A new monoclonal antibody detecting ochratoxin A at the picogram level

Á. GYÖNGYÖSI-HORVÁTH, I. BARNA-VETRÓ AND L. SOLTI. 1996. A monoclonal antibody against ochratoxin A was produced after immunization of Balb/c mice with ochratoxin A-BSA. This antibody was of the IgGl heavy chain subclass with a k type light chain. The 50% inhibition value was 0.45 ng ml^-1 in a direct competitive ELISA and the detection limit was 42 pg ml^-1. This antibody is very specific, cross-reacting only with ochratoxin B (9.3%).     

Production of antibody against ochratoxin A.

Antibody against ochratoxin A was obtained after repeated injection of different protein-ochratoxin A conjugates to rabbits. Among many protein-ochratoxin conjugates tested, bovine serum albumin-ochratoxin A was found to be the best antigen. The antibody is specific for ochratoxins A, C, and T, but is not specific for ochratoxins B, alpha, and other coumarin derivatives. The sensitivity for ochratoxin A detection using a binding assay is in the range of 0.5 to 10 ng/0.5-ml sample. Detailed methods for the preparation of protein-ochratoxin conjugates, preparation of immune serum, and methods for antibody titers are described.     

A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

A monoclonal antibody to free N-acetylneuraminic acid

A monoclonal antibody (70-A) to free N-acetylneuraminic acid was obtained by immunizing mice with its synthetic beta-glycoside, sodium O-[(5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2- nonulopyranosyl)onate]-(2----3)-1,2-di-O-tetradecyl-sn-glyce rol, followed by fusing the isolated spleen cells with mouse myeloma cells and cloning positive fusions. 70-A reacted with various synthetic beta-glycosides of N-acetylneuraminic acid and also with cytidine-5'-monophosphate-N- acetylneuraminic acid, known as its sole naturally occurring beta-glycoside. The inhibition assay showed that N-glycolylneuraminic acid had slightly lower reactivity than N-acetylneuraminic acid, but other monosaccharides tested, such as N-acetylglucosamine, N-acetylgalactosamine or N-acetylmannosamine, had no reactivity toward 70-A. Reactivity of 70-A with free N-acetylneuraminic acid was confirmed by measuring the specific binding of N-[14C]acetylneuraminic acid to the antibody. The association constant of 70-A with N-acetylneuraminic acid was determined to be 5.96.10(4) M-1 by equilibrium dialysis.     

A monoclonal antibody directed to Tn antigen

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.     

A monoclonal antibody to human insulin receptor

A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG₁ (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated ¹²⁵I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity.     

Development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody

Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.     

A monoclonal antibody to rat liver ornithine decarboxylase

A monoclonal antibody was obtained against rat liver ornithine decarboxylase by using hybridoma technology with a small amount of partially purified enzyme. The antibody, IgG1 of kappa-type, was affinity-purified to homogeneity from culture supernatants of hybridoma cells. While the antibody had no inhibitory effect on ornithine decarboxylase activity when tested alone, it precipitated up to 87 units (60 ng) of the enzyme per microgram in the presence of formalin-fixed Staphylococcus aureus Cowan I bacteria. Immunoadsorption on a column of the monoclonal antibody-Sepharose 4B was shown to be useful for the removal of ornithine decarboxylase from antizyme inhibitor preparations, an essential procedure for the accurate assay of either ornithine decarboxylase-antizyme complex or antizyme inhibitor. It was also shown that antizyme could be affinity-purified by using a column of the monoclonal antibody-Affi-Gel 10 to which ornithine decarboxylase had been bound.     

