Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody

Ixodid tick-associated spirochetes have been implicated as the etiological agents of Lyme disease. We raised a murine monoclonal antibody (H5332) against a spirochete, strain B31, isolated from Ixodes dammini ticks. In indirect immunofluorescence assays and western blot analyses, H5332 reacted with whole cells or isolated components of not only strain B31 but also spirochetes isolated from Ixodes ricinus ticks, a field mouse, a raccoon, and patients with Lyme disease. In contrast, H5332 did not bind to representative borreliae, treponemes, and leptospires. Using indirect immunofluorescence assays and immune electron microscopy, we found the H5332 determinant to be diffusely distributed over the surface of prefixed spirochetes but to be aggregated in patches when the organisms were incubated with H5332 and a second ligand before fixation. Radioimmunoprecipitation and western blot studies revealed the H5332 determinant to be either on or tightly associated with an abundant outer membrane protein with an apparent subunit molecular weight of 31,000.     

A common epitope between HLA-B27, -B13 and -B37 alloantigens defined by a monoclonal antibody

An anti-HLA-B27 monoclonal antibody produced by the hybridoma technique is described. This BD.7 reagent is a cytotoxic IgM antibody. Its reactivity was studied by lymphocytotoxicity tests, indirect immunofluorescence tests and biochemical analysis against an extensive panel of peripheral blood mononuclear cells. All HLA-B27 positive samples, either from normal subjects or from patients with Ankylosing Spondylitis, were recognized by this reagent. Moreover, a cross-reaction was observed with HLA-B13 cells, and a new unexpected reaction with all HLA-B37 cell suspensions. The interest of such a reagent is discussed.     

Pneumocystis carinii and specific fungi have a common epitope, identified by a monoclonal antibody.

Because Pneumocystis carinii may be related to fungi, we evaluated the reactivities of monoclonal antibodies raised against P. carinii with a variety of fungi. Fifty-two fungi and six protozoa were evaluated by immunofluorescence. One of three monoclonal antibodies (MAbs) tested (MAb 7D7) reacted with 15 fungi but no protozoa. Saccharomyces cerevisiae showed the strongest reactivity by immunofluorescence. The reactive antigen was characterized for four fungi by the immunoblot technique. In all cases the antigen that was reactive with MAb 7D7 was larger than the P. carinii antigens that reacted with 7D7. In further studies with P. carinii, Aspergillus species, and S. cerevisiae, we found that MAb 7D7 reacted with a carbohydrate component in all organisms. The presence of an epitope that is common to P. carinii and a number of fungi further supports the fungal nature of P. carinii.     

Carbohydrate epitope defined by an antitumor monoclonal antibody detected on glycoproteins and a glycolipid by immunoblotting

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components. A low-molecular-weight band, migrating approximately with the dye front, was also immunostained by MOv2. On the other hand, the immunostaining of high-performance thin-layer chromatography (HPTLC) of the total glycolipid extract and its neutral and acid fractions, after DEAE chromatography, showed selective reactivity with a neutral glycolipid. Reanalysis by immunoblotting of this glycolipid band scraped off the HPTLC plate indicated that it corresponds to the low-molecular-weight component. Periodate oxidation and Pronase digestion further demonstrate the saccharide nature of the determinant on both types of glycoconjugates. In conclusion, we report evidence that with a single analytical procedure, i.e., immunoblotting, it is possible to recognize the same carbohydrate determinant carried on both protein and lipid molecules.     

Identification of a common Plasmodium epitope (CPE) recognised by a pan-specific inhibitory monoclonal antibody.

A Plasmodium falciparum genomic expression library was screened with a monoclonal antibody produced from mice infected with Plasmodium yoelii. Eleven unique clones were isolated all of which contained the sequence NKND, IKND or KKND. This sequence was confirmed as the epitope of M26-32 by testing a series of overlapping peptides and the allowable substitutions determined by testing the binding of M26-32 to peptides containing all possible single amino acid replacements of NKND. Potential epitopes of M26-32 occur in many plasmodial proteins and this is consistent with the large number of proteins recognised in these parasites by Western blotting. Since this monoclonal antibody shows marked in vitro inhibition of P. falciparum growth, these data suggest that an anti-malarial vaccine may be produced by targeting such common plasmodial epitopes without necessarily identifying the corresponding antigens.     

