Production and characterization of monoclonal antibody to a melanoma specific glycoprotein

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.     

Production and characterization of a monoclonal antibody to chloramphenicol

A monoclonal antibody to chloramphenicol (CAP) was produced. After immunization of BALB/c mice with CAP base coupled to human serum albumin and incubation of the stimulated splenocytes in vitro in the presence of antigen for three days, these splenocytes were hybridized with X63‐Ag8·653 myeloma cells. The antibody, designated 6A10, proved to be IgG2b, and it had a detection limit for CAP of 10 ng/ml (0·5 ng/assay) in the direct enzyme immunoassay using horseradish peroxidase‐labelled CAP. The cross‐reactivities with CAP base, p‐nitrobenzyl alcohol, and p‐nitrophenol were 5·0, 0·94, and 0·007%, respectively. No cross‐reactivities were observed with penicillin, tetracycline and thiamphenicol, respectively.     

Production and characterization of an anti-idiotypic antibody specific for a monoclonal antibody to glycoprotein D of herpes simplex virus

A monoclonal antibody specific for glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was used to prepare an anti-idiotypic antibody in rabbits. After removal of antibody reactivity to constant region determinants by absorption with polyclonal mouse immunoglobulins and a monoclonal antibody of the same subclass as the anti-gD monoclonal, the anti-idiotypic (anti-id) antibody reacted specifically with anti-gD. Using an ELISA inhibition assay with immunoaffinity-purified gD, the anti-id D reagent inhibited the binding of anti-gD to gD, suggesting that anti-id D mimics an epitope of gD by binding the antigen-combining site of anti-gD. Immunization of mice with anti-id D could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. The possibility that anti-id D could act similarly to gD and stimulate an immune response to HSV when administered in vivo is discussed.     

Production and characterization of monoclonal antibody to gentamicin

Splenocytes from BALB/c mice immunized with a gentamicin-hemocyanin conjugate were fused with X63-Ag8.653 murine myeloma cells to produce hybridomas that secreted monoclonal antibody to gentamicin. Sixteen positive clones were obtained. The monoclonal antibody chosen for a gentamicin immunoassay has been characterized with respect to class, subclass, type of light chain, electrophoretic homogeneity, and binding affinity. Gentamicin monoclonal antibody purified from mouse ascites fluid was analyzed by immunoelectrophoresis, double immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and polyacrylamide gel electrophoresis (PAGE). The results show that the antibody is an IgG2a (kappa). Two bands were detected when the purified antibody was electrophoresed on polyacrylamide gels: a major band and a less mobile minor component (5.6% of the major band). Both were IgG2a (kappa). The major band contained antibody which bound 2.1 moles of the substrate-labeled gentamicin derivative, beta-galactosyl-umbelliferone sisomicin, per mole of IgG, whereas one mole of the minor band bound only 0.095 moles of the substrate-labeled conjugate. The antibody has an affinity constant of 1.22 X 10(10) M-1.     

Production and characterization of a monoclonal antibody to bovine estrogen sulfotransferase

A murine IgGl (k) monoclonal antibody, termed 33-11, was produced against purified bovine estrogen sulfotransferase. After the enzyme was separated into its four isoenzyme forms by native polyacrylamide gel electrophoresis, a Western blotting analysis indicated that the antibody reacted to a similar extent with each of the isoenzymes. An immunoprecipitation and SDS-poly acrylamide gel electrophoresis analysis was performed with an I-125-labeled enzyme preparation. Antibody 33-11 precipitated protein in the molecular weight range 70,000-74,000, the size of native estrogen sulfotransferase, plus additional bands with molecular weights of 29,000, 54,000, and 61,000. The lower molecular weight polypeptides may represent fragments, bearing a common epitope, produced by limited endogenous proteolysis of the intact enzyme. Solid-phase radioimmunoassay testing demonstrated strong and specific binding of the antibody to the bovine enzyme. No cross-reactivity was observed with a panel of nine other purified bovine proteins. An immunohistochemical analysis with the antibody was performed on two bovine tissues with high levels of enzyme activity. Placenta showed strong, specific staining of fetal giant cells of chorionic villi, while adrenal had weak but widespread staining which tended to be more concentrated in the inner cortex. Monoclonal antibody 33-11 has potential utility in the purification, detection, and quantitation of bovine estrogen sulfotransferase.     

