Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.     

A monoclonal antibody against a new differentiation antigen of thymocytes

B14-2-14 is a monoclonal cytotoxic IgM antibody which reacts with thymocytes of all mouse strains tested. The fraction of positive cells (by visual immunofluorescence) varies between strains from about 25-45% in A.CA to 65-85% in C57BL/6, and high levels are dominant in F1 hybrids. In the periphery, the antigen is found on a few percent of lymph node and not on splenic T cells, and it is absent in nude mice. Among thymocytes, the distribution of the B14 determinant largely overlaps with that of the TL antigen and of molecules binding peanut agglutinin. The B14 antibody reacts only minimally with hydrocortisone-resistant thymus cells. Biochemical analysis shows that B14 antibody, anti-TL antibody and peanut agglutinin bind to separate molecules. The target of the B14 antibody may be either an immature, thymic form of Thy-1, or another molecule associated with it. Two polypeptides, of 40 and 35 kDa are precipitated by both B14 and anti-Thy-1 antibodies from biosynthetically labeled thymus cell lysates, and two others, of 27 and 17 kDa, from surface-iodinated thymus cell preparations. B14-2-14 offers an additional method for identification and selection of thymocytes at different stages of differentiation, and should also be useful for studies of the Thy-1 antigen.     

Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat

The monoclonal antibody (F10–89–4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1 : 1 : 0.8 : 0.3 : 0.3 : 0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190 000 to 215 000.     

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.     

A monoclonal antibody to a rat hepato-renal membrane antigen.

A monoclonal antibody against a membrane glycoprotein of rat hepatocytes has been produced. The nature of this antibody designated as HAM.4 was analysed by cellular radioimmunoassay, flow cytofluorography and indirect immunoperoxidase procedures. The following characteristics of HAM.4 were elucidated. First, an immunohistochemical study revealed that this antibody stained preferentially the bile canalicular face of hepatocyte membrane. Secondly, HAM.4 cross-reacted with kidney, spleen and thymus as well as liver. The kidney expressed much more the antigen molecules detected by this antibody than the liver did. The antigen was located predominantly on the brush border of proximal tubules in kidney. Thus, HAM.4 would be useful for analysing one of the brush border antigens of renal tubules which has been thought to be a pathogenic antigen for inducing experimental membranous glomerulonephritis. Finally, HAM.4 failed to label the cell membrane of rat hepatoma cell lines examined, indicating that the antigen detected by HAM.4 may disappear from cell surface during the course of hepatocarcinogenesis.     

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.     

Recognition by a mouse monoclonal antibody of a glycoprotein antigen of rat brain which is expressed intracellularly by neurons

We describe a previously uncharacterised glycoprotein antigen of rat brain. The antigen was localised by immunofluorescence on 10 μm cryostat tissue sections, and was found to be present intracellularly in neurons. No other cell types or structures within the brain were stained. The antigen is recognised by a mouse monoclonal antibody called NGP41. The antibody was produced after immunising a mouse with glycoproteins purified by lentil lectin affinity chromatography of solubilised rat brain membranes. Spleen cells from the immunised mouse were fused with the myeloma P3X63Ag8. The antigen is expressed by neurons in all brain regions, and also in the dorsal root ganglion neurons of the peripheral nervous system. In all brain regions, the large projection neurons are the most intensely stained by immunofluorescence, but some small neurons also express the antigen. Although dendrites were not stained, sections of sciatic nerve were stained by NGP41, suggesting that the antigen is expressed by axonal processes. The cell bodies of neurons in the inferior olive were stained by NGP41, but their terminals on Purkinje cell dendrites in the cerebellar cortex were not stained, suggesting that the antigen is absent or expressed below the limit of detection in terminals. Both crude brain membranes and a lentil lectin affinity purified brain glycoprotein fraction absorbed the antibody, suggesting that the antigen is a membrane bound glycoprotein. In immunoblotting experiments, the antigen was detected in homogenates of brain and spinal cord membranes, where it appeared as a triplet of bands with molecular weights of 41K, 38K and 36K. Antigen was not detected by immunoblotting in homogenates of six different tissues of non-nervous origin. The antigen was enriched in glycoprotein fractions from adult and juvenile cerebellum as assessed by immunoblotting. Adult brain glycoprotein preparations had a triplet structure similar to that in the homogenates, although most of the antigenic activity of the juvenile preparation was found in a position corresponding to the upper two bands of the triplet.     

