Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Antigenic study of the differentiation of the human kidney using monoclonal antibodies

The production of monoclonal antibodies against human embryonic renal cells allowed to display on the adult human kidney some antigens typical of certain structures or tissues: the proximal convoluted tubule for EG 9-11 and EG 19-6 monoclonal antibodies, the glomerular basement membrane for EG 14-1, the urothelium for EE 24-6, the connective tissue for EK 8-1 and EK 17-1 and probably the capsular and tubular basement membranes for EK 8-1. Simultaneously, we could follow the spatial and temporal repartition of the antigens during the renal development. One of them (EI 16-1) seemed to disappear in the adult and might correspond to a foetal type-antigen.     

Demonstration of carbohydrate- and protein-determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to precipitate an la-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-la extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-la; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2,9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.— Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia. 17 shows preference for a- linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of thek haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murineI region.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Three mouse monoclonal antibodies to human differentiation antigens: reactivity with two mucin-like antigens and with connective tissue fibers

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.     

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Demonstration of carbohydrate- and protein determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to participate an Ia-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-Ia extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-Ia; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2, 9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.--Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia.17 shows preference for a beta-linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of the k haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murine I region.     

Demonstration of human alpha-L-fucosidase polymorphism by means of monoclonal antibodies

Conventional rabbit antibodies and mouse monoclonal antibodies were raised to alpha-L-fucosidase purified from human placenta. Four monoclonal antibodies were studied, of which only one (A) was able to immunoprecipitate the fucosidase activity completely. Two antibodies (B and C) precipitated 65% and one (D) 35% of the activity. The enzyme precipitated by the monoclonal antibodies remained fully active, whereas the enzyme precipitated by conventional antibodies was partly inactivated. As shown by the method of successive immunoprecipitations, the monoclonal antibodies B and C recognized the same set of placental fucosidase molecules, and D a subset thereof. The purified fucosidase also yielded two components after gel electrophoresis in nondenaturing conditions, and the slower component corresponded to the set recognized by antibodies B and C. The fucosidase extracted from different tissues and serum was studied by immunoprecipitation. In all cases, the enzyme was completely precipitated by monoclonal antibody A. Two patterns were found with B, C and D: either part of the activity was precipitated by these antibodies (leucocytes, placenta, brain, liver, spleen, thymus) or B, C and D failed to precipitate any of the enzyme (serum, heart, kidney, testes).     

Development of monoclonal antibodies that recognize antigens associated with human cervical carcinoma

Six monoclonal antibodies, generated by immunization of mice with human cervical carcinoma cells maintained in tissue culture or with cells from fresh tumor tissue, reacted specifically with the malignant cells in 71% to 90% of the tumor tissue imprints and cervical smears containing neoplastic cells but not with normal cervical epithelial cells in smears from 21 to 23 healthy donors. Antibody CE 402 bound to epithelial cells associated with regeneration in 2 of the 23 normal smears tested. Considerable heterogeneity of antibody binding by malignant cells was observed. Antibody CE 400 was the most reactive, binding to more than 50% of the tumor cells in all reactive specimens. Five of these monoclonal antibodies detected protein antigens in the 80 K to 110 K molecular weight range. Our studies demonstrate the feasibility of producing monoclonal antibodies with selected specificity for cervical carcinoma. These antibodies may be of considerable diagnostic value.     

Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.     

Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies

The distribution, structural profile and functional properties of Ia-like antigens synthesized by human monocyte-macrophages have been analyzed using monoclonal antibodies to common determinants of these antigens. Up to 45 and 70%- of monocyte-macrophages isolated from the fluid of blisters induced with cantharidin and from peripheral blood, respectively, react with monoclonal antibodies to human Ia-like antigens. The level of Ia-like antigens on monocytes-macrophages appears to be similar to that on cultured B lymphoid cells. Monoclonal antibodies to common determinants of Ia-like antigens specifically block antigen presentation by monocyte-macrophages to T lymphocytes as well as proliferative response of T lymphocytes to autologous and allogeneic monocytes-macrophages. These results indicate that common determinants of Ia-like antigens play a role in the interaction of monocytes-macrophages with T lymphocytes.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.