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol (DAS) was produced by a hybridoma, designated 2E5. It secreted antibody of the IgGl subclass and had a detection limit for DAS of 16 ng/ml with a direct enzyme immunoassay on a double antibody solid phase. The relative cross-reactivities with 3α-acetyl-DAS, diacetylverrucarol, neosolaniol, T-2 tetraol tetraacetate, fusarenon X, T-2 toxin, and HT-2 were 2224·5, 53·7, 13·9, 9·2, 6·4, 1·7, 0·6, and 0·35%, respectively.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

A monoclonal antibody specific to zeatin o-glycosyltransferases of phaseolus

Zeatin O-xylosyltransferase and zeatin O-glucosyltransferase occur in immature embryos of Phaseolus vulgaris and P. lunatus, respectively. Purified preparations of the xylosyltransferase were used as antigen to elicit the formation of antibodies in mice. Hybridoma clones were produced by fusion of mouse spleen cells with myeloma cell line Fox-NY. A clone secreting monoclonal antibody (MAb), XZT-1, capable of immunoprecipitating both enzymes was obtained. The MAb detected a unique protein band from crude embryo extracts of each species with the correct molecular mass (50 kilodaltons) and relative charge (R_F = 0.5 and 0.3) of the respective enzymes. Competition experiments with substrates indicated that the glycosyl dinucleotide binding sites of the enzymes are probably not involved in MAb-enzyme recognition. Western blotting of samples from vegetative tissues of P. vulgaris detected a low level of O-glucosyltransferase but not O-xylosyltransferase, in leaves. These findings suggest the occurrence of two genes in P. vulgaris coding for O-glycosylation enzymes with tissue-specific expression. The MAb will be used to screen expression libraries and to obtain pure enzymes for amino acid sequencing and for the production of additional MAbs.     

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.     

浅谈单克隆抗体

简要介绍了抗体及单克隆抗体的概念,并阐述了单克隆抗体的制备原理,制备方法及主要应用领域。     

A monoclonal antibody to Mycobacterium kansasii: preparation and properties

An IgG2/kappa monoclonal antibody specific for a subunit protein antigen (mol.w. 32 KD) obtained by means of gel chromatography of the cell-free sonicate of M. kansasii was generated by the fusion of spleenocytes from BALB/c mice immunized with this antigen and Ag-8 myeloma cells. Its specificity was demonstrated by ELISA and dot blot procedures. This MoAb has potential application in taxonomy and species identification of M. kansasii isolates, in studies relating to the molecular pattern of the bound antigen and in serodiagnostics of the mycobacterial disease due to M. kansasii.     

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.     

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.     

Binding of monoclonal antibody A2B5 to gangliosides

Monoclonal antibody A2B5 (Eisenbarth et al, Proc. Nat. Acad. Sci. (1979, 76:4913-4917), which reacts with neurons, thymic epithelium and peptide-hormone secreting cells of several species, was reported to react specifically with brain tetrasialogangliosides. We have found that A2B5 binds to gangliosides GQ1b, GD3, GD2, disialolactoneotetraosylceramide, and probably to GT1a, when assayed by an immunostaining procedure that detects binding of antibody to gangliosides on a thin-layer plate. Additional data obtained by complement fixation revealed that this antibody reacted most strongly with ganglioside GQ1b almost as well with disialogangliosides GD3, GD2 and disialolactoneotetraosylceramide, weakly with GD1b and GT1b, and very weakly with GM3 and GD1a. These data indicate that A2B5 cannot be regarded as a specific reagent for the recognition of tetrasialogangliosides.     

A monoclonal antibody to an oocyte-specific poly(A) RNA-binding protein

Xenopus oocyte-specific poly(A) RNA-binding proteins were isolated and used to prepare monoclonal antibodies. One antibody was used to characterize one particular antigen by immunoblot analysis. The antigen had a molecular weight of 56,000 was oocyte-specific, and decreased in amount during oogenesis. The antigen was localized in the cytoplasm throughout oogenesis and sedimented mainly at 40-60 S. The antigen also was shown to bind poly(A) RNA following chromatography of ribonucleoprotein particles on oligo(dT)-cellulose. The antibody was used to immunoadsorb nontranslating ribonucleoprotein particles. Fifty-five per cent of the poly(A) RNA sedimenting between 40-60 S was shown to be bound by the antigen. The further use of this antibody in attempting to examine other components of the ribonucleoprotein particle is discussed.     

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.