Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Epitope mapping of human monoclonal antibodies to HLA-B27 by using natural and mutated antigenic variants

The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCG10, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cells lines expressing the six HLA-B27 alleles B^*2701 to B^*2706. The results indicated that the mAbs are domain specific. TrBH12 recognizes the first external (alpha-1) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypeptide stretch 63–76 plus a critical amino acid at position 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VAL¹⁵²→GLU¹⁵²) destroyed its epitope. TrCG10 also binds the alpha-2 domain.     

Heteroclitic antibodies: differences in fine specificities between monoclonal antibodies directed against dinitrophenyl and trinitrophenyl haptens

The inhibition of binding of monoclonal antibodies by different haptens was studied using the 50% antibody binding assay. The binding of antidinitrophenyl and antitrinitrophenyl antibodies to dinitrophenylated or trinitrophenylated bovine serum albumin could be inhibited by monovalent dinitrophenyl or trinitrophenyl epsilon-aminocaproic acid. Some of the antibodies could be inhibited to a greater degree with the cross-reacting haptens than with the haptens homologous to the immunizing antigen, therefore these antibodies were heteroclitic.     

Generation and characterization of a panel of anti-phosphoprotein monoclonal antibodies directed against Mokola virus

The generation of a new panel of 21 monoclonal antibodies (MAbs) reactive with the P protein of Mokola virus (MOKV) is described. Through competitive ELISA and immunoblotting analyses, these MAbs were classified into several groups. Consistent with prior studies on lyssavirus P protein antigenic structure, many of the sites recognized by these Mabs appear to correspond to sites identified previously. Studies on the reactivity of these anti-MOKV P MAbs against a collection of lyssaviruses identified MAbs that were broadly cross-reactive to all genus members and others that bound selectively to members of different species. In particular the utility of this MAb panel for differentiation of African lyssaviruses was explored. Such a panel will be useful for further examination of the extent of functional complementation between lyssavirus P proteins.     

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.     

Antibodies to constant domains of therapeutic monoclonal antibodies: anti-hinge antibodies in immunogenicity testing

Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies.     

Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis lipopolysaccharide (LPS) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and ELISA-inhibition experiments with LPS and delipidated polysaccharide fragments (PS-1 and PS-2) prepared from B. pertussis LPS. Monoclonal antibody 9-1-H5 reacted with B. pertussis LPS only, whereas monoclonal antibodies 6-4-H6 and 9-2-A8 reacted with PS-1 and PS-2 as well as B. pertussis LPS. The antibodies did not react with LPS prepared from B. parapertussis and B. bronchiseptica in an LPS-specific ELISA. A monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis LPS. This assay had a detection limit of B. pertussis LPS in concentrations ranging from 0.16 to 0.32 microgram/ml. The assay was also shown to be specific for the detection of whole B. pertussis bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Streptococcus miteor, Haemophilus influenzae, or Legionella pneumophila. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis LPS.     

Production and characterization of monoclonal antibodies directed against guinea pig IgG subclasses

Monoclonal antibodies directed against guinea pig IgG subclass-specific epitopes have been generated and characterized. Three monoclonal antibodies designated GP1, GP2 and GP3 have been compared to rabbit polyclonal reagents for specificity against purified IgG subclass antigens and for the detection of herpes simplex-specific IgG subclasses, from infected guinea pig, by an indirect ELISA. The monoclonal antibodies were shown to have greater specificity than the corresponding polyclonal reagents.     

Recognition of HLA-B27 and related antigen by a monoclonal antibody

A monoclonal antibody that binds specifically to HLA-B27, B7, and B22 is described. Binding to B27 appeared to be slightly stronger than to B7 and stronger than to B22 in an indirect binding assay, but no difference in B7 and B27 binding could be detected by Scatchard analysis. No distinction could be made between B27 on cells from normal and from ankylosing spondylitis patients in any assay system. The antibody, which was not cytotoxic, blocked complement-dependent cytolysis mediated by human HLA typing sera specific for B7 and B27. Competitive binding studies with other monoclonal antibodies showed that ME1 could block the binding of antibodies that recognized different antigenic sites on HLA. ME1 did not bind to Klebsiella pneumoniae. This reagent will be useful in further analysis of the relationship between B27 and ankylosing spondylitis.     

Cell based radioimmunoassays to quantitate the immunoreactivity of TNT monoclonal antibodies directed against intracellular antigens

Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.     

Utility of monoclonal antibodies directed against B and T lymphocytes and monocytes in paraffin-embedded sections

Monoclonal antibodies reactive with B and T lymphocytes, monocytes, and granulocytes were applied to B-5-, Bouin's-, or formalin-fixed paraffin-embedded sections. Most antigens were destroyed or masked by fixation or embedding procedures, or both. However, T200, an antigen present in all lymphoid and hematopoietic cells, and Leu M1, an antigen in granulocytes, were well preserved in formalin-fixed tissues. Leu 1 and BA-1, antigens present on T and B lymphocytes, respectively, were preserved in Bouin's-fixed specimens. With careful selection of fixatives, identification of some T and B lymphocytes and granulocytes by monoclonal antibodies in paraffin-embedded specimens is possible.     

