Monoclonal antibodies against a 97-kilodalton antigen from Aspergillus flavus.

We prepared a panel of five monoclonal antibodies (MAbs) directed against Aspergillus flavus that all reacted against one 97-kDa antigen by western blot (immunoblot). Flow cytometry demonstrated that these antibodies bound (in increasing degrees) to all morphologic stages of A. flavus growth: conidia, swollen conidia, and hyphae. Cross-reactivity among species was examined by enzyme-linked immunosorbent assay of fungal culture filtrates. Four MAbs reacted with 10 of 11 A. flavus isolates, and the fifth one reacted with 9 of them. One MAb also reacted with A. fumigatus, two reacted with A. niger, A. wentii, and A. nidulans, and all five reacted with A. ochraceus. None reacted with A. terreus, A. glaucus, A. versicolor, or a Penicillium species. Each MAb bound to A. flavus hyphae in formalin-fixed paraffin sections of a muscle biopsy from a confirmed human case of invasive aspergillosis. In summary, these MAbs identified a 97-kDa antigen found on A. flavus that is both surface bound and an exoantigen. Either the same or a cross-reacting antigen is present in A. fumigatus and other Aspergillus species.     

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

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.     

Monoclonal antibodies that recognize a specific surface antigen of Treponema denticola.

Spirochetes have been implicated as potential etiologic agents of periodontitis in humans. Murine monoclonal antibodies (MAbs) specific for a serogroup of Treponema denticola, an oral spirochete, were developed and characterized in this study. Antibodies secreted by clone IAA11 were judged to be the most useful, since they were able to detect 8 of 15 T. denticola strains. This MAb consisted of an immunoglobulin G3 heavy chain and a kappa light chain. MAb IAA11 was found to react with an epitope target located on the outer sheath of the cell wall. This MAb should be of diagnostic and scientific value in the study of T. denticola populations in human periodontitis.     

Monoclonal antibodies to 52-kilodalton protein of Salmonella typhi.

Ten monoclonal antibodies (6 immunoglobulin G1 kappa [IgG1 kappa] and 4 IgG2b kappa) from six hybrid clones specific for Salmonella typhi antigen were produced by immunizing BALB/cJ mice with affinity-purified S. typhi proteins (Bp). The latter were prepared by passing crude S. typhi Bp through an affinity column made from Sepharose conjugated to IgG antibodies against partially purified S. typhi Bp. The eluent was subsequently used as the immunogen for the production of monoclonal antibody. All 10 monoclonal antibodies reacted specifically with a 52-kilodalton (kDa) protein of S. typhi and were species specific. The presence of IgM antibody to the 52-kDa antigen in the sera of a majority of patients with acute typhoid fever suggested that this 52-kDa protein is also a good immunogen for humans. The potential usefulness of this antigen in the early diagnosis of typhoid fever is discussed.     

Monoclonal antibodies against the 70-kilodalton iron-regulated protein of Neisseria meningitidis are bactericidal and strain specific.

When grown under iron limitation, Neisseria meningitidis expresses a number of outer membrane proteins (OMPs), one of which is a 70-kilodalton (kDa) major OMP. After immunization of mice with outer membrane preparations of iron-depleted cells of strain H44/76 (B:15:P1.7,16), hybridoma cell lines producing monoclonal antibodies against the 70-kDa OMP were obtained. Some of these monoclonal antibodies demonstrated strong bactericidal activity against the homologous strain H44/76 in the presence of human complement, suggesting potential application of the 70-kDa OMP as a vaccine component. However, none of the 10 selected monoclonal antibodies was able to recognize the corresponding protein from five heterologous strains of various serosubtyping characteristics. A polyclonal anti-70-kDa OMP serum also did not react with the other strains. This result shows that immunodominant surface-exposed epitopes of the meningococcal 70-kDa iron-limitation-inducible OMP are strain specific.     

