Light-optical and ultrastructural immunohistochemistry of antigen H-4, a common marker of epithelial cells]

Epitope localization reacting with mice monoclonal antibodies (Mabs) H 4 was investigated using the specimen of epithelium of skin, human uterine cervix as well as in the culture of epithelium cells of guinea-pig duct deferent. Mice monoclonal antibodies against antigen H 4 obtained by the hybridoma method after immunization of mice with rat colon epithelium cytoskeletal fractions were used. Mabs H 4 were shown to react with antigen of intermediate filaments of all studied normal epithelial, cancer cells as well as culture epithelial cells. Mabs H 4 are supposed to be used as a unique immunohistochemical marker of epithelium cells under normal and malignant growth conditions.     

Identification of proteolysis-resistant fragments carrying P-type antigenic determinants in chordin and immunologically related human brain antigen

An antigen immunologically related to chordin was identified in white and gray matter of large hemispheres of human brain as well as in one of glial tumours. It was shown that human and rabbit brain extract components cross-react with eight monoclonal antibodies directed against chordin-specific epitopes of P-group. Exhaustive proteolysis of giant sturgeon notochord and human brain extracts resulted in fragments interacting with anti-chordin antibodies and eluted in an equal volume during chromatography on TSK HW-40 gel. At the same time, gel electrophoresis performed under denaturing conditions revealed that the mobility of chordin subunits strikingly differs from that of brain antigen immunologically related to chordin. Thus, the cross-reactivity of antichordin antibodies with the human brain extract component is due to the presence in this antigen of a P-type determinant which, after exhaustive proteolysis of both antigens, is detected in structures (presumably, glycopeptides) having an identical molecular mass.     

一种与受精有关的人精子膜蛋白（BS－17）的分离纯化

本文用盐分级分离,ＭｏｎｏＱＦＰＬＣ及ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法从人精子ＣＨＡＰＳ抽提液中分离纯化出一种与不育病人血清中抗精子抗体发生特异反应的ＢＳ－１７人精子膜蛋白。该蛋白为一糖蛋白,分子量为１７．５５±２．１５ｋＤ,等电点为５．６５,中性己糖含量为１６．６７％。在人精子上主要分布于顶体区域,不同于已有报导的人精子膜蛋白。在体外实验中抗ＢＳ－１７多克隆血清可以显著影响人精子的获能（ｐ＜０．０２５）和对去透明带仓鼠卵的穿透（ｐ＜０．００５）,但不影响人精子运动性及与去透明酯酸带仓鼠卵的结合。小鼠体内被动免疫实验结果证明抗ＢＳ－１７多抗血清具有明显地抑制受精的功能（ｐ＜０．００１）。     

Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies.

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.     

The detection of Rickettsia prowazekii in the vectors of louse-born typhus Pediculus humanus by using monoclonal antibody-based preparations]

The results of this investigation indicate that preparations obtained on the basis of monoclonal antibodies have proved to be suitable for the detection of R. prowazekii antigens in the natural carrier of typhus when used in all types of the enzyme immunoassay; of these, the assay made by the capture method has been found to possess the highest sensitivity. The testing of the sensitivity limits has shown that this method known as ELISA-mu-capture, i.e. ELISA carried out with the use of antibodies to the mu chain of human immunoglobulin, is capable of detecting the antigen in a dose of 0.5-1 ng. Preparations based on monoclonal antibodies to species-specific R. prowazekii antigen permit the identification of the causative agent of typhus in its natural carrier within 24 hours.     

Cross--reactivity of the V3-speciflc antibodies with the human C1q

It has been previously shown that the sequence similarity between a portion of the envelope glycoprotein 120 (gp120) from the human immunodeficiency virus type-1 (HIV-1) and several types of human collagen and collagen-like molecules exists. That observation led to the suggestion that the antibodies against the third hypervariable region (V3) of HIV-1 gp120 (V3-specific antibodies) might have a role in the autoimmune phenomena observed in HIV-infected persons. In this study we have examined the cross-reactivity of the V3-specific antibodies purified from sera of HIV-infected individuals, sera obtained from the rheumatoid arthritis and systemic lupus crythematosus patients, as well as from the sera of healthy volunteers with the separate chains of a subcomponent of the first component of the human complement system, Clq. Our results show that the V3-specific antibodies are present in the sera of the HIV-infected individuals, patients suffering of the systemic autoimmune diseases as well as in the sera of healthy volunteers. Whereas these antibodies appeared in the HIV+-sera after antigen challenge, those present in the HIV- -sera probably represent the antibodies that are cross-reactive with the antigen. V3-reactive antibodies can be purified by affinity chromatography and they were highly specific for the V3-peptide. Additionally, they showed cross-reactivity with the separate chains of the human Clq as well as with the chicken collagen type VI. Possible physiological implications are discussed.     

