Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes.

Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains.     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.     

Monoclonal antibodies to HLA recognize monomorphic and polymorphic epitopes on BoLA

Fourteen monoclonal antibodies recognizing monomorphic and polymorphic epitopes on class I and class II antigens of the human MHC have been assayed on lymphocytes of a panel of 20–150 BoLA typed bovine animals from 12 different breeds. Some monomorphic antibodies cross-reacted and others did not. Two polymorphic monoclonal antibodies in man recognize a polymorphism in cows that follows allospecificities (BoLA-w3, w9) already described. Immunoprecipitation experiments with monomorphic anti-B2m and anti-HLA-DR monoclonal antibodies have shown that these cross-reactions concern BoLA antigens. They also revealed that Ia-like antigens in cattle present the same two chain features characterized in other species.     

Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.     

Monoclonal antibodies directed against human airway secretions. Localization and characterization of antigens.

Cellular mechanisms of normal airway mucus secretion and their alterations in chronic obstructive lung disease are poorly understood. To aid in their study, the authors have produced a panel of monoclonal antibodies directed against various constituents of human airway secretions. Two fusions yielded 401 hybridoma-containing cultures. Supernatants from 150 of these cultures stained human tracheal secretory cells by immunofluorescence. Twenty-nine hybridomas were selected for expansion because they selectively stained a single cell type or displayed another interesting distribution. Antigens were further characterized by their localization in glycol methacrylate sections of human trachea, sensitivity to periodate oxidations, selective affinity for fraction peaks obtained by Sepharose 4B chromatography, and reactivity with molecules of various sizes, as estimated by SDS-PAGE. These antibodies will be useful for 1) quantitative detection of antigens in sputum or lavage samples by immunoassay and 2) purification and biochemical characterization of molecular constituents of airway secretions in health and disease.     

Monoclonal antibodies selectively directed against the cell wall surface of Mycobacterium tuberculosis.

In the last few years several monoclonal antibodies against Mycobacterium tuberculosis have been described, but their use as diagnostic tools has been limited. In this study we describe eight monoclonal antibodies against M. tuberculosis for diagnostic purposes. The monoclonal antibodies were selected after enzyme-linked immunosorbent assay screening with whole bacterial suspensions of mycobacteria and other bacterial species. Four monoclonal antibodies (BS100, BS101, BS102, and BS104) reacted with a whole bacterial suspension of M. tuberculosis but not with the other mycobacteria. When tested with a cytoplasmic fraction of mycobacteria the same monoclonal antibodies showed a broad cross-reactivity. Therefore the monoclonal antibodies showed not specific but selective binding to M. tuberculosis. The molecular size of the recognized antigens ranged from 12 to 71 kilodaltons as determined by the immunoblotting technique. The ability to differentiate M. tuberculosis from mycobacteria other than tuberculosis was investigated by enzyme-linked immunosorbent assay with 131 freshly cultured strains of M. tuberculosis from patients and 36 strains of mycobacteria other than tuberculosis. The monoclonal antibody BS104 could clearly distinguish between M. tuberculosis and other mycobacterial species.     

Monoclonal antibodies to human C-reactive protein (CRP) that recognize epitopes in functional regions

Thirteen mouse hybridomas secreting monoclonal antibodies (MAbs) to human C-reactive protein (CRP) were generated and characterized. The relative avidity of the MAbs for CRP ranged from 0.005 to 8.6 micrograms/ml of purified Ig. Several of the MAbs recognized an epitope on CRP expressed only in the presence of physiological levels of Ca++. The epitope specificity of the 13 MAbs was examined by testing their cross-reactivity and allowed us to assign them to two groups. Group I MAbs recognized the Ca++-dependent phosphorylcholine (PC)-binding site of CRP since their reactivity was inhibited by PC. Group II MAbs recognized epitopes not affected by binding of PC by CRP. The results suggest that these MAbs may be suitable for assigning functional properties of CRP to specific peptide sequences.     

Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes.

Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function.     

Monoclonal antibodies defining epitopes on human IgE.

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.     

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin G1 subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoformans, since in this group the present latex agglutination test has some limitations.     

