Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.     

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.     

Ultrastructural localization of viral antigens using the unlabeled antibody-enzyme method.

Employing the unlabeled antibody enzyme method at the ultrastructural level, a comparison was made between preembedding staining and postembedding staining for the detection of viral antigens. The bacteriophage P1 absorbed to the surface of Shigella dysenteriae was used as a model system. Preembedding staining resulted in the specific deposition of peroxidase-antiperoxidase (PAP) complexes as an electron-dense coating around the viral heads. Disadvantages of the preembedding staining method included the agglutination of cells by the primary antiserum which produced a gradient of specific staining and the "bleeding" or migration of electron dense reaction product away from the sites of attached PAP complexes. The postembedding staining method had distinct advantages over the preembedding staining in that PAP complexes were deposited directly over exposed viral heads within the thin section. In addition, the specific immunostaining of viruses was uniform through the section and no artifactual migration of reaction product was observed.     

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.     

Rubella virus antigens: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against the rubella virion were used to locate epitopes involved in hemagglutination and neutralization. The MAbs exhibiting high-level hemagglutination-inhibiting activity were shown by Western blot analysis to be specific for the E1 polypeptide; this is consistent with the presence of the hemagglutinin on the E1 polypeptide. Some of the E1-specific MAbs also neutralized viral infectivity. However, hemagglutination-inhibiting activity and neutralizing activity did not always correlate. Three distinct functional epitopes were identified on the E1 polypeptide by competition analyses: one which reacted with MAbs with high-level hemagglutination-inhibiting activity and with neutralizing activity, one which reacted with MAbs with low-level hemagglutination-inhibiting activity and with neutralizing activity, and one which reacted with MAbs with only hemagglutination-inhibiting activity. A MAb specific for the E2 polypeptide exhibited neutralizing activity. This E2-specific MAb and two E1-specific MAbs with neutralizing activity were capable of precipitating intact virus which indicates that at least three epitopes involved in neutralization are accessible on the surface of the virion.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

Electron-microscopic localization of baboon acrosomal antigens using antisperm monoclonal antibody.

A sperm antigen corresponding to baboon sperm monoclonal antibody 1A9 was localized in the testis and ejaculated sperm in this animal, using the immunofluorescence technique and immunogold labelling. Immunohistochemical studies of the baboon testis showed that the antigenic determinant was localized in the late spermatid cells and spermatozoa close to the seminiferous tubules. Immunofluorescence studies indicate that the protein was localized on the acrosome region of ejaculated baboon sperm. At the electron-microscopic level, gold particles indicative of the presence of this determinant recognized by 1A9 monoclonal antibody were detected on the inner acrosomal region of ejaculated baboon sperm.     

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.     

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.     

The detection of two surface antigens on human lung macrophages using monoclonal antibodies

Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

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.     

Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.     

Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.     

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.     

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.