A rat cytotrophoblast antigen defined by a monoclonal antibody.

A monoclonal antibody that recognizes specifically a cytotrophoblast antigen was obtained. The monoclonal antibody 22H6 was tested on rat choriocarcinoma (in vivo, in vitro), normal placenta, ectoplacental cone, blastocysts, and several normal organs. The antigen was detected on frozen sections and on tissue culture by indirect immunofluorescence. The monoclonal antibody 22H6 reacts with the cytotrophoblasts of rat choriocarcinoma. The giant cells do not display a positive reaction. It is not expressed on other tumors than choriocarcinoma. In adult rats the only cells revealing a positive reaction are the hepatocytes and the epithelial cells lining the small intestine. In the pregnant rat, the antigen is expressed on the cytotrophoblasts of the junctional zone in the placenta, but not on the giant cells. The mab reacts only with the small trophoblast cells of the ectoplacental cone, but not with trophectoderm of blastocyst. The mab has an IgG2b isotype and is not cytotoxic for choriocarcinoma cells in a complement-dependent cytotoxicity test. The described monoclonal antibody is to our knowledge the only known marker of rat benign and malignant cytotrophoblast.     

Development of olfactory nerve glia defined by a monoclonal antibody specific for Schwann cells.

Although there is considerable interest in the possible role of olfactory glia in the pathfinding abilities of olfactory nerve axons, the complete development of these glia in vivo has not been described. Using a specific Schwann cell marker, the 1E8 antibody, we have found that olfactory nerve glia can be identified throughout development. These glia appear to originate in the olfactory placode and migrate initially into the periphery of the olfactory nerve, and later into the center of the nerve. Olfactory nerve glia enter the presumptive olfactory bulb with the olfactory receptor neuron axons and distribute themselves along the edge of the olfactory nerve layer. The fact that olfactory nerve glia are specifically immunostained by the 1E8 monoclonal antibody, which recognizes the Schwann cell-specific protein P0, suggests that these cells more closely resemble Schwann cells than astrocytes or enteric glia. These results support and extend previous findings suggesting that olfactory nerve glia have distinctive developmental and anatomical features which may be important to the regenerative capacity of the olfactory system.     

Astrocytes and microglia in human brain share an epitope recognized by a B-lymphocyte-specific monoclonal antibody (LN-1)

A B-lymphocyte-specific mouse monoclonal antibody, LN-1, recognizes two morphologic classes of glial cells in human brain. The nature and duration of tissue fixation and processing are critical in the detection of the two cell types. In tissue that is lightly fixed, LN-1 recognizes astrocytes. The astrocytic nature of the LN-1 reactive glial cell was confirmed by cytologic features, tissue distribution, immunoelectron microscopy, double labeling immunofluorescent microscopy, and staining of serial sections with antibodies to glial fibrillary acidic protein. In tissue that is fixed for longer periods or in Bouin's fixative, two glial cell types are recognized: astrocytes and microglia. The identity of the latter cell type as microglia was confirmed by morphologic features, tissue distribution, immunoelectron microscopy, and double staining with monoclonal antibodies or lectins to macrophage markers, including class II major histocompatibility antigens. The two cell types had different disposition in senile plaques of elderly individuals and of those with Alzheimer's disease. Astrocytes were present at the periphery of the plaques, whereas microglial cells were centrally placed, often in juxtaposition to amyloid. The results are discussed with respect to ontogeny of glial cells and the ability of monoclonal antibodies to recognize epitopes on unrelated proteins.     

A human X-linked antigen defined by a monoclonal antibody

We have constructed hybrids between human thymocytes and the mouse thymoma BW5147. These hybrids, and others, have been used to show that the expression of a thymocyte antigen is controlled by an X-linked gene.     