Monoclonal antibodies identify conserved epitopes on the polyhedrin of Heliothis zea nuclear polyhedrosis virus

Monoclonal antibodies were formed against polyhedrin from the Heliothis zea S nuclear polyhedrosis virus. The cross-reactivity of these antibodies with polyhedrins and granulins of other occluded baculoviruses indicates that group and subgroup epitopes exist. Evidence is presented that multiple copies of one epitope or a series of closely related epitopes exist on the polyhedrin molecule. The monoclonal antibodies, with the exception of one IgM-producing hybridoma clone, were all of the IgG1 isotype. Clones producing antibodies with identical patterns of reactivity with heterologous polyhedrins and/or granulins but exhibiting different affinities for the homologous antigen (HzSNPV polyhedrin) are demonstrated.     

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.     

Monoclonal antibodies directed against human fibroblast interferon: characterization and functional studies

Spleen cells from BALB/c mice, immunized with SDS-acrylamide gel purified human fibroblast interferon (HuIFN-beta) were fused with mouse myeloma cells. Culture fluids from the resulting hybrid cells were screened for HuIFN-beta specificity by the following tests: a solid-phase RIA using partially purified HuIFN-beta, a protein-transfer RIA using electrophoretically resolved and immobilized HuIFN-beta, and a bioassay which tests residual HuIFN-beta activity in the supernatant following immunoprecipitation. Six HuIFN-beta-specific hybridomas were identified; two were capable of partially neutralizing HuIFN-beta activity. All six antibodies bind to the beta 1-IFN polypeptide synthesized in E. coli cells containing a cloned beta 1-IFN DNA sequence. All six monoclonal antibodies were found to be IgG3/kappa.     

Production and characterisation of monoclonal antibodies directed against different epitopes of human thyroid stimulating hormone.

We have produced monoclonal antibodies (mAbs) to human thyroid stimulating hormone (hTSH) and selected five that specifically recognize hTSH and do not cross-react with the other human glycoprotein hormones such as luteinizing hormone (LH), chorionic gonadotropin (CG), follicle stimulating hormone (FSH). All of the antibodies were of the IgG1 subclass with affinities ranging from 5.3 X 10(8) to 1.9 X 10(10) mol-1.l; they could be assigned to two subgroups on the basis of their epitope specificity.     

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.     

Antibodies against HLA-DP recognize broadly expressed epitopes

HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies.     

Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin-producing Escherichia coli.

Hybridoma-secreting monoclonal antibodies (MAbs) against colonization factor antigen I (CFA/I) were produced by the fusion of spleen cells from immunized BALB/c mice with F/O myeloma cells. The 25 MAbs with the highest antibody titer against CFA/I, as determined by an enzyme-linked immunosorbent assay, were studied for specificity and the capacity to agglutinate CFA/I-carrying bacteria. Most of the MAbs agglutinated a majority of 16 CFA/I-positive strains tested but not any of a number of CFA-deficient mutants or strains carrying other colonization factors (CFA/II and CFA/IV). One MAb that agglutinated all the CFA/I-positive strains and one MAb that did not agglutinate any of these strains were compared for their reactivities with different preparations of CFA/I. Whereas both MAbs bound to or could be inhibited by isolated CFA/I fimbriae as well as the subunit protein, only the agglutinating MAb bound to CFA/I, as expressed on whole bacteria. The nonagglutinating MAb, on the other hand, bound considerably better than the agglutinating MAb to a peptide corresponding to the 46 N-terminal amino acid residues of the CFA/I subunit protein. These results suggest that the two MAbs are directed against different epitopes on CFA/I.     

Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo.

We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.     

Monoclonal antibodies directed against human myoglobin: characterization and application in a bideterminant radioimmunoassay

Monoclonal hybridoma cell lines secreting antibodies directed against human myoglobin were selected. Two of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for human myoglobin that uniquely recognizes two different epitopes on the same molecule.     

Monoclonal antibodies against microorganisms

The recent spread of hybridoma technology among laboratories has promoted the development of monoclonal antibodies against a wide variety of infectious disease agents. While monoclonal antibodies theoretically represent an excellent (perhaps superior) alternative to conventional antisera as diagnostic, therapeutic or laboratory reagents, traditional antisera may be preferable to monoclonal antibody in some circumstances because of the fixed affinity and specificity as well as the limited functional capacities of some antibodies. The acceptance of monoclonal antibodies by the clinical microbiologist and physician must await proof of their reliability, safety and efficacy.