Monoclonal antibody specific to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Virulent Rhodococcus equi produces 15- to 17-kDa surface protein antigens. These antigens are used as markers to identify virulent R. equi isolates from foals and their environment by Western blot (immunoblot) analysis with naturally infected foal serum. In the present study, a monoclonal antibody (MAb; 10G5) was generated against the 15- to 17-kDa antigens excised from sodium dodecyl sulfate-polyacrylamide gels to develop sensitive and specific immunoblot assays for the identification of virulent R. equi. MAb 10G5 strongly reacted with R. equi ATCC 33701 and L1, which expressed 15- to 17-kDa antigens by Western blot, colony blot, and dot immunobinding assays, but it did not react with strains ATCC 33701P- and L1P-, which lacked the antigens. For identification of virulent R. equi, clinical and environmental isolates were tested by these assays with the MAb, and all virulent strains were successfully identified; these strains possessed virulence plasmids. These results suggest that the MAb is a useful reagent for the identification of virulent R. equi.     

Monoclonal antibodies to a 28,000 mol. wt protein antigen of Mycobacterium leprae.

Monoclonal antibodies (MoAb) have been used to analyse a protein antigen from Mycobacterium leprae with a subunit mol. wt of 28,000 daltons. Three different patterns of species specificity were observed with two antibodies being specific for M. leprae, two partially specific, and one broadly cross-reactive amongst all mycobacteria. Competitive binding and sandwich assays demonstrated that the specific and partially specific antibodies recognized closely related regions of the molecule while the cross-reactive antibody recognized a spatially separate epitope on the same polypeptide chain. Identification of specific and cross-reactive epitopes on a single antigenic molecule may be of considerable importance for understanding the functioning of the cell-mediated immune system during leprosy infection and the use of MoAb for such analyses is discussed.     

Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis.

We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.     

Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications. Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B. burgdorferi antibodies. To identify antigens specific to B. burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera. One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa). These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin. Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39. All of 33 the serum specimens with immunofluorescence assay titers of greater than or equal to 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of less than or equal to 1:64 reacted with P39. Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin. Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot). Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39. Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis.     

Monoclonal antibodies to a 16-kDa antigen of Serpulina (Treponema) hyodysenteriae.

Monoclonal antibodies (MAbs) were produced to an outer-envelope preparation from Serpulina (Treponema) hyodysenteriae, the aetiological agent of swine dysentery. Three MAbs (isotype IgG1) were obtained. All three recognised a 16-kDa antigen that was common to most strains of S. hyodysenteriae of different serotypes but was absent from nonpathogenic, porcine intestinal spirochaetes. Immunofluorescence and immunogold labelling studies showed that the 16-kDa antigen was exposed on the surface of intact spirochaetes. The MAbs agglutinated freshly grown cultures of spirochaetes and inhibited growth of S. hyodysenteriae strains in vitro.     

Monoclonal antibodies to the murine Ly-2.1 cell surface antigen.

Six monoclonal anti-Ly-2.1 antibodies are described that arose from a single fusion using the spleen from a 129/ReJ mouse immunized with CBA/H thymus cells. The specificity was determined from the strain distribution, and testing of congenic strains where all six monoclonal antibodies detected the Ly-2.1 alloantigen. Furthermore, the antibodies fall into two groups: Group I (four antibodies) detected the expected number of Ly-2+ thymocytes and peripheral T cells, whereas Group II (two antibodies) detected 10% fewer peripheral T cells. Functional studies demonstrated that the Ly-2.1 determinant detected by a Group I antibody (49-31.1) was presented on cytotoxic T cells (Ly-1+2+), on concanavalin A (Con A)-induced suppressor T cells (Ly-1-2+), but absent from helper T cells (Ly-1+2-). One antibody (49-11.1) precipitated a 68,000-75,000 mol.wt glycoprotein, which on reduction yielded 30,000 and 35,000 mol.wt moieties. Thus, by functional, genetic and biochemical criteria these antibodies detected the Ly-2.1 specificity. In addition, a monoclonal, noncytotoxic, IgGl, anti-Thy-1.2 antibody is described.     