Subsets of thymopoietic rat thymocytes defined by expression of the CD2 antigen and the MRC OX-22 determinant of the leukocyte-common antigen CD45

The MRC OX-22 monoclonal antibody recognizes a restricted determinant of the rat leukocyte-common antigen (L-CA, CD45), which is expressed on most peripheral T cells and all B cells. In contrast only 2%-3% of thymocytes are OX-22+ and these are now shown to be mostly of the immature CD4-CD8- (double-negative, DN), phenotype with very few of the mature phenotype cells being OX-22+. Analysis of immunoperoxidase sections suggests that the DN OX-22+ cells are located in the cortex. Among the DN cells about 60% are OX-22+ and a similar percentage are positive for CD2 antigen. Double staining showed that OX-22+CD2-, OX-22+CD2+, and OX-22-CD2+ populations can be defined and that these three sets account for approximately 95% of the DN cells. Measurement of the thymopoietic activity of DN subsets showed that OX-22+CD2- and OX-22+CD2+ cells have regenerative capacity whilst OX-22-CD2+ cells do not.     

Detection and quantification of circulating antigen in schistosomiasis by a monoclonal antibody. I. Specificity analysis of a monoclonal antibody with immunodiagnostic capacity.

Monoclonal antibodies were obtained after immunization of mice with Schistosoma mansoni excretory/secretory antigen, previously shown to contain the circulating cathodic (M) antigen. Among these, the 40:B1 monoclonal antibody proved to be specific for the schistosome genus and to detect only adult worm-derived antigens as shown both by immunoprecipitation and with a two-site immunoradiometric assay using the monoclonal as both the solid-phase and the labelled antibody. The two-site immunoradiometric assay allows a sensitive measurement (detection limit: 5 ng) of circulating schistosome antigen in blood and in urine from patients with schistosomiasis. The amount of circulating schistosome M antigen is correlated with schistosome egg excretion in stool.     

Characterization of a monoclonal antibody against a 24KD antigen from rat testis important for fertility regulation

Monoclonal antibodies were raised against a 24KD antigen from rat testicular cytosol which was previously shown to produce antibody titres in rats with inhibitory effect on fertility. Of the 20 hybridoma clones selected for study, one clone HS-D5 was finally chosen for characterisation as it proved to be functionally promising. The clone was secreting IgM type of antibody. It produced strong agglutination of human sperm and prevented binding of hamster sperm to hamster oocyte. In localization studies it identified the acrosome of sperm of multiple species. Western blots with rat testicular cytosol and human sperm extracts showed a strong band at around 24KD with HS-D5. On Western blots from two dimensional gels, HS-D5 identified multiple spots at the region of 24KD. The mechanism of action of the antibody seemed to be at the level of interaction of the oocyte and spermatozoa.     

A monoclonal antibody to the rat Crry/p65 antigen, a complement regulatory membrane protein, stimulates adhesion and proliferation of thymocytes

A murine monoclonal antibody (mAb), 3F10, was produced by fusion of spleen cells obtained from mice immunized with a rat cortical thymic epithelial cell line (R-TNC.1) stimulated with interferon-gamma and P3X myeloma cells. 3F10 recognized an antigen expressed both on thymocytes and non-lymphoid cells in the thymus. Flow cytometry showed that 3F10 stained more than 98% thymocytes and 90% R-TNC.1 cells. Immunoprecipitation and Western blot studies demonstrated that 3F10 reacted with molecules of 55000 and 65000 MW from both thymocyte and R-TNC.1 cell lysates. 3F10 recognized the same antigen on Chinese hamster ovary cells transfected with rat Crry as did 5I2 mAb, confirming the specificity of 3F10 mAb for the rat homologue of mouse Crry/p65, a membrane-bound complement regulatory protein. 3F10 mAb induced homotypic aggregation of thymocytes and exhibited an additive effect on the aggregation evoked by phorbol myristate acetate. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of protein phosphatases 1 and 2A (as assessed by use of okadaic acid). In contrast, H-7, HA1004 and genistein partially inhibited, whereas staurosporine potentiated the aggregation of thymocytes triggered by 3F10. 3F10 mAb also stimulated binding of thymocytes to the R-TNC.1 line. Both homotypic and heterotypic adhesive interactions are mediated by leucocyte function-associated antigen-1 (LFA-1). In addition, 3F10 stimulated proliferation of thymocytes induced by suboptimal concentrations of concanavalin A. These data suggest that rat Crry/p65 might be involved in the regulation of both cell adhesion and activation of thymocytes. This is a novel, non-complement-dependent function of Crry/p65.     

