Production and use of rat monoclonal antibodies to the human myeloid cell nuclear differentiation antigen

A dot immunoblot screening assay was used to identify rat monoclonal antibodies to a human myeloid cell differentiation-specific nuclear antigen (MNDA). The selection was based on the positive reaction of hybridoma cell supernatants with a concentrated nuclear protein extract prepared from late stage human myeloid leukemia cells that express MNDA (HL-60) coincident with a negative reaction with the same extract prepared from a non-expressing more immature human myeloid leukemia cell line. The approach provided an efficient method for obtaining monoclonal antibodies to a specific low abundance nuclear antigen that has not been purified. Sixteen wells from three fusions contained antibody displaying a specific reaction with the nuclear protein fraction obtained from the HL-60 cells. Immunoblotting analysis revealed that all of the sixteen specific hybridoma cell lines produced antibody against the same Mr 55,000 nuclear antigen. Selecting hybridoma cells that produce antibody reactive with the native antigen provided antibody suitable for detecting MNDA in immunocytochemical tests. The rat monoclonal antibodies were purified and coupled to CNBr-activated agarose and carbonyldiimidazole-activated agarose. Although both antibody affinity matrices exhibited the same antigen binding capacities, the matrix prepared using carbonyldiimidazole-activated agarose bound the MNDA with a high level of specificity while the matrix prepared from CNBr-activated agarose bound numerous other nuclear proteins.     

Immunocytochemical staining of endogenous nuclear proteins with the HIS-1 anti-poly-histidine monoclonal antibody: A potential source of error in His-tagged protein detection

Histidine-tagged proteins are widely used in biochemical studies and frequently detected with antibodies specific for the histidine tag. Immunocytochemistry is widely used in studies with overexpressed proteins to determine cellular localization and in the case of histidine-tagged proteins can be carried out with anti-polyhistidine antibodies. Recent studies have suggested that polyhistidine sequences are present within a small number of human proteins and may direct expression to the nucleus and nuclear speckles compartments of the cell. In this study immunocytochemical staining of human SH-SY5Y neuroblastoma cell lines with the HIS-1 anti-polyhistidine monoclonal antibody were determined. Results showed that the HIS-1 anti-polyhistidine monoclonal antibody stained endogenous nuclear proteins in SH-SY5Y cells. The stained proteins were contained within the nuclear membrane, but were not directly linked to DNA. In a histidine-tagged catalase overexpressing cell line the HIS-1 anti-polyhistidine monoclonal antibody showed nuclear staining, whilst staining with the CAT-505 anti-catalase monoclonal antibody showed primarily cytoplasmic staining. These results suggest that anti-polyhistidine antibody staining shows significant cross-reactivity with endogenous nuclear proteins in SH-SY5Y neuroblastoma cells and may not be suitable for localization studies of histidine-tagged proteins. Immunocytochemical studies with anti-polyhistidine antibodies and localization of histidine-tagged proteins must be confirmed with protein specific antibodies or other methodology.     

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry

Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA signal. Thus, monoclonal PCNA-specific antibodies offer a convenient tool for the detection of human cell proliferation by immunofluorescence microscopy and by flow cytometry.     

An immunocytochemical method for demonstrating estrogen receptor in human uterus using monoclonal antibodies to human estrophilin

We have used monoclonal antiestrophilin antibodies to develop an improved immunocytochemical method for localizing estrogen receptors in tissue sections with an indirect immunoperoxidase technique. Rat monoclonal antibodies were raised against human estrogen receptor (estrophilin) protein derived from the cytosol of MCF-7 human breast cancer cells. These monoclonal antibodies have been shown to have extensive cross-reactivity with estrogen receptors from various primate and nonprimate tissues. We have used frozen sections of human proliferative phase endometrium and an indirect immunoperoxidase technique to establish conditions for demonstrating estrogen receptor antigenic determinants in frozen tissue sections. The method involves (a) brief fixation with formaldehyde-containing fixatives either prior to freezing or immediately after cutting cryostat sections, (b) bleaching the tissue of endogenous peroxidase activity, (c) application of primary antibody or control immunoglobulin, (d) application of an adsorbed bridging antibody (goat antirat IgG), and (e) application of rat peroxidase-antiperoxidase followed by diaminobenzidine. Specific nuclear staining for estrogen receptor antigenic determinants was observed in the vast majority of epithelial and stromal cells. No specific cytoplasmic staining was identified in cryostat sections of any of the 17 cases studied for this report.     

Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies

Monoclonal anti-DNA antibodies produced by hybridomas derived from MRL-lpr/lpr mice and human lupus patients were found to bind to the cytoskeleton of mink lung cells. When tested by indirect immunofluorescence, 17/29 human monoclonal anti-DNA antibodies reacted with the cytoskeleton; 4 of the 29 also produce antinuclear reactions with epithelial cells. The cytoskeletal staining was not inhibited by prior treatment of the cells with DNase, but it was completely blocked by prior incubation of the monoclonal antibodies with DNA and other nucleic acids. The ability of the polynucleotides to inhibit the cytoskeletal staining corresponded to their ability to bind to the antibodies in competitive immunoassays. An (Fab')2 preparation of a monoclonal antibody bound to the cytoskeleton as well as the whole immunoglobulin. The effect of colcemid on the staining pattern, the blocking effect of a monoclonal antivimentin antibody, and results with nitrocellulose blots of cellular proteins indicated that the cytoskeletal protein to which the antibodies bound was vimentin.     

Monoclonal anti-la antibody derived from a mouse with experimental SLE is similar to human anti-la antibodies

The La antigen is a highly conserved protein, originally defined by sera of patients with Sj?gren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.     

Bacterial endosymbiont-derived lipopolysaccharides and a protein on symbiosome membranes in newly infected amoebae and their roles in lysosome-symbiosome fusion.

Experimental results are presented to support the view that symbiont-derived lipopolysaccharides are involved in the prevention of lysosome-symbiosome fusion in xD amoebae harboring bacterial endosymbionts. Monoclonal antibodies against lipopolysaccharides and a 96-kDa protein present on symbiosome membranes of amoebae were used to monitor the appearance of the membrane-specific components in newly infected amoebae with endosymbionts from xD amoebae. The lipopolysaccharides and protein appeared on the newly forming symbiosome membranes within 3 to 7 days, as detected by indirect immunofluorescence staining with monoclonal antibodies. The lysosome-symbiosome fusion was followed by double staining of two antigens with different monoclonal antibodies applied to the same amoeba. Antilipopolysaccharide monoclonal antibodies were detected by staining with a fluorescein isothiocyanate-conjugated secondary antibody, and a biotinylated anti-lysosomal protein monoclonal antibody was detected by staining with Texas Red-conjugated streptavidin. In xD amoebae injected with an antilipopolysaccharide antibody, lysosomes fused with some of the symbiosomes that did not fuse with lysosomes in noninjected cells.     

Monoclonal antibodies to the Sjögren's syndrome associated antigen SS-B (La)

The nuclear autoantigen SS-B (Sjögren's syndrome B antigen) was purified from rabbit thymus extract by immunoaffinity chromatography with human autoantibodies, and used to immunise BALB/c mice for production of monoclonal antibodies. Fusion of spleen cells from an immunised mouse with NS-1 myeloma cells resulted in the isolation of 3 clones secreting anti-SS-B antibody. Subclasses were shown to be IgG2b by immunodiffusion. Specificity of the monoclonal antibodies (MCA) was determined by ELISA and indirect immunofluorescence. By immunoblotting all 3 MCA identified a single 45 K immunoreactive polypeptide in rabbit thymus, identical with the major polypeptide recognised by human sera containing anti-SS-B. Affinity columns prepared from the 3 MCA all bound SS-B from rabbit thymus extract, without binding other nuclear antigens. Immunofluorescence studies on standard substrates showed that SS-B was located predominantly in the nucleoplasm but in cells transformed by EBV or phytohaemagglutinin more prominent nucleolar and cytoplasmic staining was seen.     

Monoclonal antibody stains oligodendrocytes and Schwann cells in zebrafish (Danio rerio)

We prepared a monoclonal antibody that recognizes oligodendrocytes and Schwann cells in zebrafish. On immunoblots, the antibody mainly recognized three protein bands of 34 kDa in a membrane fraction from adult zebrafish brain. Medaka fish (Oryzias latipes) also possessed the same protein bands in a membrane fraction. The antibody did not stain neurons, but stained cells in fiber tracts and cranial and spinal nerves. In order to determine the nature of these cells, the staining pattern of the monoclonal antibody was compared with that of a myelin basic protein antiserum. Both antibodies stained oligodendrocytes and Schwann cells in fixed sections from the adult zebrafish. Both antigens were also co-localized in cultured glial cells. Taken together, these results indicate that the new monoclonal antibody recognizes myelinating glial cells in zebrafish and will be useful for the analysis of piscine glia. Accepted: 18 October 1999     

Immunohistochemical analysis of PTEN in endometrial carcinoma: a tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities.