Production and characterization of a monoclonal antibody against neopterin

We have produced and characterized the first monoclonal antibody against neopterin (D-erythro-6-(1,2,3,-trihydroxypropyl)pterin). The antibody specifically recognizes neopterin in a modified RIA. The binding capacity in this assay is 34%, the sensitivity limit of inhibition is 0.9 nmol/l. Cross-reactivity exists with monapterin (L-threo(1,2,3,trihydroxypropyl)pterin) in 30%, with other pteridines cross-reactivity has been found in less than 5%.     

Production and characterization of a monoclonal antibody to human saposin C.

A murine IgG1 monoclonal antibody, termed 68-12, was produced against purified human saposin C. Immunoprecipitation and binding analysis indicated that the antibody reacted only with saposin C. Dot blotting and Western analysis demonstrated that antibody 68-12 also reacted with prosaposin and a higher molecular weight protein(s) in murine spleen and cerebral grey matter. Solid phase competitive radioimmunoassay against 125I labeled saposin C (0.25 micrograms/ml) showed no cross reactivity for saposin A, B and D up to 15 micrograms/ml. At a concentration of 50 micrograms/ml saposin A, B and D cross reacted 21, 1.5, and 49% respectively. Monoclonal antibody 68-12 appears to have potential utility in the purification, detection and quantitation of human saposin C and its precursor.     

抗血小板糖蛋白VI单克隆抗体的制备及其功能研究

目的:制备抗血小板糖蛋白VI(GPVI)单克隆抗体,观察其在体外抗血小板黏附和聚集功能。方法:采用基因重组技术体外表达血小板糖蛋白VI胞外区重组蛋白(rGPVI)。以rGPVI免疫小鼠,经细胞融合及筛选后制备抗GPVI单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convu lxin及ADP诱导的血小板聚集的影响;利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果:正确构建了rGPVI表达载体pET-20b(+)-GPVI,rGPVI在原核细胞中有效表达。rGPVI能够被抗Penta-H is单抗和抗GPVI多抗识别。制备的抗GPVI单克隆抗体SZ118能够识别rGPVI,并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convu lxin诱导的血小板聚集,呈抗体剂量依赖性;对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明,SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论:成功制备抗GPVI单克隆抗体SZ118,该抗体与血小板有良好的结合能力,显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

抗血小板糖蛋白Ⅵ单克隆抗体的制备及其功能研究

目的：制备抗血小板糖蛋白Ⅵ（GPⅥ）单克隆抗体，观察其在体外抗血小板黏附和聚集功能。方法：采用基因重组技术体外表达血小板糖蛋白Ⅵ胞外区重组蛋白（rGPⅥ）。以rGPⅥ免疫小鼠，经细胞融合及筛选后制备抗GPⅥ单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convulxin及ADP诱导的血小板聚集的影响；利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果：正确构建了rGPⅥ表达载体pET-20b（＋）-GPⅥ，rGPⅥ在原核细胞中有效表达。rGPⅥ能够被抗Penta-His单抗和抗GPⅥ多抗识别。制备的抗GPⅥ单克隆抗体SZ118能够识别rGPⅥ，并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convulxin诱导的血小板聚集，呈抗体剂量依赖性；对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明，SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论：成功制备抗GPⅥ单克隆抗体SZ118，该抗体与血小板有良好的结合能力，显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

Production and characterization of a mouse monoclonal antibody to the glycolipid asialo-GM1

The glycosphingolipid asialo-GM1 (aGM1) is a true differentiation antigen of murine lymphoid cells. This glycolipid is highly immunogenic in the rabbit, but the antisera produced shows some cross reactivity with GM1, the naturally occurring sialylated derivative of aGM1. In the present study we examined the ability to raise anti-aGM1 antisera in the mouse. We compared the efficiency of several immunization methods in various strains of mice. The most effective procedure involved repeated intraperitoneal injections of aGM1-cholesterol rich particles in the NZB mouse. Hybrid B cell lines were generated by fusion of mouse myeloma cells with the splenocytes of an NZB mouse immunized with aGM1. The specificity of the antisera produced and of the monoclonal antibody secreted by one of these hybridomas (103HT30) was defined by ELISA and by immunostaining on thin layer chromatograms. The monoclonal antibody 103HT30 is an IgM. It reacted with aGM1 but not with any of the structurally-related ganglioside or neutral glycolipids tested. In particular, 103HT30 monoclonal antibody did not present any detectable cross-reactivity with GM1.     