Distinction of naive and memory BoCD4 lymphocytes in calves with a monoclonal antibody, CC76, to a restricted determinant of the bovine leukocyte-common antigen, CD45.

A murine IgG1 monoclonal antibody (mAb), CC76, has been produced that, based on findings of the relative molecular mass of polypeptides that it recognized, staining of leukocytes in blood and tissues, and the biological properties of the T lymphocyte subpopulations with which it reacts, is considered to identify an isoform of the leukocyte common antigen (LCA) family of molecules in cattle. The mAb is more similar to human CD45R which detect products requiring the presence of the B exon within the LCA gene and to the anti-rat mAb MRC-OX22, than to CD45RA or CD45R0. mAb CC76 reacts with an antigen expressed by subpopulations of cells in bovine blood that express BoCD2 and either the BoCD4 or BoCD8 antigens. T cells that express the gamma/delta T cell receptor identified with mAb to BoWC1 antigen did not react with CC76. The molecule detected is expressed on B cells but not on monocytes or granulocytes. Only 2% of cells in thymic suspensions stained with mAb CC76. Immature cortical thymocytes that were BoCD1+ did not react with CC76 and 90% of the cells in thymic suspensions that were CC76+ had the phenotype of mature thymocytes. These cells were primarily in the medulla. The LCA isoform detected thus appears to be acquired by mature cells shortly before emigration from the thymic medulla into the periphery. Expression of the molecule detected by mAb CC76 on cells from lymph nodes was similar to that in blood, but expression on cells from the gut mucosa was quite different. Almost all, 95% and 93% respectively, of the BoCD4+ cells in the gut mucosa or discrete Peyer's patches were CC76-. A greater proportion of BoCD8+ cells from these sites, 35% and 26%, expressed the antigen. Lymphocytes from animals that had been immunized with Trypanosoma brucei were sorted into BoCD4+, CC76+ and BoCD4+, CC76- populations and cultured in vitro with the variable surface glycoprotein antigen from the parasite. Lymphocyte transformation responses were entirely within the CC76- population indicating that the mAb distinguished naive from memory BoCD4+ T cells in cattle. Major histocompatibility complex (MHC) class I-restricted cytotoxic precursor cells that expressed the BoCD8 antigen sorted from cattle that were immune to Theileria parva were both CC76+ and CC76- indicating that different isoforms of the LCA may be expressed on MHC class I- and class II-restricted memory cells and that BoCD8 memory cells are heterogeneous with respect to the LCA isoform that they express.     

Purification and characterization of an antigen involved in neutrophil chemotaxis and degranulation using a monoclonal antibody

In a previous study we described an anti-neutrophil monoclonal antibody, which inhibited human neutrophil chemotaxis and degranulation without any detectable effect on phagocytosis or oxidative metabolism (Cotter, T. G., Spears, P. and Henson, P. M., J. Immunol. 1981. 127: 1355). This antibody was termed NCD 1. In this study we determined the number of NCD 1-binding sites per neutrophil. Approximately 25 000 NCD 1 IgG-binding sites per cell were found with an equilibrium dissociation constant (Kd) of 6.5 microM for antibody binding. NCD 1 Fab bound to approximately 39 000 sites per cell with a Kd of 16.5 microM. Affinity chromatography columns prepared by coupling NCD 1 to Sepharose 4B beads were used to purify the antigen which bound this antibody. The antigen was a 110-kDa glycoprotein which was not susceptible to reduction by 2-mercaptoethanol. The antigen was not internalized following phagocytosis of opsonized sheep erythrocytes by neutrophils.     

Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody

A monoclonal antibody to HLA-G has been generated by immunizing HLA-A2.1/human β²-microglobulin (β²m) double transgenic mice with murine L cells transfected with both human β²m and HLA-G. This monoclonal antibody, designated as G233, has been found not to cross-react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA-G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA-G in the fetal thymus raises the question as to how T-cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA-G expressed on the surface of trophoblast is complexed with p2m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA-G may also occur as free heavy chains not associated with p2m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several iso-forms of HLA-G from trophoblast similar to the characteristic spot array previously described for HLA-G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA-G.     

Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients.

Monoclonal antibodies were raised against Barber antigen (Ba) of Salmonella typhi 0901. Antibodies produced to antigen 9 of group D salmonellae were used in double- and triple-sandwich antibody enzyme-linked immunosorbent assays (ELISAs) for detecting antigen 9 in urine and plasma specimens from three groups of patients and 49 controls. The triple-antibody ELISA detected the antigen in urine samples from 11 of 18 (65%) patients with hemoculture-proven typhoid (group 1) and 12 of 39 (31%) patients with clinical features compatible with typhoid but whose hemocultures were negative (group 2). This ELISA was negative in three patients from whom Salmonella paratyphi A, Escherichia coli, and Klebsiella pneumoniae (group 3) were isolated by hemoculture and in all healthy controls. The double-antibody sandwich ELISA was positive in 41 and 15% of urine samples from patients in groups 1 and 2, respectively, and was negative with samples from two patients from group 3 and all controls. The sensitivity and specificity compared with those for healthy controls were 65 and 100%, respectively, for the triple-antibody ELISA. Although as little as 7.8 ng of homologous lipopolysaccharide could be detected, background in clinical specimens prevented accurate interpretation of the detection of this antigen in serum. Results were best with urine specimens.     

Detection of avian encephalomyelitis viral antigen with a monoclonal antibody

A monoclonal antibody (MAb), designated VR9-1, prepared against avian encephalomyelitis virus (AEV) reacted with the embryo-adapted strain, Van Roekel, two vaccine and two field strains in indirect immunofluorescent and immunoperox-idase staining of frozen sections of chicken brain, chicken embryo fibroblast and brain cells inoculated with each virus. The MAb was also used as a detector in avidin-biotin-peroxidase staining in paraffin sections where results indicate that it recognized a common epitope among strains of AEV and may be useful for detection and quantitative studies.     

抗人全T细胞单克隆抗体大鼠杂交瘤细胞株的建立

通过融合大鼠免疫细胞株IR983F和经T细胞株HPB—ALL免疫大鼠脾细胞,获得分泌单克隆抗体LO—CD6a的杂交瘤细胞株。实验证实,LO—CD6a是人全T细胞特异性的。     

Effect of interleukins and anti-CD3 monoclonal antibody on the proliferation of thymocytes from irradiated mice.

The responsiveness of thymocytes on day 8 after irradiation to mitogens or anti-CD3 monoclonal antibody was evaluated in the presence of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6) or phorbol-myristate-acetate (PMA). After irradiation, the thymocytes were poorly responsive to T cell mitogens (Concanavalin A, phytohemagglutinin) and the defect could not be overcome by exogenous IL-2, IL-4, IL-6 or by PMA. In contrast, the combination of the calcium ionophore (A23187) and PMA stimulated thymocyte proliferation to a normal level. The anti-CD3 antibody associated with PMA activated thymocytes above the control values, but this was not observed when anti-CD3 was associated with either IL-2 or IL-4. These results suggest that in the thymic populations present early after irradiation 1) the weak proliferative response to mitogens could be related to a defect at a thymocyte level associated or not to an accessory cell deficiency, 2) the intracellular mechanisms involved in T cell proliferation were not altered, 3) the T cell antigen-receptor/CD3 complex was functional.     

Production and characterization of monoclonal antibody to a melanoma specific glycoprotein

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.     

Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC. Demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells.

In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility. However, there are some potential problems in the preparation of monoclonal antibodies. A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture. Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen. Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture. The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea. HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody. This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.