Structural polypeptides of the enteropathogenic bovine coronavirus strain LY-138

Summary The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm³ in CsCl and 1.185 g/cm³ in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm³ in CsCl and 1.201 g/cm³ in sucrose.Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.With 5 Figures     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Recognition of enteropathogenic Escherichia coli virulence determinants by human colostrum and serum antibodies

Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic Escherichia coli (EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic E. coli strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic.     

Double-antigen sandwich ELISA for detection of antibodies to SARS-associated coronavirus in human serum

The study presented here was conducted to evaluate the performance of a double-antigen sandwich ELISA to detect antibodies in human serum against the coronavirus associated with severe acute respiratory syndrome (SARS). A recombinant partial nucleocapsid protein of SARS-associated coronavirus was used as a serodiagnostic antigen in the ELISA. A total of 2892 clinical serum samples were tested with the ELISA kit, which positively identified 25 of 35 (71.4%) samples of patients with confirmed SARS infection, 286 of 407 (70%) samples of patients suspected of having SARS, 229 of 302 (75.8%) samples of convalescent SARS patients, and 0 of 544 samples obtained from healthcare workers; only 1 of 1604 clinical samples obtained from patients with other diseases demonstrated a weakly positive result. These results indicate that the double-antigen sandwich ELISA is an effective screening method for the serodiagnosis of SARS-associated coronavirus.     

Reactivity of human tissues with monoclonal antibodies to myeloid activation and differentiation antigens. An immunohistochemical study

We have characterized antimyeloid monoclonal antibodies (mAbs) produced to human rheumatoid arthritis (RA) synovial tissue macrophages (MPs) (8D7) and to lipopolysaccharide (LPS)-treated U937 cells (3D8). The 3D8 antigen is upregulated with LPS stimulation of monocytes/MPs and during monocyte maturation. The 8D7 antigen is upregulated on functionally distinct subpopulations of RA synovial tissue MPs. We used immunohistochemistry to determine the spectrum of reactivity of these unique mAbs on myeloid cell suspensions, monocytes, and mature tissue inflammatory and noninflammatory MPs. The antigens identified by the mAbs were characterized biochemically, by immunoprecipitation of solubilized 125I-labelled antigens from cell surfaces, and immunohistochemically by enzymatic digestion of myeloid cells followed by a cellular ELISA. MAb 3D8, characterized as an anti-CD13 antibody, recognizes a 150-170 kd antigen, has almost exclusive myeloid reactivity, but reacts with Langerhans' cells of the skin and thymus, pointing to shared antigens between these cells and MPs. Unlike 3D8 antigen, 8D7 antigen is strongly expressed in inflammatory states, being present on MPs in granulomata as well as in sarcoid lymph nodes. Both mAbs react with frozen and methanol-Carnoy's fixed, paraffin-embedded tissues and detect antigenic differences among human mononuclear phagocytes present in different anatomical sites and in varying stages of differentiation and activation. These mAbs should prove to be a valuable tool for studying heterogenous populations of myeloid cells.     

Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.     

Analysis of bovine herpesvirus 4 (DN 599) major antigens with monoclonal antibodies and polyclonal immune serum

Summary Monoclonal antibodies (MAbs) and polyclonal immune sera were produced and used to identify the major antigens of bovine herpesvirus type 4 (BHV-4). SDS-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled lysates from infected cells resolved 24 peptide bands varying from 12kDa to over 300kDa. Six peptides were identified as major viral antigens by immunoprecipitation. Based on the pattern of radioimmunoprecipitation, MAbs were assigned into four groups. Group 1 precipitated a tunicamycinsensitive glycoprotein complex which contained six components (245, 190, 152, 123, and 48/46kDa). Deglycosylation with endoglycosidase F revealed two peptides with M_r of 93 and 38kDa as the basic peptides of the glycoprotein complex. In addition, a 115kDa glycopeptide containing glycan-peptide bonds of mixed type was identified. Group 2 precipitated a non-glycosylated protein complex consisting of three monomers (33/31/30kDa). Groups 3 and 4 reacted with single monomeric non-glycosylated peptides with M_r of 48 and 14kDa, respectively. Although none of the MAbs exhibited significant neutralizing activity, some reacted strongly in immunosorbent and/or immunohistochemical assays, suggesting they may be good candidates for use in diagnostic assays.     

Reactivity of anti-human brain serum with human lymphocytes.

Specific anti-human T-cell serum was prepared in rabbits by multiple subcutaneous injections of human brain homogenates in incomplete Freund's adjuvant. The serum was exhaustively absorbed with human RBCs, lyophilized human liver, lyophilized normal human serum, and peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL). Specificity of the antiserum for human T lymphocytes was tested by indirect immunofluorescence. It stained 70 to 80% of lymphocytes in circulation, 95% of thymus, 27 to 35% of spleen, 5 to 10% of tonsil lymphocytes, and over 90% of phytohemagglutinin-stimulated lymphocytes in vitro. Only T-dependent areas of cryostat-sectioned human lymph nodes stained with the antiserum. It did not stain circulating lymphocytes which formed HEAC rosettes, plasma cells in marrows of multiple myeloma patients or macrophages. After removal of HEAC rosettes by centrifugation in Ficoll-Hypaque, 75% of interface cells formed E rosettes and 65 to 75% stained with the antiserum. The antiserum was used in studies of lymphocytes in chronic and acute lymphocytic leukemias, lymphomas, and other lymphoproliferative diseases. Numbers and distribution in the circulation, spleen and nodes of lymphocytes bearing the T marker were significantly altered in patients with these disorders.     

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.     

