Chicken thymocyte-specific antigen identified by monoclonal antibodies: ontogeny,tissue distribution and biochemical characterization

Two monoclonal antibodies, CT-1 (IgG₁,χ) and CT-1a (IgG₃,χ), were prepared against chicken thymocytes. The antigen identified by these antibodies was found to be a glycoprotein with a major polypeptide component having an apparent molecular weight of 63000 and a minor polypeptide component of approximately 103000. Immunoprecipitation and blocking experiments revealed that the two antibodies react with different antigenic determinants of the molecule. Ontogenic studies employing immunofluorescence failed to reveal the antigenic determinants on cells from the embryo or embryonic yolk sac on days 3 and 6 of incubation. The number of embryonic thymocytes bearing the molecules detected by these antibodies increased from 6% on day 12 to 54% on day 13; the frequency of thymocytes expressing this glycoprotein reached adult levels (> 90%) by the 15th day of embryonic age. In contrast, the CT-1 and CT-1a antibodies reacted with only 2-5% of blood and splenic cells and less than 0.05% of cells from bursa or bone marrow of young adult chickens. In the quail CT-1 reacted with cortical, but not medullary, thymocytes, while the CT-1a antibody was unreactive with quail thymocytes. The surface glycoproteins detected by these discriminating monoclonal antibodies may provide an important discriminating marker for thymic lymphocytes in the chicken and the quail.     

Human tumor-associated antigens identified by monoclonal antibodies

Conclusions Monoclonal antibodies have been obtained that are specific for cell surface antigens of human tumors. Some of these antigens are tissue type specific tumor antigens, most strongly expressed on tumors of the same histological type. The antigens identified by the monoclonal antibodies are primarily normal differentiation antigens that are expressed more strongly on neoplastic cells than on most normal adult cells. For several antigens, the higher degree of antigen expression on neoplastic cells appear to be adequate for diagnostic and therapeutic purposes. Future work in this area is likely to have far-reaching consequences for the clinical handling of human cancer patients.This work was supported by grants CA 14135, 19148, 19149, CA 25558, CA 27841 from the National Institutes of Health, and IM 241A from the American Cancer Society. The authors wish to acknowledge collaboration with Drs. M.-Y. Yeh, R. G. Woodbury, S. M. Larson, and K. Nishiyama     

Human monocyte-histiocyte differentiation antigens identified by monoclonal antibodies

Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.     

Two distinct antigens on chicken thymocytes defined by monoclonal antibodies

Two distinct antigens expressed on chicken thymocytes were defined by monoclonal antibodies designated as Lc-1 and Lc-2. Lc-1 (IgGl) reacted with 80% of the thymocytes (mostly cortical and medullary thymocytes) and Lc-2 (IgM) reacted with 40% of the thymocytes (mainly cortical thymocytes). Lc-1 reacted with 1% of the spleen lymphocytes, but the antibody was nearly nonreactive with cells from peripheral blood leukocytes, bursa and bone marrow. Lc-2 reacted with only small percentages of spleen and bursa cells, and with very few cells from peripheral blood leukocytes and bone marrow. This antibody reacted with a portion of concanavalin A stimulated spleen lymphocytes. When Marek's disease-derived T-lymphoblastoid cell lines were tested for their reactivities with monoclonal antibodies, Lc-1 reacted with none of the cell lines tested, whereas Lc-2 reacted with four of the six cell lines tested. Antigens recognised by Lc-1 and Lc-2 were first found in chick embryonic thymus on day 13 of incubation, after which the number of cells positive for Lc-1 and Lc-2 rapidly increased, reaching young adult levels by days 15 and 14 of embryonic life, respectively. Lc-1 precipitated materials with apparent molecular weights of 60 and 120 kDa from radioiodinated thymocytes.     

Cyclophosphamide- and testosterone-induced alteration in chicken bursal stroma identified by monoclonal antibodies.

