Entamoeba histolytica: specific antigen recognized by a monoclonal antibody

Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.     

Immunohistochemical studies of colorectal carcinoma with a monoclonal antibody against carcinoembryonic antigen

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of colorectal carcinoma using a monoclonal antibody (MAb) against CEA. CEA has been demonstrated in 20 out of 22 rectum carcinomas (90.9%), in all of 23 colonic carcinomas, in none of 4 hyperplastic polyps and in 2 out of 6 adenomatous polyps (33.3%). CEA was found more often, and the intensity of the staining was stronger in well-differentiated carcinomas than in moderately and poorly differentiated carcinomas. No correlation was found between the presence of CEA in colorectal carcinoma and the stages of the disease. The mean values of serum CEA in patients with colorectal carcinoma and polyps with negative, weakly and strongly positive staining were 5.4 +/- 3.9 ng/ml, 28.3 +/- 23.8 ng/ml and 99.8 +/- 145.3 ng/ml respectively. Elevation of serum CEA occurred in 30 out of 39 (78.9%) cases with strongly positive CEA staining, in 4 out of 6 (66.7%) with weakly positive and in 1 out 9 (11.1%) with negative staining. A significant difference was found in serum CEA activity between the group with negative CEA staining and positive CEA staining (P less than 0.01). Our results suggest that the monoclonal antibody (MAb C27) can be used for the localization of CEA in conventionally prepared tissues of colorectal carcinomas by immunoperoxidase techniques for routine immunopathological diagnosis.     

alpha-Spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin-based cytoskeleton is present in the cortex starting from stage 1. At stages 4-6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of alpha-spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus alpha-spectrin. Furthermore, an antibody against mammalian alpha-spectrin was used to localize the protein. Our results showed a stage-dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4-6. Mol. Reprod. Dev. 55:229-239, 2000.     

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.     

Studies of a tumor-associated antigen,COX-1, recognized by a monoclonal antibody

Summary Monoclonal antibodies against an ovarian tumor cell line, OC-3-VGH, were generated using modified hybridoma technology. Among the seven that were selected for their high specificity and affinity to ovarian cancer cells and low cross-reactivity to most normal human tissues, RP 215 was shown to react specifically with a tumor-associated antigen, COX-1, from certain ovarian/cervical cancer cell lines. By Western blot assay, COX-1 was shown to have a subunit molecular mass of about 60 kDa and exist as an aggregate in the native state. COX-1 could also be detected in the shed medium of certain cultured tumor cells. A solid-phase sandwich enzyme-immunoassay procedure was designed for quantitative determinations of COX-1 in the shed medium or in patients' sera using RP 215 for both well-coating and the signal detection. Highly purified COX-1 was obtained from the shed medium of cultured OC-3-VGH tumor cells mainly by hydroxyapatite and immunoaffinity chromatography with RP 215 as the affinity ligand. At neutral pH, purified COX-1 also exists as an aggregate and is relatively stable at temperatures below 50°C. Its immunoactivity was found to decrease with time in the presence of trypsin. However, the immunoactivity of COX-1 was not affected upon incubation with carbohydrate-digestive enzymes or concanavalin A and only partially inactivated in the presence of NaIO₄ or iodoacetamide. Treatments of COX-1 with dithiothreitol and guanidine thiocyanate resulted in a complete loss of activity. Furthermore, rabbit antisera raised against purified COX-1 exhibited similar immunospecificity to that of RP 215. The results of this study suggest that COX-1 is a glycoprotein consisting of a 60 kDa subunit, which is recognized by RP 215 through its peptide determinant. Preliminary retrospective clinical studies were performed to assess the utility of a COX-1 enzyme immunoassay kit for detection and monitoring of patients with ovarian and cervical cancers.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Immunolocalization of a guinea pig sperm surface antigen recognized by a monoclonal antibody E74

