Loomis, Kristin H.; Waybright, Timothy J.; Qasba, Pradman K.

    2012-01-01

    The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using β1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant β1,4 galactosyltransferase (β1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence Activated Cell Sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods. PMID:19425533

  7. Adverse Events of Monoclonal Antibodies Used for Cancer Therapy

    PubMed Central

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  8. [Increases in pharmaceutical expenditures of PHI by monoclonal antibodies].

    PubMed

    Wild, F

    2013-06-01

    The dynamics of one of the most innovative segments of health care and its impact on pharmaceutical expenditure of private health insurance (PHI) is examined on the basis of drug prescription data from private health insurance companies. The study shows that the increase in pharmaceutical expenditure can be explained partly by the new treatment possibilities available with monoclonal antibodies. The per capita expenditure on drugs with monoclonal antibodies increased by 255% from 2006 to 2010 in private health insurance, while the corresponding expenditure of all pharmaceuticals has risen by only 19% in the same period. In the coming years, growth on this scale will be a challenge for all payers in the health system. PMID:23926705

  9. Adverse events of monoclonal antibodies used for cancer therapy.

    PubMed

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  10. Biosimilar monoclonal antibodies in lymphoma: a critical appraisal.

    PubMed

    Rioufol, Catherine; Salles, Gilles

    2015-05-01

    Rituximab, an anti-CD20 monoclonal antibody, revolutionized the treatment of lymphoma. Although newer generation anti-CD20 monoclonal antibodies are being examined, patent expiries and patient demand have fueled the development of rituximab biosimilars. The development of such agents is both an important and difficult undertaking. By definition, although they aim to have safety and efficacy comparable with their reference agents, biosimilars are not exact replicas of those agents, and small changes in nonclinical and preclinical properties may ultimately affect in vivo activity. Consideration must be given to the complex mechanisms of action, sensitive patient populations that may be treated, and appropriate clinical trial endpoints. Furthermore, extrapolation of indications is multifaceted, deserving close examination. This review represents a critical look at biosimilars in lymphoma and their safety, efficacy and long-term effects on patient outcomes. PMID:25818308

  11. Human tumor antigens identified with monoclonal antibodies

    SciTech Connect

    AlSedairy, S.T.

    1987-01-01

    MoAbLc1 (IgM) and MoAbLc2 (IgG/sub 2a/) were produced against human lung carcinoma cell line (ChaGo). Lc1 recognizes a approx. = 330-kd/approx. = 310-kd glycoprotein complexes, and Lc2 recognizes a approx. = 60-kd/approx. = 47-kd protein complex. With a panel of cell lines of different tissue origin, Lc1 showed a more restricted reactivity to ChaGo; it cross-reacted with another lung carcinoma cell line (SK-Lc-2) and two breast carcinoma cell lines, but failed to react with cell lines of fetal lung, of colon, esophageal, prostate, stomach, and ovarian carcinomas, of B and T lymphoblastoid cells, neuroblastomas, glioblastoma, astrocytoma, and human peripheral blood lymphocytes. New and improved methods were developed for the production of indium-111-labeled MoAbs for tumor imaging. To facilitate the application of bicyclic anhydride diethylenetriaminepentaacetic acid (BADTPA) to In-111 labeling of antibodies, we have modified the original method by using C-14-labeled BADTPA, which allows precise quantitation of DTPA molecules incorporated. A new heterobifunctional reagent, 2,6-dioxo-N-(carboxyl)morpholine (DCM) was synthesized for chelating In-111 to MoAbs, and demonstrated higher retention of immunoreactivity of the labeled antibody.

  12. Monoclonal antibodies to the two most basic papaya proteinases.

    PubMed

    Goodenough, P W; Kilshaw, P J; McEwan, F; Owen, A J

    1986-08-01

    The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A. PMID:3545314

  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. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1992-08-01

    This research project is developing methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). This report describes the development of methods for labeling MAbs and their fragments with positron-emitting halogen nuclides, fluorine-18 and iodine-124. These nulides were selected because of the widespread availability of F-18 and because of our extensive experience in the development of new protein radiohalogenation methods.

  15. 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

  16. Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

    PubMed

    Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

    1991-07-01

    Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

  17. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  18. Monoclonal antibodies for medical oncology: a few critical perspectives.

    PubMed

    Belda-Iniesta, Cristóbal; Ibáñez de Cáceres, Inmaculada; de Castro, Javier

    2011-02-01

    Incorporation of antibodies as weapons for cancer therapy has meant a turning point in the survival, clinical and radiological response of many oncology patients. These drugs are effective, well designed missiles that either alone or in combination with chemotherapy are unavoidable weapons for breast, lung and colon cancer as well as for haematological tumours. In addition, incoming monoclonal antibodies (mAbs) and folder-like proteins will be incorporated into clinical practice in the near future. This review aims to discuss a few imminent indications of current mAbs that are used for solid tumours and to briefly introduce future mAbs to the reader. PMID:21324795

  19. Characterization of rabbit cells by monoclonal and polyclonal antibodies.

    PubMed Central

    Ponsard, D C; Cinader, B; Chou, C T; Dubiski, S

    1986-01-01

    Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed. PMID:3489667

  20. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    PubMed Central

    Moussavou, Ghislain; Ko, Kisung; Lee, Jeong-Hwan; Choo, Young-Kug

    2015-01-01

    Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. PMID:26550566

  1. [Continuous perfusion culture hybridoma cells for production of monoclonal antibody].

    PubMed

    Mi, Li; Li, Ling; Feng, Qiang; Yu, Xiao-Ling; Chen, Zhi-Nan

    2002-05-01

    Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization. PMID:12192875

  2. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    PubMed

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. PMID:27330002

  3. Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

    PubMed Central

    Kou, Baijun; Crawford, Sue E.; Ajami, Nadim J.; Czakó, Rita; Neill, Frederick H.; Tanaka, Tomoyuki N.; Kitamoto, Noritoshi; Palzkill, Timothy G.; Estes, Mary K.

    2014-01-01

    Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive diagnostic assays are essential for rapid detection of NoV infections. We previously generated a panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivities by use of virus-like particles (VLPs) from 16 different NoV genotypes (6 from genogroup I [GI], 9 from GII, and 1 from GIV) coating a microtiter plate (direct enzyme-linked immunosorbent assay [ELISA]) and by Western blotting. MAbs were genotype specific or recognized multiple genotypes within a genogroup and between genogroups. We next applied surface plasmon resonance (SPR) analysis to measure MAb dissociation constants (Kd) as a surrogate for binding affinity; a Kd level of <10 nM was regarded as indicating strong binding. Some MAbs did not interact with the VLPs by SPR analysis. To further assess this lack of MAb-VLP interaction, the MAbs were evaluated for the ability to identify NoV VLPs in a capture ELISA. Those MAbs for which a Kd could not be measured by SPR analysis also failed to capture the NoV VLPs; in contrast, those with a measurable Kd gave a positive signal in the capture ELISA. Thus, some broadly cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles. One MAb, NV23, was able to detect genogroup I, II, and IV VLPs from 16 genotypes tested by sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time reverse transcription-PCR when the threshold cycle (CT) value was <31. Biochemical analyses of MAb reactivity, including SPR analysis, identified NV23 as a broadly reactive ligand for application in norovirus diagnostic assays. PMID:25428247

  4. Monoclonal antibodies and reagents for botulinum research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum as a dichain protein of ~ 150 kDa that blocks acetylcholine release resulting in muscular paralysis. Diagnosis of BoNT often relies on the mouse bioassay that has a detection limit of 10–20 pg/mL and can take up to four days to complet...