Production and characterization of a monoclonal antibody to human Type IV collagen.

We have produced a monoclonal antibody to human basement membrane Type IV collagen. The antibody reacts with the pepsin-resistant, collagenase-sensitive domain of Type IV collagen isolated from placental membranes, but not with human collagens of Types I, II, III, V, 1alpha, 2alpha, and 3alpha. The antibody precipitates biosynthetically labeled human Type IV procollagen, and the precipitate contains both the alpha1 (IV) and alpha2 (IV) chains, suggesting the occurrence of both of these chains within the same triple-helical molecule. When used in indirect immunofluorescence, the antibody gives brilliant staining of basement membranes from a variety of human tissues but does not stain tissues of bovine, canine, rabbit, rat, or mouse origin. It is suggested that this antibody will be of value in research on the structure of human basement membrane collagen, on the distribution of this collagen in various basement membranes, and particularly for the study of basement membranes in normal human development and pathologic processes.     

Production and characterization of a monoclonal antibody to the v-mos oncogene protein.

Valuable information about proto-oncogenes and their physiological functions has been obtained by studying their expression in normal cells. However, the protein product of the c-mos gene, the cellular homologue of the transforming gene (v-mos) of Moloney murine sarcoma virus, has not been detected in normal mouse cells or tissues. Here, we have constructed a v-mos expression vector, pRI-delta mos, which directs the synthesis of a truncated v-mos gene product, a protein A fusion protein. Using the truncated v-mos oncoprotein produced in Escherichia coli as immunogen, we prepared anti-v-mos monoclonal antibodies (MAbs). In immunoblotting assays, the MAb was reactive with v-mos oncoprotein and detected bands at 43 KDa or 39 kDa in the tissue extract of mouse testes or ovaries, respectively, in which the c-mos protooncogene mRNA is expressed. These results demonstrate that the v-mos MAb obtained is suitable for elucidating the physiological functions of v-mos gene product and may also be utilized to detect c-mos gene product at the cellular level.     

Production and characterization of a monoclonal antibody against human glomerular heparan sulfate

After immunization with heparan sulfate proteoglycan (HSPG) isolated from human glomeruli, two mouse monoclonal antibodies (mAbs) against heparan sulfate (HS) were obtained. Both mAbs were of the IgM isotype and showed identical specificity. One of these, mAb JM-13 is described in detail. In enzyme-linked immunosorbent assay and Western blotting, reactivity was found with human glomerular basement membrane HSPG and HS. No binding occurred to the core protein of HSPG obtained after removal of HS with trifluoromethanesulfonic acid. In enzyme-linked immunosorbent assay, mAb JM-13 did neither bind to other proteoglycans, nor to other basement membrane components like collagen type IV, laminin, or fibronectin. In indirect immunofluorescence on cryostat sections of human kidneys, a restricted staining of tubular basement membranes was observed along with staining of the vascular basement membranes. In the glomerulus, a weak, fine granular staining was seen along the capillary wall and in the mesangium. MAb JM-13 bound also to the basolateral cell membranes of proximal tubular cells, to the cell membranes of cultured human and rat glomerular visceral epithelial cells, rat mesangial cells, human hepatocytes in culture, and in liver cryostat sections, indicating also a recognition of cell surface-associated HS. Pretreatment of the sections with heparitinase abolished binding of JM-13, whereas treatment with chondroitinase ABC had no effect. Inhibition studies in enzyme-linked immunosorbent assay as well as in indirect immunofluorescence corroborated the HS specificity of mAb JM-13. In conclusion, mAb JM-13 binds to an epitope on the HS chains of glomerular, tubular, and cell surface-associated HSPG.     