Both cyclophosphamide (Cy) and testosterone propionate (TP) treatments ablate B cells in chickens. Essential bursal microenvironmental elements, however, are altered or lost following TP treatment, while bursae from Cy-injected birds can be reconstituted with donor precursors. These two models can thus be utilized to distinguish which bursal stromal molecules are functionally most important in the specific microenvironment of this organ. Monoclonal antibodies (mAb) reactive with non-lymphoid components of the chicken bursa of Fabricius have been used to examine bursal sections from birds treated with Cy or TP. Molecules have been identified on the epithelial buds and follicle-associated epithelium (FAE) that are enhanced following Cy treatment (MUI-52 and 58) and are absent in TP-treated birds. The expression of these molecules may correspond to the ability of Cy-treated but not TP-treated bursae to attract lymphoid precursors. Molecules have also been identified on cells in the subepithelial mesenchymal layer (MUI-63, 65 and 75). These cells interact with the surface epithelium (sEp) prior to epithelial bud formation, an interaction which appears to be TP sensitive. Additionally, two potentially important molecules have been identified in the bursal medulla (MUI-54 and 72) which may have an interactive role with developing B lymphocytes.     

Human myeloid differentiation antigens identified by monoclonal antibodies: expression on leukemic cells

Six murine monoclonal antibodies reactive with human myeloid lineage differentiation antigens are described. These antibodies, designated WM-12, WM-14, WM-15, WM-16, WM-19, and WM-20, react with normal peripheral blood neutrophils and monocytes, as well as a proportion of myeloid precursor cells in bone marrow, but fail to react with the majority of normal lymphoid cells. Immunoprecipitation studies have demonstrated binding of WM-12, -14, -16, -19, and -20, to elements of a protein multimer with sub-units of 50, 105, and 170 kilodalton molecular weight under reducing conditions. WM-15 antibody, however, reacts with a separate protein of 165 kilodaltons. These 6 antibodies reacted with 89% of cases of acute myeloid leukemia, but showed significant binding with only occasional cases of acute lymphoblastic leukemia. Cases of AML (FAB M1 and M2) were more frequently positive with WM-15 (60% of cases positive) than with the other 5 antibodies, whereas the converse was true for leukemias with monocytic differentiation (FAB M4 and M5; 82-100% of cases positive with WM-12, -14, -16, -19, -20). These antibodies appear useful for diagnosis and classification of acute leukemia.     

Expression of myeloid differentiation antigens on normal and chronic granulocytic leukemia erythroid progenitor cells identified by monoclonal antibodies

The antigenic phenotype of erythroid progenitor cells (BFU-E and CFU-E) in bone marrow and peripheral blood from normal individuals and patients with chronic granulocytic leukaemia (CGL) was studied using 14 myeloid monoclonal antibodies (Mc Abs) in a complement dependent cytotoxicity assay followed by culture in methylcellulose. Mc Abs which reacted with normal CFU-GEMM and CFU-GM antigens usually reacted strongly with normal and CGL-BFU-E. In contrast, the majority of myeloid Mc Abs used in the study reacted poorly or did not recognize the antigens on normal and CGL CFU-E. HLA-DR antigens recognized by L243 Mc Ab were expressed on the majority of BFU-E from blood and bone marrow of normal individuals and CGL patients. On the other hand, those antigens were not expressed on normal and leukaemic CFU-E. Two Mc Abs, R1.B19 and WGHS29.1, which recognised the antigens on "late" CFU-GM and not on "early" CFU-GM reacted with a higher proportion of BFU-E from the marrow and blood of CGL patients than of normal subjects. These results indicate that BFU-E in CGL are more differentiated than their normal counterparts. The Mc Ab 54/39, frequently expressed on platelets, recognized a higher percentage of BFU-E and CFU-E from CGL patients than from normal individuals. This may suggest that in CGL there exists a tendency for the expression of megakaryocytic markers on erythroid progenitor cells.     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell-mediated cytotoxicity and mitogen-induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65- and 45-kDa proteins from detergent extracts of ¹²⁵I surface-labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)-induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33–35-kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A-induced proliferative response and significantly reduced both major histocompatibility complex (MHC)-restricted and non-MHC-restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA 1 and 6 immunoprecipitated a 65-kDa protein. mAb CTLA l and 6 had no effect on the Con A-induced blastogenesis and CTLA 6 caused no decrease in virus-specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA 5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA 3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA l and 6 identify the avian homologue of CD4 antigen.     