Conformation dependence of a monoclonal antibody defined epitope on free human kappa chains

Chemically and enzymatically modified kappa chains were tested by inhibition radioimmunoassay for their ability to block the binding of antibody K-1-21 with native kappa chains. Complete reduction and carboxymethylation of intrachain disulphide bonds destroyed the free kappa-chain epitope, a result confirmed by Western blotting of unreduced and reduced kappa monomers and dimers. Purified V kappa fragments failed to block the homologous interaction while inhibition was obtained with a pepsin digest yielding predominantly the C kappa region. Dimeric kappa chains were less effective than monomers in the inhibition assay, although HPLC analysis of immune complexes demonstrated the binding of two antibody molecules per molecule of dimer. Thus, the epitope on free kappa chains recognized by K-1-21 is dependent upon conformational integrity of the C kappa domain, the decreased binding activity of dimeric chains possibly being due to minor conformational changes induced by C-domain interactions.     

A monoclonal antibody reactive with a common epitope of Moraxella (Branhamella) catarrhalis lipopolysaccharides.

A hybrid cell line producing a monoclonal antibody (MAb) against Moraxella (Branhamella) catarrhalis lipopolysaccharide (LPS) was established. The specificity of the MAb 1B12 to purified rough LPSs from six strains of M. catarrhalis was ascertained by enzyme-linked immunosorbent assay (ELISA), competitive-inhibition ELISA, and immunoblotting. MAb 1B12 bound to live bacterial cells and culture supernatants from a total of 34 strains of M. catarrhalis, including 12 strains with different LPS serotypes. No cross-reactions with smooth and rough LPSs from selected enterobacterial and nonenterobacterial strains, with other respiratory pathogens, or with Neisseria species were observed. These data suggest that MAb 1B12 recognizes a common epitope of M. catarrhalis LPS which differs from serotype determinants.     

A monoclonal antibody against an epitope common to HLA-B locus antigens

A monoclonal antibody G4 that appears to be directed against a determinant common to akl HLA-B locus antigens is described. This antibody reacted with a large panel of B and T lymphocytes and cell lines, but it did not react with two lines that do not serologically express HLA antigens (Daudi and K562) and two lines that expressed A-locus but not B-locus antigens (8402 and HPBMLT). The F(ab′)₂ fragment of G4 blocked B-locus but not A-locus HLA alloantisera. By immunoprecipitation and SDS-polyacrylamide gel electrophoresis G4 reacted with a dimer consisting of a heavy chain of 44,000 daltons and a light chain of 12,7000 daltons.     

Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHL1.

The monoclonal antibody UCHL1 identified an antigen present on most thymocytes, a subpopulation of resting T cells within both the CD4 and CD8 subsets, and on mature activated T cells. The UCHL1 determinant is also present on cells of the myeloid lineage, but not normal B cells or NK cells. Functionally, UCHL1 identifies a subpopulation of T cells which proliferates maximally to soluble antigen and provides maximum help for PWM-stimulated immunoglobulin synthesis. In contrast, the UCHL1- cells do not induce immunoglobulin synthesis and do not proliferate in the presence of soluble antigen, although both the UCHL1- and the UCHL1+ fractions of T cells proliferate well in the presence of PHA. By standard immunoprecipitation techniques and SDS page, the antigen recognized by UCHL1 was found to have a molecular weight of 180,000-185,000. Preclearing experiments using antibodies identifying the leucocyte common antigen, LCA, and the lymphocyte function-associated antigen, LFA-1, which have similar molecular weights to UCHL1, showed that the UCHL1 determinant is not biochemically related to these antigens.     

Pancreatic islet-cell epitope recognized by an anti-sulphatide monoclonal antibody.