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=-0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.     

Immunomorphological characterisation of antinuclear antibodies in chronic liver disease.

Two immunofluorescence procedures to evaluate antinuclear antibodies were compared in a series of 221 patients with chronic liver disorders of various aetiologies. The use of HEp-2 cells allowed us to discriminate with more confidence between the homogeneous and speckled patterns, to show the presence of associated patterns in the same serum, and, above all, to identify two specificities, unrecognizable on tissue sections. The anticentromere antibody was found in 10% of cases of primary biliary cirrhosis and occasionally in other conditions; the antibody staining multiple nuclear dots was strictly confined to primary biliary cirrhosis (17%). With the exception of autoimmune chronic active hepatitis the prevalence of antinuclear antibodies increased in all groups, particularly in primary biliary cirrhosis. Homogeneous antinuclear antibody was associated by both immunofluorescence procedures with autoimmune chronic active hepatitis. The multiple nuclear dot antinuclear antibody turned out to be an additional marker of primary biliary cirrhosis, helpful for the positive diagnosis of primary biliary cirrhosis in a proportion of cases negative for antimitochondrial antibody. Absorption experiments showed that multiple nuclear dot and antimitochondrial antibody are antigenically distinct. Moreover, multiple nuclear dot antinuclear antibody was associated with the finding of a dry Schirmer's test.     

Localization of proliferating cell nuclear antigen (PCNA/Cyclin) in workshop cases of Hodgkin's disease and non-Hodgkin's lymphoma.

Proliferating cell nuclear antigen (PCNA/Cyclin) is a 36-kD protein that is present in cycling cells but not in resting cells, and therefore represents a marker of tumor proliferation. Application of anti-PCNA/Cyclin monoclonal antibodies has shown that this protein is localized to the nucleus of cycling cells, with the exception of cells in mitosis, which demonstrate faint cytoplasmic reactivity. Recently, Benjamin and Gown found that Reed-Sternberg cells and variants show nuclear and cytoplasmic staining with anti-PCNA/Cyclin antibody 19A2, and suggested that this feature may be useful in distinguishing Hodgkin's disease from other tumors. This report describes the reactivity of 42 workshop cases that were stained with anti-PCNA/Cyclin antibodies 19A2 and/or PC10. Thirty-three (79%) of the 42 cases showed adequate reactivity to allow for interpretation of staining localization. In the group of reactive cases, 26 (79%) showed nuclear and cytoplasmic staining. The localization of PCNA/Cyclin was compared with the consensus diagnosis in each case. Eighty percent of cases classified as Hodgkin's disease, 67% of cases classified as non-Hodgkin's lymphoma, and 100% of unresolved cases showed both nuclear and cytoplasmic staining. The incidence of cytoplasmic PCNA/Cyclin was not different between Hodgkin's disease and non-Hodgkin's lymphoma in this study.     

Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.     

Production of a monoclonal antibody to a membrane antigen of human T-cell leukaemia virus (HTLV1/ATLV)-infected cell lines from a systemic lupus erythematosus (SLE) patient: serological analyses for HTLV1 infections in SLE patients.

Human T-cell leukaemia virus (HTLV1/ATLV), which causes adult T cell leukaemia (ATL), is an infectious, lymphotrophic retrovirus unique for humans. The present study was undertaken to determine whether HTLV1 had any pathogenetic role for systemic lupus erythematosus (SLE). The incidence of antibodies to ATL cell-associated antigens (ATLA) in sera from patients with SLE and other collagen diseases was investigated by an indirect immunofluorescent cytoplasmic staining of an HTLV1-infected cell line (MT-1). A radioimmunoassay was also performed to detect antibodies to HTLV1 protein and crude membrane fraction derived from an HTLV1-producing cell line MT-2. Furthermore, an Epstein-Barr virus (EBV)-transformed B cell line (ES-1) was constructed from an SLE patient, which produced a monoclonal antibody (IgG, lambda) reactive to an HTLV1-related cell-membrane antigen expressed on MT-1 and MT-2 cells. The specific reactivity of the monoclonal antibody was analysed by an indirect immunofluorescent cell-membrane staining and a microcytotoxicity test. The incidence of anti-ATLA antibodies was not different among SLE and other collagen diseases. The monoclonal antibody produced by ES-1 stained and killed HTLV1-infected cell lines specifically, but did not react with other human lymphoid cell lines. This monoclonal antibody failed to react with peripheral blood mononuclear cells (PBMC), mitogen-induced T cell blasts, and iododeoxyuridine-treated T cells from SLE patients. Thus, a possible role of HTLV1 in the aetiology of SLE was not established.     