  5. Thrombus detection with a radiolabeled antiplatelet monoclonal antibody: Comparison with In-111-oxine technique in coronary and peripheral thrombi in dogs

    SciTech Connect

    Srivastava, S.C.; Esekowitz, M.D.; Meinken, G.E.; Lange, R.C.; Smith, E.; Carbo, P.; Scudder, L.E.; Coller, B.

    1985-05-01

    Platelets (P) labeled with low concentrations of an I-123 or In-111 labeled anti-P monoclonal antibody (7E3) were shown earlier to successfully image in-vivo thrombi. This study was carried out to compare this technique with In-111-oxine-P for localizing coronary and venous thrombi (CT and DVT) in dogs. Thrombi were induced using either transcatheter placement of a dacron pug, mechanical trauma, or electrocoagulation. 7E3 was labeled with I-131 in 70 +- 10% yield (labeling conditions: 100 ..mu..g 7E3; 5 ..mu..g chloramine T; I/sup -//7E3 less than or equal to1; 2 min reaction; 200 ..mu..l pH 7 phosphate buffer; Sp. act. 10-30 ..mu..Ci/..mu..g). Binding of I-131-7E3 to dog P was 75 +- 10% after 1 hr incubation with whole blood. The dogs were injected with 0.5 mCi In-111-oxine-P and 1 mCi I-131-7E3-P in quick succession and data (blood clearance and imaging) were collected for 4 hr. In one experiment, DVT were clearly imaged within 10 min and CT in 30-60 min with I-131-7E3-P. In contrast, In-oxine-P required at least 30 min for imaging DVT and CT were only faintly visualized at 3-4 hr. The DVT to blood and CT to blood ratios at 2.4 hr were 16 and 12.3 for I-131-7E3-P and 9.4 and 6.5 for In-111-oxine-P respectively. In these preliminary studies, I-131-7E3-P thus appears to be superior to In-oxine-P for imaging venous and coronary thrombi. Additionally, 7E3 can be labeled with Tc-99m and I-123 for reduced dose and better imaging properties.

  6. Isolation of human monoclonal antibodies from peripheral blood B cells.

    PubMed

    Huang, Jinghe; Doria-Rose, Nicole A; Longo, Nancy S; Laub, Leo; Lin, Chien-Li; Turk, Ellen; Kang, Byong H; Migueles, Stephen A; Bailer, Robert T; Mascola, John R; Connors, Mark

    2013-10-01

    Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity. PMID:24030440

  7. 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.

  8. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    PubMed Central

    Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

    2015-01-01

    Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

  9. Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

    PubMed

    Doorbar, J; Ely, S; Coleman, N; Hibma, M; Davies, D H; Crawford, L

    1992-03-01

    The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer. PMID:1371027

  10. Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

    PubMed

    Handman, E

    1990-07-01

    Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified because, unlike the parasite LPG as expressed on promastigotes or amastigotes, it could not be radiolabeled by galactose oxidase or periodate treatment of infected cells followed by reduction with 3H-labeled sodium borohydride. Some LPG epitopes displayed on the macrophage may be anchored with glycosylphosphatidylinositol, and some may be in the water-soluble phosphoglycan form bound to macrophage integrins involved in its specific recognition. The water-soluble population could be released from the infected macrophage by gentle protease treatment. PMID:1694823

  11. Distribution of monoclonal antibody-defined monosialoganglioside in normal and cancerous human tissues: an immunoperoxidase study.

    PubMed

    Arends, J W; Verstynen, C; Bosman, F T; Hilgers, J; Steplewski, Z

    1983-01-01

    The immunoreactivity of a monosialoganglioside antigen defined by monoclonal antibody 116NS19-9 (19-9) was studied in neoplastic and normal glandular and mucosal epithelia using an indirect immunoperoxidase method. In neoplastic mucosae, the antigen was detected in the majority of colorectal and endometrial carcinomas, predominantly in a focal staining pattern. A substantial proportion of gastric and pancreatic tumors and an occasional breast carcinoma also reacted with the monoclonal antibody. Expression of the monosialoganglioside in normal colonic mucosa appeared to be restricted to areas adjacent to tumor tissue. In gastric mucosa, the antigen was confined to some areas showing intestinal metaplasia. The antigen was also detected in the epithelium of normal mucosa of the gall bladder and endocervix, as well as in some ductal epithelia of the pancreas and salivary glands. Most other mucosae were negative for antigen expression. PMID:6381289

  12. Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

    PubMed Central

    Jacob, L; Tron, F; Lety, M A; Bach, J F

    1986-01-01

    An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera. PMID:3486065

  13. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

    PubMed Central

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  14. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  15. 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

  16. [Progress in preparation of small monoclonal antibodies of knock out technique].

    PubMed

    Liu, Jing; Mao, Xin-min; Li, Lin-lin; Li, Xin-xia; Wang, Ye; Lan, Yi

    2015-10-01

    With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research. PMID:26975094

  17. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  18. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    PubMed Central

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.

    2015-01-01

    ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more

  19. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

    PubMed Central

    Emini, E A; Jameson, B A; Lewis, A J; Larsen, G R; Wimmer, E

    1982-01-01

    Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites. PMID:6183443

  20. 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

  1. Endotoxin reduction in monoclonal antibody preparations using arginine.

    PubMed

    Ritzén, Ulrika; Rotticci-Mulder, Joke; Strömberg, Patrik; Schmidt, Stefan R

    2007-09-01

    A monoclonal antibody preparation was found to be contaminated with endotoxin. Several commercial endotoxin removal steps were attempted but failed to produce a significant reduction due to the fact that the endotoxin was associated with the antibody. Here, several methods for endotoxin removal based on immobilizing monoclonal antibodies to chromatographic media have been evaluated. A crucial step in this process was to dissociate the endotoxin from the protein surface for subsequent removal. This was accomplished by introducing different buffer additives in the mobile phase. In agreement with previous reports, non-ionic detergents efficiently removed endotoxin, but it was also found that 0.5M arginine performed equally well. Since arginine is a non-toxic common amino acid that can be readily removed, it was selected and successfully used in large-scale experiments. With this method, endotoxin could be reduced to <0.2 EU mg(-1) with recovery of the target protein being >95%. Since this procedure is easily integrated into the existing processes of mAb purification, it offers advantages in speed, cost and effort. PMID:17644450

  2. Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A

    SciTech Connect

    Goldman, A.; Vivian, G.; Gordon, I.; Pritchard, J.; Kemshead, J.

    1984-08-01

    The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered.

  3. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    PubMed

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-01

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. PMID:21315722

  4. 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

  5. Monoclonal antibodies: longitudinal prescribing information analysis of hypersensitivity reactions.

    PubMed

    Kleyman, Konstantin; Weintraub, Debra S

    2012-01-01

    Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy's life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3-4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use. PMID:22531444

  6. Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies.