Cross-reactivity of anti-human immunodeficiency virus type 1 gp41 antibodies with human astrocytes and astrocytoma cell lines.

An antigen expressed by astrocytes in human brain tissue and by various human astrocytoma cell lines was shown to cross-react with a monoclonal antibody generated against amino acids (aa) 584 to 609 of the transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1). This region is an immunodominant segment of gp41, and high levels of antibodies against this epitope have been detected in both serum and cerebrospinal fluid of HIV-infected individuals at all stages of HIV infection. Immunohistochemistry with this monoclonal antibody demonstrated the presence of a cross-reactive antigen in human brain tissue, with an increased frequency and intensity of staining in HIV-positive individuals when compared with HIV-negative controls. By using a panel of HIV-positive and -negative sera, we show that antibodies in HIV-positive serum specifically bound to the surfaces of human astrocytoma cells. HIV-positive sera depleted of antibodies recognizing gp41 aa 584 to 609 showed a significant diminution in cell surface binding. Conversely, the serum antibodies that bound to and were eluted from the aa 584 to 609 peptide also bound to the astrocyte cell surface. To identify the target antigen, the immunoreactivity of three astrocytoma cell lines was examined. By immunoprecipitation of metabolically labeled cell lysates and Western blot (immunoblot) analysis, we identified a protein of approximately 100 kDa as the target antigen. Cross-reactive antibodies between HIV proteins and astrocyte epitopes, such as this 100-kDa protein and others previously reported, suggests that an autoimmune response against these target antigens may disrupt the normal functions of astrocytes.     

Polyclonal antibodies against human cell-surface antigen CD34

An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.     

Purification and characterization of a protein antigen (p94) from human brain and its identification in other species.

A protein antigen was chromatographically purified from human brain by its immunoaffinity to 44E3 monoclonal IgG and its chemical nature was investigated. The yield of antigen was estimated at 71%, and a 3160-fold purification was achieved relative to the homogenate. The antigen preparation from brain showed a very high degree of purity when analysed by SDS/polyacrylamide-gel electrophoresis and was composed of a single polypeptide of Mr 94,000. Amino-sugar and neutral-sugar analyses indicated that the protein was not glycosylated. The amino acid composition of the purified protein from brain was compared with that of the analogous protein purified from an acute-lymphoblastic-leukaemic cell line, HOON. The compositions were very similar, suggesting that the two proteins were closely related. Both purified proteins were equivalent in their ability to inhibit the reactivity of monoclonal antibodies 44E3 and 44H4 with leukaemic cells. These two antibodies appear to recognize spatially related, if not identical, epitopes on the same molecule. The antibodies were shown to cross-react with a polypeptide of Mr 94,000 in homogenates of human, bovine and guinea-pig brain white matter. Indirect immunoperoxidase staining of human grey- and white-matter acetone-fixed tissue sections incubated with either antibody indicated that the antigen was present on neuronal and glial cells; the staining was seen as clusters in the cytoplasm, starting at the plasma membrane, but leaving the nucleus unstained. The concentration of the protein in human brain was shown to be similar throughout postnatal development and aging.     

β淀粉样蛋白人源性抗体的制备

目的:应用噬菌体展示及抗体库技术,制备并鉴定β淀粉样蛋白(Aβ)人源性抗体。方法:应用固相筛选方法,以人工合成的Aβ1-15肽为靶标分子,在大容量全合成人源性噬菌体抗体库中筛选抗Aβ人源性抗体,并进行特异性鉴定。结果:经过3轮筛选,单克隆鉴定获得噬菌体抗体F11,竞争性ELISA表明抗体对Aβ1-42的结合位点位于1～15氨基酸残基内,ELISA结果证实抗体特异性良好。结论:以Aβ1-15肽为靶标分子获得了特异性良好的人源性抗体。     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Molecular organization of glutamate-sensitive chemo-stimulated nerve cell membranes. Interaction of monoclonal antibodies with glutamate-binding membrane proteins in the rat brain, human neuroblastoma and molluscan neurons