Immunohistochemical characterization of fibrin(ogen)-related antigens in human tissues using monoclonal antibodies

A new approach to study the distribution of fibrin(ogen)-related antigens was investigated using three different monoclonal antibodies (MAbs) and the avidin-biotin complex immunoperoxidase technique. MAb I8C6 recognizes B beta 1-42 peptide and can react with either fibrinogen or fibrin I; MAb T2G1 recognizes B beta 15-42 peptide and detects fibrin II but does not cross-react with fibrinogen; MAb GC4 reacts with Fragments D/DD derived from plasmin degradation of fibrinogen or fibrin but not with intact fibrinogen. The method can be applied to frozen or Bouin's fixed paraffin-embedded tissues obtained at biopsy, surgery, and autopsy. The distribution of the three antigens observed with the three MAbs was compared with that obtained with a polyclonal antiserum to fibrinogen and with the more conventional histochemical stains used in pathology to demonstrate fibrin deposits in tissues (Lendrum and PTAH). The staining observed with the three monoclonals clearly detected three different populations of fibrin(ogen)-related antigen in the tissues examined. The staining with MAb T2G1 specifically detected fibrin II with greater sensitivity than did conventional stains. The results of this study suggest that this method allows the molecular form of fibrin(ogen)-related deposits in tissues to be determined and this information may help to elucidate the role of fibrin in various disease states, such as atherosclerosis and renal disease, and in tumor growth and metastasis.     

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.     

Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies

Stage-specific Trypanosoma cruzi surface antigens were characterized by using monoclonal antibodies (MAb) which bind specifically to the metacyclic trypomastigotes derived from either the insect vector or acellular cultures. A protein with an apparent molecular weight of 90,000 was detected by the MAb 5E7 on the surface of cultured metacyclics of four strains of T. cruzi: G, CL, Y and Tulahuen. The MAb 1G7, which binds to an epitope of the 90 kDa antigen distinct from that recognized by the MAb 5E7, reacted with metacyclics of the G and Tulahuen strains but not of the Y or CL strain. A polypeptide of approximately 82 kDa, identified by the MAb 3F6, was found in the metacyclics of all four T. cruzi strains. The MAb 3F6 also detected a 75 kDa antigen in the G strain metacyclics. The stage-specific MAb and the polyclonal antibodies from mice protected against acute T. cruzi infection by immunization with killed G metacyclics identified the same set of major surface proteins of G metacyclic trypomastigotes.     

Mononuclear phagocytes of normal and rheumatoid synovial membrane identified by monoclonal antibodies.

The presence of cells bearing epitopes of the mononuclear phagocyte series was studied immunohistochemically in synovium removed from joints involved by trauma (T), osteoarthritis (OA) and rheumatoid arthritis (RA). Mononuclear phagocytes were the most consistent feature of the inflamed rheumatoid synovium. They shared at least five epitopes expressed by mononuclear phagocytes in other tissues. In OA/T samples, cells bearing markers of the less mature monocyte were present at the surface of the synovial membrane, namely the intimal layer, while those bearing macrophage epitopes were apparent throughout the intimal layer and subintimally. This suggested that maturation of the monocyte population takes place after the monocytes have entered the synovial tissues, settled at the surface, then moved downward into the subintimal layer. The synovial monocytes accounted for all the HLA-D region positive cells in the lining layer.     

Induction of Marek's disease virus antigens by IdUrd in a chicken lymphoblastoid cell line.