Monoclonal antibodies to detect markers specific for tumors

Monoclonal antibodies to different markers can facilitate the diagnosis of T and B cell lymphomas, histiocytic lymphomas, malignant histiocytosis, and Hodgkin's disease. The B-cell lymphomas can be identified specifically by monoclonal anti-idiotype antibodies. Monoclonal antibodies are produced to defined markers like carcinoembryonic antigen (CEA) in colon carcinomas and other antigens especially of breast and ovarian carcinomas. When conjugated with 123I, monoclonal antibodies can be used to detect tumors by emission computerized tomography. Chromogranins are markers for neuroendocrine tumors, and they can be identified by monoclonal antibodies. More recently monoclonal antibodies have been produced to ras gene product p21, present in breast and colon carcinoma cells.     

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.     

Multiple antigen peptides for specific detection of antibodies to a malaria antigen in human sera.

Multiple antigen peptides (MAP), consisting of a number of peptide copies synthesized on a branching lysyl core, offer a novel approach for rendering small peptides immunoreactive in solid-phase immunoassays. An octameric MAP, carrying 6 repeats of the sequence -N-A-A-G-, tandem repeated in the immunodominant region of the circumsporozoite (CS) protein of Plasmodium malariae, was used as a model to evaluate the suitability of the MAP system in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against a parasite antigen in individuals exposed to natural infection. The reaction of endemic sera in ELISA on MAP8-(NAAG)6 was related to that obtained in immunofluorescence on sporozoites, indicating the specificity of the antibody-MAP interaction. The reactivity of immune sera was found to be directed only against the (NAAG)6 moiety of the MAP and not against the lysyl core, since antibody binding to MAP8-(NAAG)6 was completely inhibited by (NAAG)6-NA monomer, but remained uninfluenced when lysyl core was used as competing ligand. The levels of antibodies to MAP8-(NAAG)6, in two groups of individuals naturally exposed to malaria infection, appeared to be related to their respective exposure to the parasite.     

Monoclonal antibodies specific for dengue virus type 3.

Mouse lymphocyte hybridomas were prepared by polyethylene glycol-mediated fusion of cells from a mouse plasmacytoma line with lymphocytes from a mouse hyperimmunized with dengue virus type 3 (dengue-3). Media from 50 hybrid colonies were screened; 46 of them showed antibody activity against dengue-3-infected cells as determined by an indirect immunofluorescent antibody technique. Dengue monoclonal antibody obtained after cloning one of these colonies demonstrated activity in hemagglutination inhibition and indirect immunofluorescent antibody assays with dengue-3 antigen, but not type 1, 2, and 4 antigens. In addition, this antibody activity could be removed from culture media only by absorption with dengue-3 antigen.     

Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host.

The importance of superoxide dismutase (SOD) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial SOD and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial SOD antibody inhibited the effect of nocardial SOD on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial SOD, another monoclonal antinocardial antibody (not reactive with SOD), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at 3, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-SOD antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-SOD antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for SOD. The data demonstrate that surface-associated SOD protects N. asteroides for oxidative killing in vivo during all stages of infection.     

Monoclonal antibodies to tissue-specific cell surface antigens. I. Characterization of an antibody to a prostate tissue antigen

Monoclonal antibodies were raised to PC-3 human prostate adenocarcinoma cells, and one hybridoma, designated F77-129, was extensively purified and used to characterize a PC-3 antigen. The F77-129 antibody also showed serological reactivity with the Du-145 prostate cancer line and with three of four breast carcinoma lines tested; it showed variable binding to a colon carcinoma line. Several other lines tested, including melanomas, fibrosarcomas, and leukemias, were completely negative. Immunoperoxidase staining of frozen surgical specimens showed binding to both normal and malignant prostate and breast tissue. Injection of radioiodinated F77-129 into tumor-bearing nude mice showed specific in vivo targeting to prostatic cancer implants. The antigen also showed surface modulation by bound antibody, suggesting possible clinical utility of this antibody in delivering immunotoxins to tumors.     

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.