    PubMed

    Gutiérrez-Aguirre, Ion; Hodnik, Vesna; Glais, Laurent; Rupar, Matevž; Jacquot, Emmanuel; Anderluh, Gregor; Ravnikar, Maja

    2014-02-15

    Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins. PMID:24220292

  7. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-12-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. PMID:2903125

  8. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed Central

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-01-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. Images PMID:2903125

  9. Characterization of group II avian adenoviruses with a panel of monoclonal antibodies.

    PubMed Central

    van den Hurk, J V; van Drunen Littel-van den Hurk, S

    1988-01-01

    The interaction between a panel of ten monoclonal antibodies and hemorrhagic enteritis virus, a group II avian adenovirus, was determined. The monoclonal antibodies reacted with all nine isolates of group II avian adenoviruses, but not with any of five types of group I avian adenoviruses. All ten monoclonal antibodies recognized antigenic determinants on the hexon protein of hemorrhagic enteritis virus when analyzed by immunoprecipitation and immunoblotting. They reacted only with the native hexon protein and not with protein denatured by sodium dodecyl sulfate or guanidine-HCl/urea treatment combined with reduction and carboxymethylation. Based on the results of competitive binding assays, the panel of monoclonal antibodies could be subdivided into two groups, which recognized different antigenic domains of the hemorrhagic enteritis virus hexon protein. The monoclonal antibodies in group 1 neutralized hemorrhagic enteritis virus infectivity while the monoclonal antibodies of group 2 did not. Group 1 consisted of eight monoclonal antibodies which could be further subdivided into subgroups 1A, 1B, 1C and 1D. The subdivision of the monoclonal antibodies was based on the degree of blocking in the competitive binding assays and differences in their ability to induce enhancement. In general, the monoclonal antibodies had a higher avidity for the virulent isolate of hemorrhagic enteritis virus than for the avirulent hemorrhagic enteritis virus isolate. Images Fig. 1. Fig. 2. Fig. 4. PMID:2461793

  10. Monoclonal antibody that preferentially binds polylysine, polyarginine, and histones and selectively decorates nuclei and chromosomes.

    PubMed Central

    Morgan, J L; Dennis, D D

    1984-01-01

    A monoclonal antibody, designated J-57, selectively and uniformly decorates the interphase nuclei and mitotic chromosomes of a variety of eucaryotic cells as determined by indirect immunofluorescence. As determined by enzyme-linked immunosorbent assay, however, this monoclonal antibody is not monospecific. It reacts weakly with cytochrome c, RNase A, and brain tubulin. By these tests monoclonal antibody J-57 has broad cross-reactivity similar to that of antisera directed against polylysine. The differential reactions of this monoclonal antibody suggest that it may be a useful immunohistochemical probe for nuclei and chromosomes in whole cells. Images PMID:6490815

  11. Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.

    PubMed

    Rothwell, C E; Khazaeli, M B; Bernstein, I A

    1985-08-15

    A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b. PMID:3935002

  12. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R. . Dept. of Radiology)

    1989-12-01

    The overall objective of this research project is to develop methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). Both diagnostic and therapeutic applications of labeled MAbs could be improved as a result of knowledge obtained through the exploitation of the advantageous imaging characteristics associated with PET. By labeling MAbs with positron-emitting nuclides, it should be possible to quantitate the dynamics of their three-dimensional distribution in vivo. Our long-term goals are to apply this approach. 3 tabs.

  13. Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

    PubMed Central

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

    Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens. Images PMID:2405017

  14. [Production of the monoclonal antibodies to the rabies virus nucleoprotein].

    PubMed

    Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

    2013-01-01

    Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate. PMID:24640170

  15. Immunosuppression associated with novel chemotherapy agents and monoclonal antibodies.

    PubMed

    Morrison, Vicki A

    2014-11-15

    The introduction of novel agents to the therapeutic armamentarium for oncologic, rheumatologic, and neurologic disorders has resulted in major clinical advances. These agents impact immune function, resulting in a discrete spectrum of infectious complications. Purine analogues and alemtuzumab alter cell-mediated immunity, resulting in opportunistic viral/fungal infections. Herpes zoster incidence increases with bortezomib. Hepatitis B reactivation may occur with rituximab. Cases of progressive multifocal leukoencephalopathy have occurred following monoclonal antibody therapy. Tumor necrosis factor-α inhibitor therapy is complicated by tuberculosis reactivation and fungal infections. We summarize the impact of these therapies on pathogenesis and spectrum of infection complicating their usage. PMID:25352632

  16. Development of monoclonal antibodies in China: overview and prospects.

    PubMed

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Hu, Hao; 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. PMID:25811022

  17. 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. PMID:25811022

  18. Boronated monoclonal antibody conjugates for neutron capture therapy

    SciTech Connect

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    Monoclonal antibodies (MoAbs) to tumor-associated antigens are attractive for concentrating /sup 10/B in cancer tissue, in part because neutron capture therapy (NCT) is not disadvantaged by the hours to days required to optimize tumor:background concentration ratios of MoAbs or their F(ab')/sub 2/ or Fab fragments. Since direct coupling of /sup 10/B compounds in amounts sufficient for radiotherapy appears to inactivate MoAbs, the authors used dextran intermediate carriers to provide high levels of /sup 10/B per MoAb while modifying fewer amino acid residues.

  19. Monoclonal Antibodies in Cancer Therapy: Mechanisms, Successes and Limitations

    PubMed Central

    Coulson, A; Levy, A; Gossell-Williams, M

    2014-01-01

    ABSTRACT Rituximab was the first chemotherapeutic monoclonal antibody (CmAb) approved for clinical use in cancer therapeutics in 1997 and has significantly improved the clinical outcomes in non-Hodgkin's lymphoma. Since then, numerous CmAbs have been developed and approved for the treatment of various haematologic and solid human cancers. In this review, the classification, efficacy and significantly reduced toxicity of CmAbs available for use in the United States of America are presented. Finally, the limitations of CmAbs and future considerations are explored. PMID:25803383

  20. Therapeutic monoclonal antibodies and derivatives: Historical perspectives and future directions.

    PubMed

    Rodgers, Kyla R; Chou, Richard C

    2016-11-01

    Biologics, both monoclonal antibodies (mAbs) and fusion proteins, have revolutionized the practice of medicine. This year marks the 30th anniversary of the Food and Drug Administration approval of the first mAb for human use. In this review, we examine the biotechnological breakthroughs that spurred the explosive development of the biopharmaceutical mAb industry, as well as how critical lessons learned about human immunology informed the development of improved biologics. We also discuss the most common mechanisms of action of currently approved biologics and the indications for which they have been approved to date. PMID:27460206

  1. 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.

  2. Specific biodetection of B16 mouse melanoma in vivo by syngeneic monoclonal antibody

    SciTech Connect

    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 /sup 125/I-labeled monoclonal antibody. Tumor location was specifically visible without subtraction or enhancement methods 3-5 days after injection of the radiolabeled antibody.