Production and characterization of a rat monoclonal antibody against leu5 enkephalin

A rat X mouse hybridoma line producing a monoclonal antibody against leu5 enkephalin has been obtained. The monoclonal antibody belongs to the IgG2b class and has an affinity constant of 8.0 X 10(8) M-1 at 4 degrees C. This antibody exhibits approximately 40% cross-reactivity with 1-6 dynorphin but very weak cross-reactivities with met5 enkephalin (1.4%), 1-13 dynorphin (1.3%) and beta-endorphin (0.0045%). The detailed study, by competitive assay, of the interaction between this antibody and various enkephalin derivatives shows that the carboxy-terminal part of the molecule and, particularly, the leu side chain constitutes the immunodominant group. Nevertheless, the tyrosyl residue also contribute considerably to the binding, probably via a peculiar conformation effect, although we can not exclude the tyrosyl residue acting as a secondary contact residue. This monoclonal antibody has been used in a radioimmunoassay to determine leu5 enkephalin like immunoreactive material present in rat brain and hypothalamus.     

Production of a monoclonal antibody to bovine kappa-casein

Caseins (including alpha s1, alpha s2, beta, kappa and gamma casein), a family of phosphoproteins which binds to calcium, are the major proteins in mammalian milk. Kappa-casein, in addition to its calcium binding capacities has an important role in the stabilization of the micelle structure of milk. In the course of studies to investigate the immunologic effects of ingested bovine kappa-casein in IgA deficiency, a hybridoma has been produced that secretes a monoclonal antibody, (IgG1 kappa isotype), which is specific for bovine kappa -casein. The antibody has been characterized by ELISA and it has been shown to bind specifically to bovine kappa -casein. In a sandwich radioimmunoassay, as little as 0.3 x 10(-4) nM/ml of kappa-casein. could be detected. This antibody does not bind to other bovine milk proteins, nor to human casein.     

Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein

Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA.     

Isolation and characterization of a chimpanzee monoclonal antibody to the G glycoprotein of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.     

Production and characterization of a monoclonal antibody to the complement fixation antigen of Coccidioides immitis.

Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgG1) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Ag14 myeloma cells, and the fusion products were screened for IgG antibody to coccidiodin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgG1 Mab was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60 degrees C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, alpha-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.     

Production and characterization of a monoclonal antibody specific to the human 70-kDa heat shock protein

Heat shock protein 70 (hsp 70) plays major roles in apoptosis prevention and thermotolerance as well as molecular chaperoning. It is also expressed on the surface of human tumor cells, but not on normal cells, suggesting that hsp70 may be some tumor-associated antigen. To investigate the diverse functions of the protein species, various types of transgenic mice or cell models overexpressing human hsp70 have been made. In these models a monoclonal antibody (MAb) specific for the human hsp70 is highly desirable to distinguish the human from the endogenous mouse hsp70. It proved difficult to make this species-specific MAb, because the hsp70 homologues are members of a family of highly conserved, abundant, and ubiquitous proteins expressed in organisms ranging from bacteria to humans. In the present study, we prepared four MAbs against human hsp70. Three, HD 5, HD 7 and HD 11, recognize human and mouse hsp70. One, though, HD 8, recognizes human hsp70, but not mouse hsp70. By Western blot analysis of hsp70 deletion mutants, the epitope of the HD 8 MAb was determined as the 585-616 amino acid region of the human hsp70, a region with relatively low homology to mouse hsp70.     

Production and characterization of a monoclonal antibody to partially purified estrogen receptor from human breast tumor

A hybridoma cell line that secretes monoclonal antibody, MAb-ER-Br-1-15-4-18 is established. The MAb is highly specific for estrogen receptor (ER) from human breast tumor cells. In order to raise the antibody, the ER was first isolated from human breast tumor. Mice were immunized with the partially purified ER and the fusion of the spleen cells from the mouse, showing the highest serum titer, with the cells of the NS-1 mouse myeloma line, produced hybrid cells which continuously secreted antibodies specific for ER. Three of the hybridoma cultures which tested strongly positive were cloned using limiting dilution method and one of the cell lines was selected for further study. The recovery of the MAb from the cell culture was done by ammonium sulfate precipitation followed by dialysis and then hydroxylapatite liquid chromatography using linear gradients. The purity of the antibody was checked by polyacrylamide gel electrophoresis. The MAb was isotyped and found to be IgG1. When checked against other antigens the MAb showed a minimal cross-reactivity to ER from rabbit uterus and none to ovalbumin or rat liver ferritin. Further experiments showed that the MAb recognized the ER bound to the hormone and ER in the nucleus of breast tumor cells.