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.     

Isolation and purification of two immunodominant antigens from Nocardia brasiliensis.

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.     

Monoclonal antibodies to immunodominant surface-exposed protein antigens ofTreponema pallidum

Specific murine monoclonal antibodies directed against immunodominant surface-exposed protein antigens ofTreponema pallidum with molecular weights of 15,500, 33,000, 44,000, and 46,000 were isolated. Of seventeen monoclonal antibodies characterized by Western blotting, three were directed against 15,500, three against 33,000, nine against 44,000, and two against 46,000 molecular weight protein antigens ofTreponema pallidum. Three of the monoclonal antibodies were reactive in the haemagglutination assay, 11 in the in vitro immobilization assay, and 13 in the immunofluorescence assay. It is suggested that the different monoclonal antibodies could be useful in isolating immunodominant protein antigens ofTreponema pallidum and in obtaining information on their biological relevance for use in the diagnosis of syphilis.     

Monoclonal antibodies to immunodominant epitope of Tropheryma whipplei.

Recent isolation of Tropheryma whipplei (formerly Trophyrema whippelii), the agent of Whipple's disease, from the cardiac valve of a patient with Whipple's disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinant T. whipplei antigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) against T. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive with T. whipplei, but they did react with two other strains of T. whipplei isolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection of T. whipplei within infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple's disease.     

Monoclonal antibodies to HLA-DR antigens

Monoclonal antibodies (Mabs) have been invaluable in the study of human MHC class-11 (or la-like) antigens. For this to continue some pooling of resources was needed and in 1982 the British Medical Research Council's Clinical and Population Cytogenetics Unit in Edinburgh initiated an information exchange scheme on anti-class-11 Mabs in an attempt to classify and group at least some of these reagents. Over 100 Mabs from 25 laboratories were screened by several centres in various serological, biochemical and functional assays and the data were discussed in Edinburgh on 2–6 September 1983.     

Monoclonal antibodies to human glomerular antigens

Summary Using cultured human fetal kidney cortical cells as antigen, two monoclonal antibodies (moAbs) against human glomeruli were produced. One of these moAbs, H-4, recognized the cell surface of glomerular epithelial cells, and the other, H-13, recognized the extracellular matrix present in the mesangial area. Both also reacted with liver, H-4 recognizing antigen present on the hepatocyte, and H-13 recognizing antigen distributed along the sinusoid. Species specificity for these moAbs was examined using mouse, rat, guinea pig and rabbit glomeruli, which revealed that H-4 reacted with rat glomerular epithelial cells and H-13 stained guinea pig glomerular mesangium. In the human fetal kidney, H-13 reacted with the mesangium, glomerular and tubular basement membrane and Bowman's capsule, and H-4 with the glomerular and tubular epithelial cells. Dot immunobinding assay of fibronectin purified from glomerular culture supernatant and plasma revealed that H-13 recognized both plasma and cellular fibronectin. Immunoblot analysis of 2.0 M guanidine HCl extract after dissociation in sodium dodecyl sulfate and electrophoresis demonstrated binding of H-4 to a 125 kd polypeptide. Immunoblot analysis of thermolysin-digested fibronectin exhibited binding of H-13 to 145 kd and 110 kd fragments, but not to 38 kd – 29 kd fragments. In renal biopsy specimens from patients with membranous nephropathy, H-13 stained the glomerular basement membrane (GBM), but not the mesangium, whereas anti-fibronectin antisera stained both the GBM and the mesangium. In those from patients with minimal change nephrotic syndrome (MCNS), IgA glomerulonephritis (IgAGN) and membranoproliferative glomerunephritis (MPGN), the staining pattern with H-13 was similar to that with polyclonal anti-fibronectin antisera. These results indicate that H-4 recognizes a 125 kd polypeptide constituent of the glomerular epithelial cell membrane and that H-13 recognizes the cell binding domain of fibronectin as well as revealing structural alterations in the mesangium and GBM.This work was supported in part by research grant (61480136) from the Ministry of Education, Science and Culture, Japan (1986).     

Monoclonal antibodies to rat renal antigens

We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis.     

Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis.

A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease.     

Monoclonal antibodies to a specific 54-kilodalton antigen of Nocardia spp.

Two monoclonal antibodies (MAbs) of the immunoglobulin G2A isotype, reacting with a Nocardia-specific 54-kDa antigen, were generated. As determined by Western blot (immunoblot), both MAbs reacted only with the 54-kDa band. As determined by indirect immunofluorescence or enzyme immunoassay with whole microorganisms, the MAbs did not react with Nocardia cells. One of the MAbs showed weak cross-reactivity with mycobacterial antigens, while the other showed no cross-reactivity.     

Monoclonal antibodies to outer membrane antigens of Vibrio cholerae.