  3. Differentiation of Trypanosoma cruzi, T. cruzi marinkellei, T. dionisii and T. vespertilionis by monoclonal antibodies.

    PubMed

    Petry, K; Baltz, T; Schottelius, J

    1986-03-01

    Anti-T. dionisii and anti-T. vespertilionis monoclonal antibodies secreted by 17 hybridoma clones were tested against various strains of T. dionisii, T. vespertilionis, T. cruzi and T. cruzi marinkellei. Strain and species specific antigens were detected for the homologous immunizing strains. The common antigenic determinants of the tested trypanosome species include a component of the flagellum and different cell structures. Seventeen T. cruzi strains could be classified into two groups when tested with anti-T. dionisii monoclonal antibodies. The cross reactions between T. dionisii and T. cruzi demonstrate a strong correlation between T. dionisii and T. cruzi group 2. On the other hand T. cruzi group 1 and T. cruzi marinkellei show very similar antigenic character. PMID:2424290

  4. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR.

    PubMed

    Bösmüller, Hans; Fischer, Anna; Pham, Deborah L; Fehm, Tanja; Capper, David; von Deimling, Andreas; Bonzheim, Irina; Staebler, Annette; Fend, Falko

    2013-03-01

    Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content. PMID:23089489

  5. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

    PubMed

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  6. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

    PubMed Central

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  7. 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

  8. A review of monoclonal antibody therapies in lymphoma.

    PubMed

    Teo, Esmeralda Chi-yuan; Chew, Yveline; Phipps, Colin

    2016-01-01

    Monoclonal antibodies (moAb) represent a novel way of delivering therapy through specific target antigens expressed on lymphoma cells and minimizes the collateral damage that is common with conventional chemotherapy. The paradigm of this approach is the targeting of CD20 by rituximab. Since its FDA approval in 1997, rituximab has become the standard of care in almost every line of therapy in most B-cell lymphomas. This review will briefly highlight some of the key rituximab trials while looking more closely at the evidence that is bringing other antibodies, including next generation anti-CD20 moAbs, and anti-CD30 moAbs, among others to the forefront of lymphoma therapy. PMID:26318093

  9. 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.

  10. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.

    PubMed

    Xie, Qing; Moore, Benjamin; Beardsley, Richard L

    2016-01-01

    Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. PMID:26651858

  11. Monoclonal antibody-based immunotherapy for multiple myeloma.

    PubMed

    Danylesko, Ivetta; Beider, Katia; Shimoni, Avichai; Nagler, Arnon

    2012-09-01

    Multiple myeloma (MM) is a life-threatening hematological malignancy. High-dose chemotherapy followed by autologous stem cell transplantation is a relatively effective treatment, but disease recurrence remains a major obstacle. Allogeneic transplantation may result in durable responses and cure due to antitumor immunity mediated by donor lymphocytes. However, morbidity and mortality related to graft-versus-host disease remain a challenge. Recent advances in understanding the interaction between the immune system of the patient and the malignant cells are influencing the design of clinically more efficient study protocols for MM. This review will focus on MM antigens and their specific antibodies. These monoclonal antibodies are an attractive therapeutic tool for MM humoral immunotherapy, with most promising preclinical results. PMID:23046236

  12. Clearance of persistent hepatitis C virus infection using a claudin-1-targeting monoclonal antibody

    PubMed Central

    Mailly, Laurent; Wilson, Garrick K.; Aubert, Philippe; Duong, François H. T.; Calabrese, Diego; Leboeuf, Céline; Fofana, Isabel; Thumann, Christine; Bandiera, Simonetta; Lütgehetmann, Marc; Volz, Tassilo; Davis, Christopher; Harris, Helen J.; Mee, Christopher J.; Girardi, Erika; Chane-Woon-Ming, Béatrice; Ericsson, Maria; Fletcher, Nicola; Bartenschlager, Ralf; Pessaux, Patrick; Vercauteren, Koen; Meuleman, Philip; Villa, Pascal; Kaderali, Lars; Pfeffer, Sébastien; Heim, Markus H.; Neunlist, Michel; Zeisel, Mirjam B.; Dandri, Maura; McKeating, Jane A.; Robinet, Eric; Baumert, Thomas F.

    2015-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer1. Cell entry of HCV2 and other pathogens3-5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model6 we show that a monoclonal antibody specific for TJ protein claudin-17 eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection via host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy. PMID:25798937

  13. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    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.

  14. Monoclonal antibodies to native noncollagenous bone-specific proteins.

    PubMed Central

    Stenner, D D; Romberg, R W; Tracy, R P; Katzmann, J A; Riggs, B L; Mann, K G

    1984-01-01

    Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J.A., Nesheim, M.E., Hibbard, L.S. & Mann, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 162-166; and Foster, W.B., Katzmann, J.A., Miller, R.S., Nesheim, M.E. & Mann, K.G. (1982) Thromb. Res. 28, 649-661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibody-bound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 125I-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10(-6) for anti-bone-Gla protein and (I50 = 10(-5) for antiosteonectin. Both of the antibovine antibodies are cross-reactive with the corresponding human protein. Immobilized specific anti-bone-Gla protein has been used to isolate human bone

  15. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  16. Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

    PubMed

    Ozyörük, F; Cheevers, W P; Hullinger, G A; McGuire, T C; Hutton, M; Knowles, D P

    2001-01-01

    Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. PMID:11139194

  17. 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

  18. 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

  19. High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

    PubMed

    Li, Xiaoning; Geng, Steven B; Chiu, Mark L; Saro, Dorina; Tessier, Peter M

    2015-03-18

    Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 μg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery. PMID:25714504

  20. 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

  1. Examination of HER3 targeting in cancer using monoclonal antibodies

    PubMed Central

    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

    2015-01-01

    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

  2. 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

  3. Screening individual hybridomas by microengraving to discover monoclonal antibodies.

    PubMed

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

    2009-01-01

    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 (approximately 10(5) 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

  4. Characterization and immunotherapeutic potential of a monoclonal antibody against a ras oncogene transformed cell line

    SciTech Connect

    Ames, R.S. Jr.

    1986-01-01

    Transformed cells express cell surface antigens not present, or present in diminished amounts on normal cells. Monoclonal antibodies can be used to identify and biochemically characterize tumor-associated antigens. Monoclonal antibody (MoAb) 45-2D9 was produced by immunization of BALB/c mice with a transformed cell line (45-2D9) induced by transfection of NIH 3T3 cells with a c-H-ras oncogene in DNA isolated from a human lung carcinoma. By immunoperoxidase staining, this antibody binds to the 45-342 cells as well as to the ras transformed primary and 3 secondary transfectants, including the one used to induce 45-342, but not to other ras transformed cell lines. Murine tumors as well as human fetal and most normal adult tissues are not stained. This antibody does bind to a variety of human tumors, including lung adenocarcinomas, as well as breast, colon and esophageal carcinomas. The ability of MoAb 45-2D9 to target ricin toxin A chain (RTA) and radio-isotopes to gp74 expressing cells was investigated. An immunotoxin generated by conjugating RTA to MoAb 45-2D9 inhibits protein and DNA synthesis by the 45-342 cells. Radiolabeled antibody specifically localizes to and can be used to image subcutaneous and pulmonary gp74 expressing tumors in nu/nu mice. Monoclonal antibodies against oncogene transformed cell lines may be useful for the detection and characterization of tumor-associated antigens as well as for the development of new tumor therapeutic and diagnostic reagents.