Comparison of monoclonal versus polyclonal calretinin antibodies for immunohistochemical diagnosis of malignant mesothelioma.

Of putative specific markers for diffuse malignant mesothelioma, nuclear staining with Zymed polyclonal calretinin antibody has shown the best specificity to date for epithelial diffuse malignant mesothelioma versus adenocarcinoma. We compared specificity and sensitivity of this polyclonal antibody for diagnosis of diffuse malignant mesothelioma with a new monoclonal antibody from DAKO. One hundred eighteen adenocarcinomas and 111 diffuse malignant mesotheliomas-70 epithelial, 22 sarcomatous, and 19 biphasic-were immunostained with calretinin antibodies from Zymed (polyclonal rabbit, prediluted, PAD:DC8) and DAKO(monoclonal mouse, 1:100, clone DAK Calret 1) using manufacturer-recommended procedures. Cases were blinded and assessed for nuclear versus cytoplasmic staining, percent positive cells, and background. Both antibodies showed similar positive predictive values for diffuse malignant mesothelioma by nuclear staining (Zymed=95%; DAKO=97%). False positives in 4 (3.4%) and 2 (1.7%) adenocarcinomas, respectively, stained greater than 10% of cells. Sensitivity for epithelial malignant mesothelioma was slightly less for DAKO antibody (Zymed=80%; DAKO=73%). Neither antibody performed well on sarcomatous malignant mesothelioma (Zymed=2/22; DAKO=1/22). Both antibodies are useful in the diagnosis of epithelial malignant mesothelioma, although monoclonal antibody is slightly less sensitive.     

Monoclonal antibody to human beta interferon: characterization and application

Seven stable mouse hybridomas secreting monoclonal antibodies to human fibroblast beta interferon (IFN-beta) were isolated, all seven of which belonged to IgGl subclass and kappa type. While neutralizing the antiviral activity of human fibroblast IFN-beta, they failed to neutralized both that of human IFN-alpha and human IFN-gamma. These monoclonal antibodies neutralized the antiviral activity of human fibroblast IFN-beta but not that of human IFN-alpha and IFN-gamma. Two of the seven monoclonal antibodies, YSB-1 and YSB-2, showed particularly high neutralization titers in the ascitic fluid. Monoclonal antibodies were purified from cultures of hybridoma grown in a serum-free medium. The purified monoclonal antibodies, YSB-1(5 micrograms/ml) and YSB-2(1 microgram/ml), neutralized IFN-beta from 10EU/ml to 1EU/ml. Human fibroblast IFN-beta was purified to 3.5 X 10(7) IU/mg protein (1.0 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-2) affinity column chromatography, whereas human recombinant IFN-beta obtained from E.coli was also purified to 6.5 X 10(7) IU/mg protein (1.1 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-1) affinity column chromatography.     

Rat monoclonal antibodies to mouse lung components for analysis of fibrosis

Six rat monoclonal antibodies to mouse lung membrane fraction have been characterized. Each has unique binding properties and can be used to stain particular lung components in paraffin sections. One antibody, 133-13A, recognizes a 100-kDa glycoprotein on lung tumor cells, but stains only macrophage-like cells in normal or fibrotic lung sections as determined by immunogold electron microscopy. The monoclonal antibody 273-34A binds to a 112-kDa protein on the surface of normal mouse lung fibroblasts. Immunogold electron microscopy demonstrates antibody binding to capillary endothelial cells, but not to fibroblasts. Type I, or Type II cells. Staining of fibrotic sections with MoAb 273-34A is dramatically enhanced over staining of normal lung. A third antibody, 370-8A, gives a general staining pattern throughout normal lung that is intensified in fibrotic lung. Another MoAb 328-41A mediates intense nuclear fluorescence of lung tumor cells and cells in lung sections. It binds to 14- and 17-kDa proteins that may be high mobility group (HMG) nuclear proteins. Enzyme inhibition studies and immunofluorescence staining patterns on normal lung indicate that elastin may be the target of MoAb 328-35B. MoAb 327-5B binds to normal mouse lung fibroblasts and red blood cell membranes. These last three MoAbs stain macrophages in fibrotic lung, but give a general pattern of light epithelial cell stain in normal lung sections indicating macrophage engulfment of the normal cell antigen during fibrosis. These antibodies should be useful in identifying cell types and molecular mechanisms involved in early stages of fibrosis induced by different chemical insults.     