Hybridoma-derived monoclonal antibodies were prepared against outer membrane antigens of four strains of Vibrio cholerae that were cultivated under iron-limited conditions, and these antibodies were partially characterized. We established a library of 66 hybridomas which produced monoclonal antibodies defining 16 different V. cholerae antigens. Two antigens (molecular weights, 18,000 and 112,000) were heat modifiable, whereas the reacting epitope of a third antigen (40,000-dalton-18,000-dalton doublet) was completely destroyed when it was heated at 100 degrees C. The 112,000-dalton heat-modifiable protein was an iron-regulated outer membrane protein. This protein bound 59Fe in vitro when it was combined with the V. cholerae siderophore-iron complex 59Fe-vibriobactin; it was also found in in vivo grown V. cholerae, as were three other antigens. A total of 26 hybridomas produced antibody to V. cholerae lipopolysaccharide. Of these, 12 were cross-reactive with lipopolysaccharides of other gram-negative bacteria, including 2 which recognized lipid A. Several of these anti-lipopolysaccharide monoclonal antibodies appeared to be lipopolysaccharide region specific. Some membrane antigens were strain specific, whereas others were common to both O group 1 and non-O group 1 vibrios.     

Nocardia asteroides and Nocardia brasiliensis infections in mice.

A model for Nocardia asteroides and Nocardia brasiliensis infections in Swiss white mice has been established without the addition to the inocula of any form of adjuvant. Serial histopathological studies revealed that these two actinomycetes cause lesions that are quite different in their features. An acute suppurative abscess characterizes the lesions of N. asteroides. In the case of N. brasiliensis infections a granuloma is produced in which a striking feature is the presence of large numbers of foam-laden macrophages, although occasional exceptions to this pattern were noted. Electron microscopic studies demonstrated that these macrophages contain within their cytoplasm organisms in varying stages of degeneration. Repeated mortality studies in mice failed to demonstrate differences in mortality rates produced by N. asteroides and N. brasiliensis. Thus, despite relatively trivial biochemical and antigenic differences between these two species of Nocardia, the local pathogenic response is quite different. The presence in the "brasiliensis lesion" of foamy macrophages with intracellular organisms is reminiscent of the histopathological features of lepromatous leprosy and of disseminated Myocobacterium bovis infection when this occurs in the immune suppressed situation. It is possible that N. brasiliensis infection produces a depression of cellular immunity that modifies the local host response to the organism.     

Monoclonal antibodies to human macrophage and leucocyte common antigens

Three monoclonal antibodies have been made to identify cells of the human mononuclear phagocyte system in fluids and tissues. The first, PHM 1, recognises a surface antigen common to all leucocytes. The other 2 antibodies, PHM 2 and PHM 3, bind to monocytes and macrophages but not to polymorphonuclear cells (PMN). PHM 2 labels all monocytes, macrophages and a small population of T-cells. PHM 3 labels most monocytes and macrophages but no other blood cells. The application of these monoclonal antibodies to the identification of mononuclear phagocytes in blood and liver using an unlabelled antibody immunoperoxidase (PAP) technique is demonstrated.     

Monoclonal antibodies against Rh and Rh related antigens.

MAB 114 and MAB 120 appeared to have the same or a very similar specificity to BRIC 125 (Avent et al., 1988). MAB 116, and possibly MAB 115, had a similar specificity to R6A (Anstee & Edwards, 1982; Avent et al., 1988). MAB 117, 118 and 119 demonstrated the MB-2D10 specificity and were, in fact, the antibodies reported by von dem Borne et al. (1990). MAB 121 was not anti-Rh34-like since it reacted with RH: 34 red cells. MAB 122 was not anti-HR since it reacted with RH: 18 cells. The specificities of MAB 121 and MAB 122 were not identified and although they probably detect different epitopes insufficient MAB 122 was available to confirm this suspicion. None of the MABS tested appeared to have the same specificity as 1D8 (Miller et al., 1987) or BS58 (Sonneborn et al., 1990). Competitive binding studies with MAB 121 provided evidence of a close association of BRIC 125 polypeptide with Rh polypeptides.     

Monoclonal antibodies to bovine major histocompatibility system antigens

Of 89 monoclonal antibodies screened for anti-class I activity in a cytotoxic assay against bovine peripheral-blood lymphocytes, 6 reacted with all lymphocytes from all cattle tested, 72 failed to react at all and 11 reacted with polymorphic determinants. The reactivity of some of the 11 polymorphic monoclonal antibodies was dependent upon the bovine major histocompatibility system (BoLA) class I type. Eight monoclonal antibodies selected for putative anti-class II activity reacted with B-enriched lymphocytes from all cattle tested.     

Monoclonal antibodies defining shared human macrophage-endothelial antigens.