  5. Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein.

    PubMed

    Mao, Shanping; Xiong, Guoxiang; Zhang, Lei; Dong, Huimin; Liu, Baohui; Cohen, Noam A; Cohen, Akiva S

    2016-01-01

    A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60. PMID:27242450

  6. Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein

    PubMed Central

    Mao, Shanping; Xiong, Guoxiang; Zhang, Lei; Dong, Huimin; Liu, Baohui; Cohen, Noam A.; Cohen, Akiva S.

    2016-01-01

    A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60. PMID:27242450

  7. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach.

    PubMed

    Sohn, Catherine S; Cheng, Tim T; Drummond, Michael L; Peng, Eric D; Vermont, Sarah J; Xia, Dong; Cheng, Stephen J; Wastling, Jonathan M; Bradley, Peter J

    2011-01-01

    Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen. PMID:21483743

  8. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    PubMed

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection. PMID:26691263

  9. Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk

    PubMed Central

    Moineau, Sylvain; Bernier, Denis; Jobin, Marie; Hébert, Jacques; Klaenhammer, Todd R.; Pandian, Sithian

    1993-01-01

    The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 107 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages. Images PMID:16348980

  10. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed Central

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-01-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. Images PMID:2893776

  11. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-03-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. PMID:2893776

  12. 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.

  13. Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species.

    PubMed Central

    Fukushi, H; Nojiri, K; Hirai, K

    1987-01-01

    A total of 77 Chlamydia psittaci strains of avian, human, and mammalian origin were grouped into four serovars with 11 monoclonal antibodies recognizing the lipopolysaccharide and the major outer membrane protein antigens. The avian and human strains, which were closely related to each other, were distinct from the mammalian strains. Immunological typing of C. psittaci with monoclonal antibodies seems practical. PMID:3667918

  14. 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 ...

  15. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. PMID:27284048

  16. New monoclonal antibodies specific for 1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole.

    PubMed

    Nakayama, Hiroshi; Kenjyou, Noriko

    2015-02-01

    1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole (AM694) is one of the synthetic cannabinoids and an illegal drug in Japan. It is important to generate a monoclonal antibody (MAb) against AM694 for use in the rapid and sensitive detection of the drug. Two monoclonal antibodies, named HN0124 (IgG1) and NK0504 (IgG1), were obtained, which were possibly effective for detecting AM694 and its derivatives. The cross-reactive ability of these MAbs was evaluated using a competitive enzyme-linked immunosorbent assay. In the results, both of these antibodies recognize 1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole, 1-(5-fluoropentyl)-3-(3-iodobenzoyl)indole, 1-(5-fluoropentyl)-3-(4-iodobenzoyl)indole. Forty nmol/L AM694 can be detected using HN0124 MAb. Thus, MAbs produced in this study could be considered a useful tool for the detection of AM694. PMID:25723285

  17. Generation and characterization of novel conformation-specific monoclonal antibodies for α-synuclein pathology.

    PubMed

    Vaikath, Nishant N; Majbour, Nour K; Paleologou, Katerina E; Ardah, Mustafa T; van Dam, Esther; van de Berg, Wilma D J; Forrest, Shelley L; Parkkinen, Laura; Gai, Wei-Ping; Hattori, Nobutaka; Takanashi, Masashi; Lee, Seung-Jae; Mann, David M A; Imai, Yuzuru; Halliday, Glenda M; Li, Jia-Yi; El-Agnaf, Omar M A

    2015-07-01

    α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including β-syn, γ-syn, β-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer's disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies. PMID:25937088

  18. Immunoperoxidase inhibition assay for rabies antibody detection.

    PubMed

    Batista, H B C R; Lima, F E S; Maletich, D; Silva, A C R; Vicentini, F K; Roehe, L R; Spilki, F R; Franco, A C; Roehe, P M

    2011-06-01

    An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available. PMID:21458492

  19. Therapy of a murine sarcoma using syngeneic monoclonal antibody

    SciTech Connect

    Kennel, S.J.; Lankford, T.; Flynn, K.M.

    1983-01-01

    Syngeneic monoclonal antibodies (MoAb) to Moloney sarcoma cells were produced by fusion of spleen cells from MSC regressor mice to myeloma SP2/0. MoAb 244-19A, an immunoglobulin G2b, bound to MSC cells and did not bind to two other sarcomas (K-BALB and Ha2), a carcinoma (Line 1), a fibroblast (A31) or a fibroblast infected with C-type virus (A31) or a fibroblast infected with C-type virus (A31-Moloney leukemia virus). In contrast, MoAb 271-1A bound to the MSC and Ha2 sarcoma and line 1 carcinoma as well as to the normal and infected fibroblast cultures. Antibodies were tested for therapeutic effect using three schedules of antibody injection. Injection i.p. of ascites fluid containing 244-19A MoAb given on Days -1, 0, and +1 relative to tumor cell injection increased life span significantly over that of control animals given injections (P3, immunoglobulin G, or MoAb 271-1A) and produced some seven of 19, one of five, and one of five long-term survivors in three separate experiments. Antibody given to animals with established tumors (4 days after implantation) also prolonged life span significantly and produced three of nine long-term survivors. Antibody given to animals with very large tumor burdens (10 days after implantation) did not prolong life span significantly. Optimal dose, schedule, and mechanism studies concerning this therapy are in progress.

  20. Production and characterization of monoclonal antibodies to Newcastle Disease Virus.

    PubMed

    Kumar, G Ravi; Saxena, Shikha; Sahoo, A P; Chaturvedi, Uttara; Kumar, Satish; Santra, Lakshman; Desai, G S; Singh, Lakshyaveer; Tiwari, Ashok K

    2016-03-01

    Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot. PMID:27145631

  1. Removal of drugs from the circulation using immobilized monoclonal antibodies

    SciTech Connect

    Brizgys, M.V.

    1987-01-01

    High-affinity monoclonal antidigoxin antibodies (dig-Ab) were immobilized to a pellicular microbead and characterized in terms of antibody affinity, specificity for other glycosides, and binding capacity. Determination of digoxin binding revealed that the binding capacity decreased to 25% of theoretical capacity. Attempts to improve the binding capacity were ineffective. A guinea pig animal model was developed to determine the efficacy of removing digoxin in vivo from the circulation using an antibody column. Male guinea pigs were hemoperfused with either a dig-Ab or bovine Y-globulin control column 16 h after a single i.v. injection of digoxin. Pre- and postcolumn plasma concentrations were obtained to evaluate the extraction efficiency. Hemoperfusion continued for 3 h at flow rates of 1.0-2.0 mL/min. Bound digoxin was eluted as described earlier and concentrations determined by (/sup 125/I) digoxin RIA. Amounts of digoxin removed represented less than 1% of the total body content. After several studies with the same column, the dig-Ab had lost most of its activity. A freshly prepared dig-Ab column removed approximately 20% of the total body content. Most of the measured constituents of the blood were unaffected by the procedure.