Oligonucleotide fingerprints of antigenically related bovine coronavirus and human coronavirus OC43

Summary Virion RNAs from the bovine enteric coronavirus and the human respiratory coronavirus OC43 were compared by one dimensional gel electrophoresis and by oligonucleotide fingerprinting. For each virus, approximately 55 per cent of the RNA migrated as a 6.8 Md species, 10 per cent as a 0.68 Md species, and 15 per cent as heterogeneous small molecular weight RNA. A sequence homology of greater than 96 per cent was observed between the 6.8 Md species from the two viruses. The 0.68 Md RNA is apparently an intravirion, subgenomic, polyadenylated molecule based on RNAse studies, oligo (dT)-cellulose chromatography, and hybridization to a cDNA clone of the 3 terminal 1.19 Kb region of the bovine coronavirus genome.With 2 Figures     

Characterization of sperm antigens reacting with human antisperm antibodies

Antibodies reacting with human spermatozoa have been detected by various immunological techniques in the sera of subfertile men. Different patterns of sperm agglutination are observed with different sera, either head-to-head, tail-to-tail, or tail-tip-to-tail-tip. Differences have been detected between the clinically relevant antibodies in spontaneously infertile males and the less important antibodies in males who have undergone reversal of vasectomy. It has been suggested that the variations in agglutination patterns are due either to different classes of antibody or to binding of antibody to different antigens. In the present study immunoblotting techniques were used to characterize the reactivity of solubilized sperm proteins with serum samples exhibiting different modes of sperm agglutination. This involved the electrophoretic transfer of proteins from SDS gels to nitrocellulose sheets followed by overlay with serum antibody. Using these techniques we have attempted to characterize the antigens of spermatozoa which react with sera from both spontaneously infertile and vasovasostomized men. The results showed that although antisperm antibodies bind to discrete and sperm-associated antigens, there is no substantial difference between the antigenic patterns observed with antibodies producing different types of sperm agglutination. Neither the antigens detected, nor the intensity of reaction showed significant differences although there was a tendency for head-to-head agglutinating antibodies to react more strongly with the higher molecular weight antigens. Moreover, although with sequential serum samples the patterns of agglutination may change, the antigenic pattern remains unchanged.     

Study of an antigenic site of human serum albumin with monoclonal antibodies

Two monoclonal anti-HSA antibodies, HA1 and HA2, have been shown to be specific for a univalent fragment of 6000 mol. wt, F1, located near the C-terminus of HSA (Doven, Pesce & Lapresle, Immunology Letters 3, 365-370, 1981). Both monoclonal antibodies have been shown to react with the same site, which includes the following components: the last small loop of HSA (558-567) the disulfide bridge 514-559 and the residue 570. This site is as available on HSA and F1, but partially masked on the 'Inhibitor' fragment from which F1 derives. Polyclonal anti-F1 antibodies, purified from rabbit sera or mouse ascites by affinity chromatography, react with the same site as HA1 and HA2. However, polyclonal antibodies are heterogenous, most probably because they consist of anti-F1 specific antibodies and of antibodies specific against other parts of the albumin molecule which cross-react with F1. In addition, monoclonal antibodies can recognize the mutation of a single amino acid residue in the albumin molecule.     

Comparison of bovine coronavirus (BCV) antigens: monoclonal antibodies to the spike glycoprotein distinguish between vaccine and wild-type strains

Monoclonal antibodies (MAbs) against two major structural proteins of the cell-adapted Mebus strain of bovine coronavirus (BCV-L9) were produced and characterized. Seven MAbs reacted with the peplomeric glycoprotein, gp 100/S, while three MAbs reacted with the nucleoprotein p53/N in Western blot analysis of BCV polypeptides. MAbs to gp 100/S reacted with discontinuous epitopes of gp 100/S in Westerns under mild but not under standard denaturing conditions. In contrast, MAbs to p53/N reacted in both types of Westerns, and those epitopes were thus continuous. MAbs to p53/N failed to neutralize BCV infectivity, while 4 MAbs to gp 100/S neutralized BCV effectively. Cross reactivity of MAbs to gp 100/S specified by five virulent wild-type strains and two high passage, cell-culture-adapted strains in mildly denaturing Westerns and neutralization assays indicated that two epitopes were conserved in all seven strains, while two epitopes of the avirulent strains were not detected in the wild-type strains. Non-neutralizing MAbs of gp 100/S reacted with all seven strains in Westerns with the exception of one MAb that was specific for the highly cell-adapted strain BCV-L9.     

Reactivity of human trophoblast monoclonal antibodies with marmoset monkey trophoblast cultures

The aim of this study was to determine whether cultured trophoblast tissues, derived from the trophectoderm of marmoset monkey blastocysts, contain homologues of human trophoblast antigens. This is an essential prerequisite to determine whether the marmoset may be a suitable model for preclinical testing of a human antitrophoblast antigen for fertility regulation. Previously evaluated monoclonal antibodies from the Flinders University laboratory, which reacted with human trophoblast with a high degree of specificity, were tested for immunohistochemical reactivity using an immunoperoxidase detection method on both frozen and paraformaldehyde-fixed sections of the cultured marmoset monkey trophoblast. All monoclonal antibodies raised against human placenta reacted positively, when compared to controls, suggesting that human and marmoset trophoblast cells share common epitopes. The specificity of the monoclonal antibodies was investigated by determining whether there was cross-reactivity with other marmoset monkey tissues, including adrenal, spleen, kidney, liver, muscle, ovary and testis. The specificities of the monoclonal antibodies on these marmoset tissues were similar to those previously found on the corresponding human tissues. We have concluded that marmoset monkey trophoblast exhibits homologues of human trophoblast antigens. The findings also suggest that marmoset monkeys should be evaluated further as a primate model to test suitable target antigens for antitrophoblast vaccines that may be useful contragestation agents in humans.      

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).