We have selected several monoclonal antibodies (mAbs) producing using human rheumatoid arthritis (RA) synovial macrophages (m phi s) as immunogen. Of these, mAbs 8H2, 10G7 and 10G9 showed cross reactivity with endothelium, suggesting common antigens between these cell types. We have determined the spectrum of reactivity of these mAbs on hematopoietic cell lines, peripheral blood cells, and inflammatory and non-inflammatory tissues by immunohistochemistry. MAb 8H2 does not react with the myeloid cell lines HL60 (myelocytic), U937 (histiocytic lymphoma), and K562 (erythroleukemia), or with peripheral blood cells. In normal and inflamed tissue sections, mAb 8H2 reacts with m phi s and endothelial cells. In contrast, mAb 10G7 does not react with peripheral blood cells, but reacts with HL60, U937, and K562 cell lines, as well as with m phi s and endothelial cells in inflamed and noninflamed tissues. MAb 10G9 does not react with myeloid cell lines, but reacts with monocytes and platelets in peripheral blood. In both normal and inflamed tissues, mAb 10G9 reacts with m phi s and endothelial cells. The antigens identified by these three mAbs were characterized biochemically, by enzymatic digestion of RA synovial tissue m phi s followed by a cellular ELISA, as well as by reactivity of the mAbs with NIH-3T3 cells genetically engineered to express known myeloid antigens. These mAbs reacted with protein or glycoprotein antigens distinct from the known myeloid antigens CD13, CD14, CD33, CD34, CD36, and c-fms. These mAbs should prove to be a valuable tool for studying m phi s and endothelial cells and their shared antigenic determinants.     

Monoclonal antibodies to human glomerular antigens. II. Using human adult kidney components as antigens

Using human kidney cortical homogenates and long-term cultured glomerular cells as antigens, the author produced three monoclonal antibodies to glomerular components; 25C reacted with the glomerular basement membrane (GBM) and the wall of blood vessels but with neither the tubular basement membrane (TBM) nor the Bowman's capsule, 33G reacted predominantly with the mesangium, and 34F reacted with glomeruli and the tubular brush border in a granular pattern. Both 25C and 33G exhibited the species-restricted property, and 34F reacted with glomeruli and tubular brush border of all the species examined. Overnight incubation of the kidney sections with 4.0 M urea revealed the reactivity of 25C to the TBM and Bowman's capsule. Dot immunobinding assay revealed that 25C did not react with the known extracellular matrices examined in this study, but rather with collagenase-digested GBM fraction. Also, 33G recognized fibronectin. Western blotting revealed the binding of 34F to the 145-kDa polypeptide solubilized from the kidney with 0.5 M NaCl, and also showed the binding of 25C to 210-kDa polypeptide of collagenase-digested GBM. These findings revealed structural variations in the basement membrane and the existence of a common antigen between the glomeruli and tubular brush border in the human kidney.     

Monoclonal antibodies to surface antigens of a pathogenic Mycoplasma hominis strain.

Three monoclonal antibodies (MAbs) were prepared against an arthritogenic strain of Mycoplasma hominis isolated from the joint aspirates of a patient with chronic septic arthritis. Immunoblots of polyacrylamide gel-electrophoresed proteins before and after surface proteolysis showed that the predominant antigenic determinants were on surface-exposed polypeptides. These polypeptides have extensive hydrophobic characteristics, as demonstrated by Triton X-114 phase partitioning. The electrophoresed proteins from cells grown in medium containing [14C]palmitate were blotted onto nitrocellulose which was both reacted with the MAbs and exposed to X-ray film. Superimposable bands on both the immunoblots and the exposed film suggested that the proteins might be acylated. The MAbs were further tested for reactivity with 16 other strains of M. hominis isolated from patients with septic arthritis (1 strain), septicemia (10 strains), or nongonococcal urethritis (1 strain); from the cervix (1 strain), rectum (1 strain), or surgical wound (1 strain) of patients; and from a contaminated cell culture. No single protein was consistently recognized from strain to strain, although a 94-kDa protein from 16 of the 17 strains tested was bound by at least one of the MAbs. The apparent antigenic heterogeneity among strains of M. hominis, including those isolated from the same tissue source and/or from patients with the same type of clinical disease, might be misleading in that all strains express epitopes associated with a discrete number of proteins to which one, two, or all three MAbs bind. The expression of the epitopes on multiple proteins from the same or different strains may reflect a mechanism for generating antigenic diversity.     

Monoclonal antibodies against surface antigens of Bacteroides gingivalis.

Monoclonal antibodies against the various surface antigens of Bacteroides gingivalis were obtained by the fusion of murine myeloma cells (SP2/0-Ag14) with spleen cells of BALB/c mice immunized with the whole cells. Two monoclonal antibodies reacted with lipopolysaccharide, and the other two reacted strongly with capsule antigen. One showed reactivity with the hemagglutinin of the cells. The five monoclonal antibodies reacted with sonicated antigen from all B. gingivalis strains tested. No cross-reactivity of the monoclonal antibodies with antigens from nine species of other black-pigmented Bacteroids strains was observed. An immunoblotting test involving the use of these monoclonal antibodies indicated that the epitope of B. gingivalis lipopolysaccharide was polysaccharide with a high molecular weight of 40,000 to 60,000. The immunoblotting test also demonstrated that the epitopes of capsule antigen and of hemagglutinin were 27,000- and 40,000-molecular-weight proteins, respectively.