E74 is a mouse monoclonal antibody raised against the acrosome-reacted guinea pig spermatozoa. This study describes immunolocalization of the E74 antigen in guinea pig spermatozoa. Immunoelectron microscopy of guinea pig spermatozoa shows that the E74 antigen is localized on the equatorial segment plasma membrane following the acrosome reaction but not associated with the surface of the acrosome-intact spermatozoa. Immunoblot analysis of Triton X-100 extract of cauda epididymal guinea pig spermatozoa following one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that E74 antibody recognizes a protein with an apparent molecular weight of 45,000 dalton. Immunoblot of sperm extracts separated by two dimensional gel electrophoresis indicates a broad spot of 45,000 dalton in the 5 to 7.5 isoelectric focusing range.     

Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.     

Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody

A mouse mAb, which recognized a rat T cell surface Ag responsible for the T cell activation, was produced by a regular hybridoma method using F344 rat T cells stimulated with PMA and a calcium ionophore, as the Ag. The mAb termed 1F4 (kappa-IgM) was reactive with rat T cells but not with B cells and immunohistochemically it stained rat thymus tissues strongly at medulla and weakly rat cortex. Addition of 1F4 mAb to a culture of T cells resulted in the proliferation of T cells by a help of PMA or a solid support. 1F4 mAb also caused the modulation of the corresponding Ag but not other T cell markers such as CD5, CD2, and OX-52-defined Ag. The 1F4 mAb immunoprecipitated a cell surface component having an apparent m.w. of 25,000 from rat T cells which could be associated with a disulfide-linked heterodimer (m.w. 92,000) consists of subunits having m.w. of about 52,000 and 43,000. These results strongly suggest that the 1F4 mAb recognizes a rat T cell Ag homologous to the human and mouse CD3.     

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.     

从噬菌体表面展示肽库中筛选衣原体单克隆抗体识别的抗原表位

利用抗体捕获法,经三轮淘洗,从表面展示随机肽序列的噬菌体文库中筛选到与衣原体单克隆抗体C17特异结合的噬菌体克隆,其一致序列为：（L／I）PGGS（P／W）,竞争抑制实验表明含特异序列的克隆能与天然抗原竞争。据此,我们认为此序列为衣原体的B细胞抗原表位。     

Trypanosoma lewisi: stage-specific surface antigens and exoantigens recognized by monoclonal antibodies

The stage-specific expression of surface antigens by Trypanosoma lewisi was investigated using monoclonal antibodies directed against this parasite. Hybridomas secreting monoclonal antibodies were produced by the fusion of SP2/0-Ag 14 mouse plasmacytomas with spleen cells from rats infected previously with the Taliaferro strain of T. lewisi. Additivity enzyme-linked immunosorbent assays and indirect immunofluorescent antibody tests indicated the determinant recognized by monoclonal antibody TL40.3 (IgM) was different from those recognized by monoclonal antibodies TL40.1 (IgA), TL40.2 (IgM), and TL40.6 (IgG2 alpha). Monoclonal antibody TL40.3 agglutinated trypanosomes collected 3 days after parasite inoculation while monoclonal antibodies TL40.1, TL40.2, and TL40.6 agglutinated trypanosomes collected 6 days after inoculation. Since agglutinin titers against trypanosomes from irradiated (700 rad from a 60Co source) and nonirradiated rats were similar, expression of the antigens recognized by the monoclonal antibodies appeared to be independent of the immunological state of the host and the morphology of the parasite. The reproduction of T. lewisi in in vitro trypanostatic assays was inhibited only when the monoclonal antibodies were present in concentrations greater than or equal to those needed to agglutinate the trypanosomes. Monoclonal antibodies TL40.1 and TL40.3, but not TL40.2 and TL40.6, agglutinated erythrocytes collected later in the infection from irradiated, infected rats. None of the monoclonal antibodies agglutinated erythrocytes from nonirradiated, infected rats, from irradiated, noninfected rats or from nonirradiated, noninfected rats. This suggests that immunocompetent rats may make blocking antibodies against the exoantigens recognized by monoclonal antibodies TL40.1 and TL40.3.     