Hybrid cells obtained by fusion of myeloma PX63-Ag8-653 with immune splenocytes of BALB/c mice were found to produce monoclonal antibodies with a high degree of specificity to rat and human brain. The kinetics of specific IgG binding to purified fractions of glutamate-binding membrane proteins from rat and human brain were analyzed in Scatchard plots. The presence of a single type of binding sites with Kd = 100 nM was demonstrated. The monoclonal antibodies were shown to inhibit the specific binding of tritium-labeled L-glutamate to different brain synaptic membranes. Addition of monoclonal antibodies to the incubation medium induced a modulating effect of physiological responses to L-glutamate in Planorbarius corneus neurons. The possible use of specific antibodies to glutamate-binding proteins as immunochemical markers for the study of glutamate receptor topography on membrane surface was demonstrated with the aid of neuroblastoma cells N18 Tg2a and rat brain tissue slices. An analysis of glutamate receptor binding sites with the use of monoclonal antibodies revealed that these antibodies specifically recognize the active center in the receptor molecules which have identical antigen determinant sites in different biological systems.     

Five epitopes of a differentiation antigen on human inducer T cells distinguished by monoclonal antibodies

A series of mouse monoclonal antibodies reacting with human T cells of the helper/inducer subclass, OKT4, 4A, 4B, 4C, and 4D, have been reported. Using double-fluorescent staining and complement-mediated depletion, it was shown that the antigen(s) recognized by OKT4, 4A, 4B, 4C, and 4D antibodies are present on the same cell. Using FITC-labeled OKT4, 4A, 4B, 4C and 4D, a lack of competition between the antibodies for their epitopes was shown. Immunoprecipitation of the antigen recognized by each antibody yielded a molecule of approximately 60,000-62,000 Da. Sequential precipitation with several antibodies resulted in a minimum of additional precipitated antigen following removal of the first antigen. Capping of cell surface antigen with OKT4, followed by staining with OKT4, 4A, 4B, 4C, or 4D, indicated that the epitopes for all five antibodies co-cap. A sandwich ELISA assay using OKT4 and the other antibodies showed that molecules binding to OKT4A, 4B, 4C, and 4D also bound OKT4. It can therefore be concluded that monoclonal antibodies OKT4, 4A, 4B, 4C, and 4D recognize distinct epitopes present on a molecule of approximately 60-62,000 Da on human helper/inducer T cells.     

Molecular and antigenic nature of isolated Sm

The Sm antigen was isolated and purified from calf thymus nuclear extract by affinity chromatography. The affinity columns were made with serum antibodies from an SLE patient or an anti-Sm monoclonal antibody derived from a hybridoma cell line. Proteins eluted from these two columns had m.w. of 58,000 and 35,000 by SDS polyacrylamide gel electrophoresis. The natural conformation of this antigen appears to be 95,000 in m.w. with the 58,000 particle containing the Sm antigenic determinant. The affinity column-purified antigen detected by the human anti-Sm antibodies is also recognized by anti-Sm antibodies in murine lupus serum, as shown by solid-phase radioimmunoassay. This study 1) demonstrates the molecular and antigenic nature of the Sm antigen and 2) compares the anti-Sm binding capabilities of antibody populations present in sera from SLE patients and from MRL lpr/lpr mice.     

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体。     

Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this artificial Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.Abbreviations HuGph human erythrocyte glycophorin - HoGph horse erythrocyte glycophorin - OSM ovine submaxillary mucin - mAb monoclonal antibody - ELISA enzyme-linked immunosorbent assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.01m Na₂HPO₄/0.15m NaCl, pH 7.2) - BSA bovine serum albumin - TBS 0.05m Tris-HCl/0.15m NaCl, pH 7.2 - TGr transformation grade     

Production and characterization of DR xenoantisera: use for detection of serum DR antigens.

Immunoadsorbents consisting of rabbit anti-DR antibodies bound to Staphylococcus aureus were used to bind DR antigens from NP40 cellular extracts of cultured B lymphoid cells. When injected into rabbits, these DR antibody absorbents elicited the production of antibodies that were shown by both serologic and immunochemical techniques to react specifically with the DR antigen molecule. A rosette inhibition assay with B lymphoid cells and anti-DR xenoantisera was used to detect DR antigens in human serum. DR serum antigens could be found in an enriched preparation of serum lipoproteins obtained by centrifugation in KBr. In addition, sera from some patients with neoplastic diseases contained higher levels of DR serum antigens than those found in normal individuals.     

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.