Marek's disease virus (MDV) antigens, as detected by immunofluorescence, were induced in a lymphoblastoid cell line, MSB-I, in the presence of IdUrd. When treated with 20 microng/ml of IdUrd there was no increase in the number of cells producing virus particles. If IdUrd was removed, an increase in virus production followed. Activation of the MDV genome appeared to require incorporation of IdUrd into cellular DNA and occurred during the first 12 h of culture. Expression of the activated genome required de novo protein synthesis and occurred during the next 12 h. The MDV genome in high producer MSB-I cells could be activated with low concentrations of IdUrd, whereas low producer MSB-I cells could not be activated with IdUrd to any great extent.     

Immunoprecipitation of Marek's disease virus-specific polypeptides with chicken antibodies purified by affinity chromatography

The sulfhydryl-containing polypeptides of vaccinia virus strain IHD-J were studied in an attempt to gain further insight into their roles in virus assembly. The virus showed 39 diagonally oriented spots in the profile of two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In spite of the reducing conditions in the electrophoretic system, two dimer spots appeared in the profile. An extraordinary highly reactive sulfhydryl residue(s) of VP17K converted the polypeptide reciprocally into monomer and dimer forms (VP17K-VP25K) due to the disulfide-sulfhydryl exchange reaction. In order to detect the structural proteins linked with disulfide bond(s), vaccinia virus was solubilized and electrophoresed under nonreducing conditions in the first dimension and then under reducing conditions in the second dimension. The viral polypeptides linked originally with disulfide bond(s) were separated into subunits. The complexes were dimers or oligomers of some polypeptides (VP133K, VP61K, VP21K, VP17K) and heterocomplexes such as VP57K + VP16K, VP63K + VP32K, VP27K + VP13K. VP57K and VP22K were not soluble in the nonreducing SDS solution. The apparent molecular sizes of VP37K, VP29K, and VP17K-25K changed significantly according to the composition of the denaturing agent used, suggesting that the differences in their molecular sizes were due to conformational changes produced by reduction of their intramolecular disulfide bonds and also by breakage of their hydrogen bonds.     

Two closely related antigens expressed on granulocytes, macrophages and some reticular elements in rat lymphoid tissues: characterization by monoclonal antibodies

Two distinct novel antigen systems preferentially expressed in rat granulocytes and macrophages were detected using two different monoclonal antibodies (R2-1A6 and R2-2B1). These two antibodies reacted with approximately 50% of rat bone marrow cells, most granulocytes, blood monocytes, alveolar macrophages and peptone-elicited peritoneal macrophages, but not with red blood cells, platelets, thymocytes and T lymphocytes. In addition, R2-2B1 but not R2-1A6 antibody cross-reacted weakly with rat B cells. These two monoclonals also reacted with some reticular elements in rat lymphoid organs including epithelial reticular cells in the thymic medulla and follicular dendritic cells in the lymphoid germinal centre, as well as with the specialized endothelium in the marginal sinuses of the spleen and the post-capillary venules of the lymph node, where lymphocyte recirculation takes place. These antibodies, however, did not label so-called 'dendritic cells' bearing Ia antigens on their cell surfaces, which were found to be located in the thymic medulla, thymus-dependent areas of rat lymphoid tissues and the interstitium of various non-lymphoid organs, suggesting that these dendritic cells, presumably ascribed to those associated with accesory cell function, are separable from the mononuclear phagocyte system in rats by their different reactivities with R2-1A6 and R2-2B1 antibodies.     

Stage-specific, strain-specific, and cross-reactive antigens of Leishmania species identified by monoclonal antibodies.