Immunohistochemical demonstration of surface antigen of human lymphocytes with monoclonal antibody in acetone-fixed paraffin-embedded sections

Although the majority of reported studies have used fresh-frozen sections in detecting surface antigen of lymphocytes in tissue via monoclonal antibody, detailed histological figures can not be obtained by this method. Nor can the antigenicity be preserved for any length of time. A new method for detecting the surface antigen of lymphocytes using fixed and embedded material is presented. Human spleens were fixed in cold acetone, embedded in low melting point paraffin wax, and the thin sections treated with hyaluronidase. Anti-T lymphocyte monoclonal antibody (anti-Leu-1, anti-Leu-2, anti-Leu-3) and anti-HLA-DR were applied on these sections, and the antigen was detected by the ABC (avidin-biotin-peroxidase complex) method. The results were then compared with those of fresh-frozen sections. There was no great difference in detecting T and B cells or their subsets, but the histological figures were substantially better preserved in sections prepared by the present method. Furthermore, the antigenicity was retained in the materials fixed and embedded for more than two years.     

Epitopes on proliferating cell nuclear antigen recognized by human lupus autoantibody and murine monoclonal antibody

The immune epitopes of proliferating cell nuclear antigen (PCNA), also called cyclin, were analyzed by determining the reactivity between PCNA peptide fragments and anti-PCNA antibodies from lupus patients, murine monoclonal antibody (19A2), and rabbit anti-NH2-terminal peptide antibody. Limited digestion of PCNA/cyclin with Staphylococcus aureus V8 protease resulted in several peptide fragments. Five fragments of 30, 20, 15, 14, and 13 kDa were reactive with rabbit anti-NH2-terminal peptide antibody denoting that they contained the NH2-terminal peptide. The 30- and 20-kDa fragments reacted with 19A2 but the others did not. Lupus sera reacted with 17- and 15-kDa peptide fragments allowing their classification into three groups. Two of eight sera (type A) reacted only with the 17-kDa fragment. Two others (type B) reacted with both the 17- and 15-kDa fragments and the remaining four sera (type C) reacted only with the 15-kDa fragment. The sera reacting with the 15-kDa fragment also reacted with the 20-kDa fragment, but the sera reactive only with the 17-kDa fragment did not, indicating that the 17-kDa fragment was not a degradation product of 20-kDa fragments. The 19A2 epitope resided in the region between 15 and 20 kDa from the NH2 terminus, whereas there was at least one distinct epitope on each 15- and 17-kDa peptide, which were recognized by lupus autoantibodies.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.     

SIVp27单克隆抗体的制备和鉴定

[目的]建立SIVp27杂交瘤细胞株,并对其分泌的SIVp27单克隆抗体进行初步鉴定。[方法]使用基因重组的SIVp27蛋白免疫BALB/c小鼠,采用杂交瘤技术使用半固体培养基法建立杂交瘤细胞株,制备单克隆抗体。通过染色体核型对杂交瘤细胞株进行鉴定;采用Westernblot、免疫荧光法、酶联免疫吸附法确定单克隆抗体的交叉反应性、相对亲和力、抗原识别表位、免疫球蛋白的类型和亚类,对单克隆抗体进行鉴定。[结果]获得四株可稳定分泌SIVp27单克隆抗体的杂交瘤细胞,1C3、2B6为IgG1类,2E12为IgG2b类,3G3为IgG2a类。四株单抗均能识别SIV的p27蛋白,与逆转录病毒SRV、STLV无交叉反应,2B6、2E12与HIVp24有交叉反应。免疫荧光法检测腹水效价为1:10240~40960。1C3、2B6、2E12、3G3染色体平均数分别为103、97、96、101。2E12与3G3识别不同的抗原表位。[结论]成功地制备出四株SIVp27单克隆抗体,均具有良好的特异性和亲和力,为进一步建立免疫分析方法,进行SIV/SAIDS及其艾滋病相关研究,奠定了基础。