Insulin-dependent (Type 1) diabetes mellitus is recognized as an autoimmune disease and islet-cell antibody (ICA) is present in the majority of patients at diagnosis. ICA labels both beta and alpha cells and is believed to be directed against a glycolipid. In this study we examine the presence of sulphatide (3'-sulphogalactosylceramide) or closely related structures (sulpholactosylceramide and seminolipid) in islet cells by means of a monoclonal antibody, Sulph I. Histological examination of pancreatic tissue from Lewis and BB rats, and BALB/c and NOD mice showed a pronounced labelling of the islets of Langerhans with Sulph I. No staining of the exocrine pancreatic tissue, the heart, the liver, the adrenals, the thymus, the spleen or lymph nodes was seen, but staining of some tubular cells and glomerular cells in the kidney as well as of myelin in nerve cells was found. Cytological examination of isolated Lewis islet cells and their cell subpopulations, separated using a fluorescence-activated cell sorter (FACS), showed positive surface labelling of 97.3 +/- 2.2% (SD) of the beta cells and 84.4 +/- 3.0% of the non-beta cells. Thus, the epitope on the glycolipid sulphatide or closely related structures is--with the exception of neural and certain kidney tissue--specifically present in islet cells. Furthermore, the staining pattern of the antibody used, Sulph I, was equivalent to that of ICA.     

A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody.

Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.     

A Borrelia-specific monoclonal antibody binds to a flagellar epitope.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.     

Identification of Moraxella bovis by using a monoclonal antibody to a lipopolysaccharide epitope.

A monoclonal antibody to the lipopolysaccharide of Moraxella bovis is described. In an indirect fluorescent-antibody test, this monoclonal antibody reacted with 39 of 39 strains of M. bovis tested and did not react with 26 nonfermenting gram-negative coccobacilli other than M. bovis. When used in an indirect fluorescent-antibody test, it proved useful for rapid and easy identification of M. bovis.     

A murine monoclonal antibody defines a unique epitope shared by Klebsiella lipopolysaccharides.

A hybridoma secreting a monoclonal antibody (MAb) directed against Klebsiella lipopolysaccharide (LPS) was derived from spleen cells of mice immunized a smooth, nonencapsulated Klebsiella strain (Friedländer 201; serogroup O1). The MAb, called V/9-5 (immunoglobulin G2a), cross-reacted with LPS preparations produced from reference strains for the Klebsiella O serogroups O1, O2ab, O2ac, O3, O4, O5, and O12. Furthermore, the MAb reacted with LPSs from serogroup reference strains O6/O8, O9, and O11, which are regarded as being identical to O1, O2, and O4, respectively. When testing the supernatant of clinically isolated Klebsiella strains by means of an inhibition enzyme-linked immunosorbent assay, we found that 86 (92.4%) of 93 Klebsiella pneumoniae subsp. pneumoniae isolates and 24 (96.0%) of 25 K. oxytoca isolates harbored the cross-reactive epitope. By contrast, two laboratory strains of K. pneumoniae subsp. rhinoscleromatis did not react with MAb V/9-5. The MAb proved to be specific for the genus Klebsiella, since it did not react with any of a total of 73 strains belonging to other gram-negative bacterial genera. In conjunction with other LPS-specific MAbs, MAb V/9-5 might become a useful reagent for rapid identification of klebsiellae in clinical specimens. Furthermore, the epitope recognized by MAb V/9-5 might serve as a target epitope for the production of human MAbs for immunotherapeutic purposes.     

Interaction of Haemophilus influenzae with human erythrocytes and oropharyngeal epithelial cells is mediated by a common fimbrial epitope.

The role of fimbriae in the adherence of Haemophilus influenzae to oropharyngeal epithelial cells and the hemagglutination (HA) of human Anton-positive erythrocytes was examined. HA of bacteria was lost after shearing. Fimbriae purified from the extracellular fluid caused HA and bound to oropharyngeal epithelial cells, as analyzed with immunoperoxidase staining, in a way which was similar to the adherence of bacteria to these cells: binding was over the entire surface of the cells and showed cell-to-cell variation. The specific role of fimbriae in HA and adherence was further examined by inhibition experiments with monoclonal antibodies elicited against the isolated fimbriae. These monoclonal antibodies bound along the entire length of the fimbriae, as seen by immunogold electron microscopy. The monoclonal antibodies and their Fab fragments inhibited HA (reduction in titer from 1:512 to 1:128 and 1:64, respectively) and inhibited the adherence of the homologous H. influenzae strain and of three of eight heterologous H. influenzae strains to oropharyngeal epithelial cells. These results indicate that fimbriae are involved in adherence and HA and that the binding site for the monoclonal antibodies on the fimbriae is not common on all strains.