  2. Potential of palladium-109-labeled antimelanoma monoclonal antibody for tumor therapy

    SciTech Connect

    Fawwaz, R.A.; Wang, T.S.T.; Srivastava, S.C.; Rosen, J.M.; Ferrone, S.; Hardy, M.A.; Alderson, P.O.

    1984-07-01

    Palladium-109, a beta-emitting radionuclide, was chelated to the monoclonal antibody 225.28S to the high molecular weight antigen associated with human melanoma. Injection of the radiolabeled monoclonal antibody into nude mice bearing human melanoma resulted in significant accumulation of the radiolabel in the tumors: 19% injected dose/g; 38:1 and 61:1 tumor-to-blood ratios at 24 and 48 hr, respectively. The localization of the radiolabeled antibody in liver and kidney also was high, but appreciably lower than that achieved in tumor. These results suggest Pd-109-labeled monoclonal antibody to tumor-associated antigens may have potential applications in tumor immunotherapy.

  3. Development of Human Monoclonal Antibodies Against Respiratory Syncytial Virus Using a High Efficiency Human Hybridoma Technique.

    PubMed

    Alvarado, Gabriela; Crowe, James E

    2016-01-01

    Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules, and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past, however, isolation of human monoclonal antibodies was difficult and inefficient. Here, we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines, especially lines secreting neutralizing antibodies. PMID:27464688

  4. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16.

    PubMed

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkafi-Mesrati, Lobna; Jaoua, Samir; Stephan, Dietrich

    2015-03-01

    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies. PMID:25492687

  5. Analysis of Campylobacter jejuni antigens with monoclonal antibodies.

    PubMed Central

    Kosunen, T U; Bång, B E; Hurme, M

    1984-01-01

    To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp. PMID:6365954

  6. [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

  7. Monoclonal antibodies and the transformation of blood typing

    PubMed Central

    Marks, Lara

    2014-01-01

    Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics. PMID:25484059

  8. Target Therapy in Hematological Malignances: New Monoclonal Antibodies

    PubMed Central

    Szymczyk, Agnieszka; Pawlowski, Johannes

    2014-01-01

    Apart from radio- and chemotherapy, monoclonal antibodies (MoAbs) represent a new, more selective tool in the treatment of hematological malignancies. MoAbs bind with the specific antigens of the tumors. This interaction is a basis for targeted therapies which exhibit few side effects and significant antitumor activity. This review provides an overview of the functional characteristics of MoAbs, with some examples of their clinical application. The promising results in the treatment of hematological malignancies have led to the more frequent usage of MoAbs in the therapy. Development of MoAbs is a subject of extensive research. They are a promising method of cancer treatment in the future.

  9. Monoclonal antibody capture and viral clearance by cation exchange chromatography.

    PubMed

    Miesegaes, G R; Lute, S; Strauss, D M; Read, E K; Venkiteshwaran, A; Kreuzman, A; Shah, R; Shamlou, P; Chen, D; Brorson, K

    2012-08-01

    Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step. PMID:22488719

  10. Enantioselective hydrolysis of naproxen ethyl ester catalyzed by monoclonal antibodies.

    PubMed

    Shi, Zhen-Dan; Yang, Bing-Hui; Zhao, Jing-Jing; Wu, Yu-Lin; Ji, Yong-Yong; Yeh, Ming

    2002-07-01

    This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer. PMID:11983513

  11. Enhancing tumor-targeting monoclonal antibodies therapy by PARP inhibitors

    PubMed Central

    Yélamos, José; Galindo, Miguel; Navarro, Judith; Albanell, Joan; Rovira, Ana; Rojo, Federico; Oliver, Javier

    2016-01-01

    ABSTRACT Monoclonal antibodies (mAbs) have become a successful therapeutic approach in cancer. However, some patients do not achieve long-term clinical benefit and most mAbs only exert modest effects as monotherapies. Therefore, combinations with chemotherapy are currently being investigated. Emerging studies have shown a synergistic therapeutic effect of PARP inhibitors and mAbs in cancer. PARP enzymes catalytically cleave β-NAD+ and transfer the ADP-ribose moiety to acceptor proteins, modifying their function. In here, we update recent data about the therapeutic effect of the combination of PARP inhibitors with mAbs in cancer treatment and discuss the molecular mechanisms involved in this synergy. PMID:26942084

  12. Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

    PubMed

    Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

    1998-11-01

    The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur. PMID:9806864

  13. Expanded clinical and experimental use of SOX11 - using a monoclonal antibody

    PubMed Central

    2012-01-01

    Background The transcription factor SOX11 is of diagnostic and prognostic importance in mantle cell lymphoma (MCL) and epithelial ovarian cancer (EOC), respectively. Thus, there is an unmet clinical and experimental need for SOX11-targeting assays with low background, high specificity and robust performance in multiple applications, including immunohistochemistry (IHC-P) and flow cytometry, which until now has been lacking. Methods We have developed SOX11-C1, a monoclonal mouse antibody targeting SOX11, and successfully evaluated its performance in western blots (WB), IHC-P, fluorescence microscopy and flow cytometry. Results We confirm the importance of SOX11 as a diagnostic antigen in MCL as 100% of tissue micro array (TMA) cases show bright nuclear staining, using the SOX11-C1 antibody in IHC-P. We also show that previous reports of weak SOX11 immunostaining in a fraction of hairy cell leukemias (HCL) are not confirmed using SOX11-C1, which is consistent with the lack of transcription. Thus, high sensitivity and improved specificity are demonstrated using the monoclonal SOX11-C1 antibody. Furthermore, we show for the first time that flow cytometry can be used to separate SOX11 positive and negative cell lines and primary tumors. Of note, SOX11-C1 shows no nonspecific binding to primary B or T cells in blood and thus, can be used for analysis of B and T cell lymphomas from complex clinical samples. Dilution experiments showed that low frequencies of malignant cells (~1%) are detectable above background using SOX11 as a discriminant antigen in flow cytometry. Conclusions The novel monoclonal SOX11-specific antibody offers high sensitivity and improved specificity in IHC-P based detection of MCL and its expanded use in flow cytometry analysis of blood and tissue samples may allow a convenient approach to early diagnosis and follow-up of MCL patients. PMID:22738398

  14. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization.

    PubMed

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-10-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts. PMID:23851482

  15. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization

    PubMed Central

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-01-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts. PMID:23851482

  16. Cell line profiling to improve monoclonal antibody production.

    PubMed

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  17. Visualization of metastases from colon carcinoma using an iodine 131-radiolabeled monoclonal antibody

    SciTech Connect

    Leyden, M.J.; Thompson, C.H.; Lichtenstein, M.; Andrews, J.T.; Sullivan, J.R.; Zalcberg, J.R.; McKenzie, I.F.