The fusion of NS-1 myeloma cells with spleen cells from mice chronically infected with Leishmania tropica resulted in nine clones of hybridomas producing monospecific antibodies to membrane antigens of L. tropica. One of the antibodies (L-5-85) bound specifically to the promastigote form of the parasite, and the remaining eight recognized antigens shared by the promastigote and amastigote of L. tropica. Four of the antibodies (L-5-16, L-5-34, L-5-44, and L-2-3F11) detected parasite antigens on the surface of L. tropica-infected macrophages. Common antigens shared by L. tropica, L. mexicana, and L. donovani were identified as well as one antigen apparent on most Leishmania spp. and present also in Crithidia fasciculata. Two monoclonal antibodies (L-5-27 and L-5-41) were found to bind only to strains of L. tropica from simple cutaneous leishmaniasis. A special property shared by these two antibodies was the inhibition of parasite growth in macrophages in vitro.     

抗鸡γ-干扰素单克隆抗体的研制及初步鉴定

目的:制备抗鸡γ-干扰素单克隆抗体(mAb)。方法:应用淋巴细胞杂交瘤技术,以重组菌BL21(DE3)(pET-ChIFN-γ)表达产物的包涵体作为抗原免疫BALB/c鼠,以纯化的GST-ChIFN-γ作为检测抗原,制备抗鸡γ-干扰素mAb;采用ELISA、Dot-ELISA和Westernblot鉴定mAb的特异性。结果:获得2株可稳定分泌抗鸡γ-干扰素mAb的杂交瘤细胞株1G5、5E3,其Ig亚类均为IgG2a,腹水mAb1G5、5E3的ELISA效价分别为1∶1600000,1∶120000。在Dot-ELISA试验中,这2株mAb均只与表达His-ChIFN-γ及GST-ChIFN-γ的重组大肠杆菌反应,与未表达这2种IFN-γ的其他8种菌株均不发生反应。在蛋白质印迹试验中,2株mAb均能与融合蛋白GST-ChIFN-γ、His-ChIFN-γ发生反应,出现特异性条带。结果表明,mAb1G5、5E3是针对鸡γ-干扰素的特异性mAb。结论:成功地制备抗鸡γ-干扰素的mAb,它们在免疫检测、免疫细胞功能分析和免疫调节研究等方面有应用价值。     

Identification and tissue distribution of rabbit leucocyte antigens recognized by monoclonal antibodies.

Three monoclonal antibodies which recognize rabbit leucocytes have been characterized by immunofluorescence staining of a variety of cell populations and also by immunochemical techniques. The evidence obtained suggests that these antibodies recognize the rabbit equivalents of the CD58/LFA-3 (VC21), CD43/leukosialin (L11/135) and CD9 (MM2/57) antigens. A fourth antibody, RPN3/57, recognizes an antigen expressed strongly on T cells, thymocytes and neutrophils and at lower levels on platelets. It has not, however been possible to characterize the antigen recognized by RPN3/57 in molecular terms. Both L11/135 and RPN3/57 are useful reagents for the detection of T cells both by flow cytometry and by immunohistochemistry.     

Immunocytochemical distribution of a breast carcinoma associated glycoprotein identified by monoclonal antibodies.

A glycoprotein, BCA-225 (Mr 225,000-250,000), has been identified in cells and spent medium of clone 11 T47D breast carcinoma cells by three murine monoclonal antibodies, CU18, CU26, and CU46. The antigen was localized in paraffin sections of 167/178 (94%) Bouin's-fixed human breast carcinoma tissues and few other carcinomas (1/8 lung [squamous], 4/4 uterine cervix) in an intracellular pattern, whereas an apical or glycocalyx distribution was seen in several normal tissues, benign lesions, and malignant tumors. Although the immunocytochemical staining patterns observed with these antibodies have many similarities to those described with other previously reported monoclonal antibodies, notable differences include the lack of reactivity of CU18, CU26, and CU46 with lactating mammary gland and with gastrointestinal malignancies. BCA-225 binds to wheat germ lectin, not to concanavalin A, but monoclonal antibody binding does not appear to involve the carbohydrate component of the molecule. The frequency of the immunocytochemical detection of BCA-225 in breast carcinomas and its restricted distribution in other human tissues suggest considerable clinical potential for this antigen and its corresponding monoclonal antibodies.