The isoforms of C protein and their distribution in mammalian skeletal muscle

Summary A monoclonal antibody that is specific for the slow skeletal muscle isoform of C protein of rabbit muscle has been prepared by immunizing mice with a crude preparation of human myosin. It reacted with the X protein fraction of rabbit skeletal muscle and stained all type I cells in this tissue. It also stained a fraction of the type II cells with varying intensities. The type II cells staining with antibody to slow C protein also stained with a polyclonal antibody prepared against rabbit fast muscle C protein. The type II cells not staining with antibody to slow C protein stained strongly with antibody to fast C protein. In the human skeletal muscle antibody to slow C protein stained all cells whereas antibody to fast C stained only type II cells. It is concluded that the distribution of the isoforms of C protein in adult vertebrate skeletal muscle is more complex than is the case with proteins such as components of the troponin complex.     

Characterization of a Sperm Nuclear Protein

PROBLEM: The molecular identity of sperm DNA-binding structural proteins contributing to the integrity of a sperm residual high salt/nuclease resistant nuclear structure is studied by cDNA cloning and monoclonal antibodies to the recombinant polypeptide. This structure, which is likely to be transferred unimpaired in the oocyte and is anticipated as a molecular correlate of the nuclear scaffold (nuclear matrix/envelope) in somatic cells, may be essential with respect to its DNA organization for the recovery and assembly of somatic-type chromatin in the zygote. Recently, a cDNA encoding one of these proteins has been cloned and the recombinant polypeptide expressed in E. coli as a β-galactosidase fusion protein. The main objective of the present study is the identification of the native sperm antigen by monoclonal antibodies raised against the recombinant molecule. METHODS: We evaluated the possibility of immunizing by direct intrasplenic deposition in BALB/c mice of the recombinant fusion protein available as transblotted on nitrocellulose membrane carriers or as nitrocellulose protein-bearing particles. Isolated sperm DNA/tight binding protein complexes were used in ELISA and Western blotting for selection of monoclonal antibodies specific to self epitopes of the nuclear antigen, as well as immunofluorescence of swollen human spermatozoa subjected to in situ extraction with high salt/β-mercaptoethanol/DNase I and proteolysis, and of a cultured fibroblast cell line L-929. RESULTS: A monoclonal antibody, Mab 2C4, was selected which recognized a 52 kDa protein in the fraction of sperm high salt/urea resistant proteins. The target polypeptide was detected on swollen spermatozoa primarily to the post-acrosomal and/or equatorial regions whereas in nonextracted sperm cells the epitope was exceedingly unavailable. The somatic cell location of the cognate epitope was confined to the nuclear envelope displaying a caplike pattern of staining, and also in a juxtanuclear cytoplasmic randomly coiled filamentous network and in compact bodies. CONCLUSIONS: A nuclear protein salt-stably bound to the sperm residual structure has been identified. The antigen appears localized in sperm exclusively to perinuclear subacrosomal sites that may be anchored at the male nuclear envelope, given the occurrence of the target epitope in somatic cells as well in nuclear and cytoplasmic sites adjacent to the nuclear membrane.     

Identification of parietal cells in gastric body mucosa with HMFG-2 monoclonal antibody.

AIMS--To identify parietal cells in the upper gastrointestinal tract by an immunoperoxidase method, using commercially available monoclonal antibodies. METHODS--Routine surgical biopsy specimens of gastric body mucosa were examined using the avidin-biotin peroxidase method with the monoclonal antibodies HMFG-1 and HMFG-2 to identify parietal cells. Double immunoperoxidase labelling with HK12.18, a well characterised monoclonal antibody directed against an epitope on the alpha (catalytic) subunit of H+ translocating, K+ stimulated adenosine triphosphatase (H,K-ATPase), was used to confirm that HMFG-1 and -2 stained parietal cells. RESULTS--HMFG-1 and HMFG-2 showed consistent parietal cell staining patterns in the gastric body mucosa. HMFG-2 gave a more intense staining pattern of the secretory canaliculi. This was confirmed by double immunolabelling with HK12.18. CONCLUSIONS--HMFG monoclonal antibodies are recommended as highly specific markers of human gastric parietal cells.