    1986-03-15

    A murine monoclonal antibody that reacts with human colonic cancer (250-30.6) was labeled with radioactive iodine (131I) and the antibody was injected intravenously into 15 patients with known metastases originating from carcinoma of the colon (10 cases), malignant melanoma (1), breast (1), pancreas (1), hepatocellular carcinoma (1), and adenocarcinoma of unknown origin (1). Of the patients with metastatic colon carcinoma, there were 19 known deposits as judged by the techniques of clinical examination, x-rays, and scans obtained using sulpha-colloid. Of these 19 deposits, 17 (90%) were found using the 131I-labeled monoclonal antibody. In one case, the primary tumor, previously undiagnosed, was found. In only 1 of the 10 patients was tumor not found and this was due to the subsequent finding that the undifferentiated tumor did not react with antibody. Of the five patients who did not have carcinoma of the colon, three had negative scans, but two were positive. Thus, the technique of immunoscintography can readily detect both primary and metastatic tumors.

  18. Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies

    PubMed Central

    Inamdar, Shivangi M.; Hsu, Shu-Chan; Yeaman, Charles

    2016-01-01

    Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used

  19. Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies.

    PubMed

    Inamdar, Shivangi M; Hsu, Shu-Chan; Yeaman, Charles

    2016-01-01

    Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used

  20. Separation of Oxidized Variants of a Monoclonal Antibody by Anion-Exchange

    PubMed Central

    Teshima, Glen; Li, Ming-Xiang; Danishmand, Rahima; Obi, Chidi; To, Robert; Huang, Carol; Lahidji, Vafa; Freeberg, Joel; Thorner, Lauren; Tomic, Milan

    2010-01-01

    Monoclonal antibodies are subject to a variety of degradation mechanisms, therefore orthogonal techniques are required to demonstrate product quality. In this study, the three individual antibodies comprising a multi-antibody drug product, XOMA 3AB were evaluated by both cation-exchange (CEX) and anion-exchange chromatography (AEX). In contrast to CEX analysis which showed only a single, broad peak for the force-oxidized antibodies, AEX analysis of Ab-A (pI=7.6) revealed two more basic peaks. Ab-B (pI=6.7) bound but exhibited only a single major peak while Ab-C (pI=8.6) flowed through. Peptide mapping LC/MS analysis of the isolated Ab-A fractions demonstrated that the basic peaks resulted from oxidation in a complementary determining region (CDR). Differential scanning calorimetry (DSC) analysis of the oxidized Ab-A species showed a decrease in the Fab melting point for the oxidized species consistent with unfolding of the molecule. Greater/lesser surface exposure of ionic residues resulting from a conformational change provides a likely explanation for the dramatic shift in retention behavior for the Ab-A oxidized variants. Peptide mapping analysis of the Ab-B antibody showed, in contrast to Ab-A, no detectable CDR oxidation. Hence, the lack of separation of oxidized variants in Ab-B can be explained by the absence of CDR oxidation and the associated changes in secondary/tertiary structure which were observed for oxidized AbA. In summary, anion-exchange HPLC shows potential as an orthogonal analytical technique for assessing product quality of monoclonal antibody therapeutics. In the case of the XOMA 3AB drug product, two of the antibodies bound and one, Ab-A, exhibited separation of CDR oxidized variants. PMID:21145555

  1. 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.''

  2. Monoclonal antibody therapeutics with up to five specificities

    PubMed Central

    LaFleur, David W.; Abramyan, Donara; Kanakaraj, Palanisamy; Smith, Rodger G.; Shah, Rutul R.; Wang, Geping; Yao, Xiao-Tao; Kankanala, Spandana; Boyd, Ernie; Zaritskaya, Liubov; Nam, Viktoriya; Puffer, Bridget A.; Buasen, Pete; Kaithamana, Shashi; Burnette, Andrew F.; Krishnamurthy, Rajesh; Patel, Dimki; Roschke, Viktor V.; Kiener, Peter A.; Hilbert, David M.; Barbas III, Carlos F.

    2013-01-01

    The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model. PMID:23575268

  3. 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

  4. Comparison of the direct platelet immunofluorescence test (direct PIFT) with a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) in detection of platelet-associated IgG.

    PubMed

    Joutsi, L; Kekomäki, R

    1997-01-01

    Glycoprotein (GP)-specific platelet-associated IgG (PA-IgG) may be demonstrable in autoimmune-mediated thrombocytopenia. We studied 159 consecutive patients with histories of thrombocytopenia by a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) assay, which immobilizes GP IIb/ IIIa, GP Ib/IX and GP Ia/IIa simultaneously. This modification requires smaller quantities of platelets than standard measurements performed separately. PA-IgG was present in 84/159 (53%) patients, as shown by the direct platelet immunofluorescence test (PIFT) with flow cytometry as a reference. PA-IgG against GP IIb/IIIa and/or GP Ib/IX and/or GP Ia/IIa was noted in 46 patients (29%), of whom 93% (43/46)-were also PA-IgG positive. The amount of PA-IgG detected by PIFT correlated directly with that detected by direct MAIPA (r = 0.71; P < 0.001). Only three patients 12548 with negative direct PIFT had GP-specific PA-IgG. GPV-specific PA-IgG was detected in 13 (10%) of the 125 patients, in whom further studies could be performed. In the subgroup of patients with GP-specific PA-IgG, the median fluorescence intensities of direct PIFT were higher than in patients with no GP-specific PA-IgG (P < 0.001). Direct PIFT and direct MAIPA divided the patients into asymmetric subgroups. However, the relative roles of these tests in the diagnosis of autoimmune-mediated thrombocytopenia await further studies. PMID:9012711

  5. Immunoassay of red dyes based on the monoclonal antibody of β-naphthol.

    PubMed

    Qi, Yong H; Zhang, Hui C; Xu, Rui T; Liu, Jin; Zhang, Lei; Wang, Jian P

    2015-01-01

    The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1-4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%-79% (with β-naphthol as 100%), and the limits of detection were in the range of 1.3-1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%-115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods. PMID:26079338

  6. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases

    SciTech Connect

    Kanner, S.B.; Reynolds, A.B.; Vines, R.R.; Parsons, J.T. )

    1990-05-01

    Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, the authors previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60{sup src}, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, they have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60{sup src} were also detected by several of the monoclonal antibodies.

  7. Monoclonal Antibodies Inhibit the Adhesion of Mouse B 16 Melanoma Cells in vitro and Block Lung Metastasis in vivo

    NASA Astrophysics Data System (ADS)

    Vollmers, H. Peter; Birchmeier, Walter

    1983-06-01

    Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.

  8. Influence of unlabeled monoclonal anti-mouse antibody on the clearance rate of radiolabeled mouse monoclonal antibody

    SciTech Connect

    Wahl, R.L.; Laino, L.; Jackson, G.; Fisher, S.; Beierwaltes, W.H.