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.     

Characterization of monoclonal antibodies and polyclonal immune sera directed against human cytomegalovirus virion proteins

Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp58-gp130, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotein, pp71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, pp155, and (4) a virion-associated phosphoprotein, pp29. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 X 10(3) PFU/ml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation.     

Mixed precipitation in gel: a new method of identification and characterization of monoclonal antibodies

A mixed precipitation in the gel (MPG) technique is suggested for detection and characterization of monoclonal antibodies (MAbs). The MPG is based on the formation of a mixed precipitate composed of an antigen, the corresponding MAb and precipitating polyclonal antiserum. MAb incorporated into the precipitate is revealed by Fab'-peroxidase conjugate added to polyclonal antiserum. The MPG technique was applied to hybridoma screening as well as for the antigen and epitope specificity analysis of different MAbs. The MPG is a one-step, simple, inexpensive technique and valuable for the study of any antigen which could be revealed by immunodiffusion.     

Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters

Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P<0.0001) in the primary disease hamster model and from 78% to 32% (P<0.0001) in the less stringent relapse model.     

Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-Hodgkin's lymphoma

A study comparing the usage of monoclonal and polyclonal antibodies specific for immunoglobulin light and heavy chains was performed on frozen-tissue sections of 30 B-cell non-Hodgkin's lymphomas. In 16 cases, monotypic staining for an immunoglobulin light chain was demonstrated with monoclonal antibodies using a three-step avidin-biotin peroxidase complex (ABC) method; 13 cases were positive for kappa. In 14 cases, no immunoglobulin light-chain production was demonstrated. Repeat staining of 11 of these 14 cases with polyclonal anti-sera by a direct immunoperoxidase method demonstrated monotypic staining for light chain in 10 cases, 9 of which were positive for lambda. In 22 of 30 non-Hodgkin's lymphomas, an immunoglobulin heavy chain was identified using monoclonal anti-sera. In eight cases, however, no heavy chain was found. Repeated staining with polyclonal sera of additional sections in three of eight cases demonstrated heavy-chain production in each case. Decreased sensitivity, especially for the detection of the lambda light chain, rendered this particular lot of monoclonal antibodies unsuitable for immunophenotyping non-Hodgkin's lymphomas. Variability of antigenic sites on the immunoglobulin molecule seems a likely explanation for these observations.     

Development of monoclonal antibodies reactive with methamphetamine raised against a new antigen

Monoclonal antibodies (McAbs) specific to methamphetamine (MA) were produced using p-amino MA coupled to bovine serum albumin (BSA) with glutaraldehyde (GA) as an immunogen and with conventional hybridoma techniques. Hybridoma clones secreting the McAbs were selected by an enzyme-linked immunosorbent assay (ELISA) system using both the above conjugate and BSA modified with GA as screening antigens. In the ELISA system were used avidin and biotinyl-alkaline phosphatase which converts nicotinamide adenine dinucleotide phosphate (NADP) into NAD. The final enzyme activity was determined using diformazan of nitroblue tetrazolium formed together with the NAD produced, alcohol dehydrogenase and phenazine methosulfate. The McAbs from 9 clones were characterized by a crossreactivity test using the ELISA. The McAbs recognized MA (100%), methoxyphenamine (8.0%), ephedrine (2.3%), but did not react with metylephedrine, amphetamine, OH-amphetamine, dimethylamphetamine, beta-phenylethylamine, norephedrine, phentermine and ranitidine. An inhibition curve for MA was obtained in the range of 0.75 to 50 ng.     

Properties of two monoclonal antibodies directed against the Fc and Fab' regions of rat IgE

Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.     

Reactivity of rat and rabbit intrafusal fibers with monoclonal antibodies directed against myosin heavy chains

Serial cross and longitudinal sections from the intracapsular portions of intrafusal fibers of rat and rabbit tibialis anterior muscles were examined by fluorescence microscopy with a library of monoclonal antibodies directed against different epitopes on myosin heavy chains. Intrafusal fiber types were identified with the histochemical reactions for acid-stable and alkali-stable actomyosin ATPase. Three antibodies, known to react with avian heart and slow-tonic myosins, produced fluorescent staining in intrafusal fibers. Nuclear bag2 fibers reacted with all three antibodies, chain fibers with two, and nuclear bag1 fibers with only one. These results indicate that in rat and rabbit tibialis anterior muscle spindles nuclear bag2 fibers and chain fibers contain more than one myosin isoform. They also demonstrate that, in addition to the histochemical actomysin ATPase reaction, nuclear chain fibers and the two types of nuclear bag fibers can be identified by the selective reactivities of their myosin heavy chains.