    1985-05-01

    High blood background levels of intact radiolabeled monoclonal antibody (MoAb) after intravenous (iv) injection are problematic. The injection of unlabeled polyclonal antimouse Abs following injection with labeled MoAbs produces accelerated MoAb clearance. This study evaluates a Mo antimouse Ab for efficacy of accelerating radio MoAb clearance. HB-58 is a rat/mouse MoAb which binds strongly to mouse kappa light chains present in 95% of murine monoclonals. It is unreactive with rat, rabbit or human kappa chains. Six rats were injected iv with 30 ..mu..Ci (approximately 6 ..mu..g) of I-125 UPC-10, a non-specific IgG2ak MoAb that is bound to well by HB-58. No alteration was seen in the clearance of UPC-10 in any of the animals, regardless of the injection type or amount on the second day. In addition, no increase in liver or spleen activity was seen in those rats that received HB-58. The lack of change in rate of clearance and biodistribution of UPC-10 after the iv injection of a purified, specific, anti-mouse MoAb is in marked contrast to the accelerated clearance reported following polyclonal anti-mouse antibody administration. This may be due to the inability of MoAbs to cross link. These preliminary studies suggest that Mo anti-mouse Abs, at these dose levels, are not useful in achieving increased rates of radiolabeled murine MoAb clearance.

  9. Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

    PubMed

    Lin, Gigi L; Pathak, Jai A; Kim, Dong Hyun; Carlson, Marcia; Riguero, Valeria; Kim, Yoen Joo; Buff, Jean S; Fuller, Gerald G

    2016-04-14

    Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization. We demonstrate that dilatational surface deformations are more important to antibody stability when compared to constant-area shear of the A/S interface. We have constructed a dilatational interfacial rheometer that utilizes simultaneous pressure and bubble shape measurements to study the mechanical stability of mAbs under interfacial aging. It has a distinct advantage over methods utilizing the Young-Laplace equation, which incorrectly describes viscoelastic interfaces. We provide visual evidence of particle ejection from dilatated A/S interfaces and spectroscopic data of ejected mAb particles. These rheological studies frame a molecular understanding of the protein-protein interactions at the complex-fluid interface. PMID:26891116

  10. Tumor size: effect on monoclonal antibody uptake in tumor models

    SciTech Connect

    Hagan, P.L.; Halpern, S.E.; Dillman, R.O.; Shawler, D.L.; Johnson, D.E.; Chen, A.; Krishnan, L.; Frincke, J.; Bartholomew, R.M.; David, G.S.

    1986-03-01

    Studies were performed to determine the effect of tumor size on the incorporation of radiolabeled monoclonal antitumor antibodies (MoAbs) into human tumors growing in nude mice. The colon tumors ranged in size from 0.03-1.6 g, the melanoma from 0.1 to 6.7 g, and the lymphoma from 0.06 to 10.2 g. Indium-111 was primarily used as the radiolabel, however, both 125I and 111In were used as tracers for the MoAb in one experiment. The per g radiopharmaceutical uptake by tumors was inversely proportional to tumor size when tumor specific MoAb was administered. This finding was independent of the radiolabel and was demonstrable when the mice bore two tumors of differing size. When the MoAb was not specific for the tumor, the data were less well defined and a statistically significant correlation with size did not occur. These data are strong evidence for a decrease in per g uptake of labeled tumor specific antibodies as tumors increase in size.

  11. Tregalizumab – A Monoclonal Antibody to Target Regulatory T Cells

    PubMed Central

    König, Martin; Rharbaoui, Faiza; Aigner, Silke; Dälken, Benjamin; Schüttrumpf, Jörg

    2016-01-01

    Regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation. PMID:26834751

  12. Evidence of a saturable hepatic receptor for mouse monoclonal antibodies

    SciTech Connect

    De Nardo, G.L.; De Nardo, S.J.; Peng, J.S.; O'Grady, L.F.; Mills, S.L.; Epstein, A.L.; Cardiff, R.D.

    1985-05-01

    Monoclonal antibodies (MAb) can be labeled with I-123 at high specific activities, so that large amounts of radioactivity attached to small amounts of protein can be injected for radioimmunoimaging. This conserves antibody and decreases the opportunity for foreign protein reactions and target tissue binding site saturation. In order to assess the effects on pharmacokinetics and imaging, the authors administered microgram amounts of I-123-MAb (Lyn-1, IgG2a or B6.01, IgGl with and following 4-5 milligram preloading with MAb on separate occasions to 4 patients with a target tumor (B cell lymphoma) and 2 patients without a target tumor (breast cancer). Pharmacokinetics were observed in blood and urine by counting whole samples and HPLC fractions of these samples and in organs by serial imaging. Early blood clearance and urinary excretion were faster after injection of microgram amounts of MAb, but subsequently were comparable to those obtained after preload. This paper concludes that the amount of administered MAb dramatically influences the pharmacokinetics of mouse MAb. Saturable hepatic Fc receptors are probably the source of these observations. Reports of accelerated deiodination of MAb are related to this phenomenon. Optimal imaging and treatment with MAb requires saturation of these hepatic receptors.

  13. Defining process design space for monoclonal antibody cell culture.

    PubMed

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. PMID:20589669

  14. Hierarchical Cluster Formation in Concentrated Monoclonal Antibody Formulations

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Zarzar, Jonathan; Zarraga, Isidro Dan; Porcar, Lionel; Falus, Peter; Wagner, Norman; Liu, Yun

    Reversible cluster formation has been identified as an underlying cause of large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations. As high solution viscosity prevents the use of subcutaneous injection as a delivery method for some mAbs, a fundamental understanding of the interactions responsible for high viscosities in concentrated mAb solutions is of significant relevance to mAb applications in human health care as well as of intellectual interest. Here, we present a detailed investigation of a well-studied IgG1 based mAb to relate the short time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. Using a combination of experimental techniques, it is found that upon adding Na2SO4, these antibodies dimerize in solution. Proteins form strongly bounded reversible dimers at dilute concentrations that, when concentrated, interact with each other to form loosely bounded, large, transient clusters. The combined effect of forming strongly bounded dimers and a large transient network is a significant increase in the solution viscosity. Strongly bounded, reversible dimers may exist in many IgG1 based mAb systems such that these results contribute to a more comprehensive understanding of the physical mechanisms producing high viscosities in concentrated protein solutions.

  15. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  16. Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

    PubMed

    Scott, M G; Cuca, G C; Petersen, J R; Lyle, L R; Burleigh, B D; Daughaday, W H

    1987-11-01

    We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h. PMID:2445506

  17. A technetium-labeled monoclonal antibody for imaging metastatic melanoma

    SciTech Connect

    Frytak, S.; Creagan, E.T.; Brown, M.L.; Salk, D.; Nelp, W. )

    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.

  18. Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

    PubMed

    Spanier, Justin A; Frederick, Daniel R; Taylor, Justin J; Heffernan, James R; Kotov, Dmitri I; Martinov, Tijana; Osum, Kevin C; Ruggiero, Jenna L; Rust, Blake J; Landry, Samuel J; Jenkins, Marc K; McLachlan, James B; Fife, Brian T

    2016-01-01

    Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes. PMID:27292946

  19. Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment

    PubMed Central

    Spanier, Justin A.; Frederick, Daniel R.; Taylor, Justin J.; Heffernan, James R.; Kotov, Dmitri I.; Martinov, Tijana; Osum, Kevin C.; Ruggiero, Jenna L.; Rust, Blake J.; Landry, Samuel J.; Jenkins, Marc K.; McLachlan, James B.; Fife, Brian T.

    2016-01-01

    Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide–MHCII complexes. PMID:27292946

  20. Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

    SciTech Connect

    Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M. )

    1991-03-01

    Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternative approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.