1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。     

Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels—against these antigens from subjects with O. viverrini-positive HBD—higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA > severe HBD > mild HBD > healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.     

金黄色葡萄球菌肠毒素C3单克隆抗体的制备与鉴定

目的制备稳定分泌抗金黄色葡萄球菌肠毒素C3（SEC3）单克隆抗体的杂交瘤细胞株,并对单克隆抗体的性质进行鉴定。方法以SEC3重组蛋白免疫Balb／c小鼠,应用细胞融合技术将小鼠的脾细胞与sR／0骨髓瘤细胞进行融合,经间接ELISA法检测筛选及2次有限稀释法克隆化培养,获得目的杂交瘤细胞株,并对其所产生的单克隆抗体进行效价、亲和常数及抗原识别表位等相关性质的鉴定。结果最终获得了两株能分泌单克隆抗体的杂交瘤细胞1C12和2A2,两者细胞培养上清的效价分别为1：3200和1：1600。经分析可知1C12细胞株的亲和力高于2A2细胞株,同时相加实验表明两个单克隆抗体识别抗原表位相同。结论单克隆抗体制备成功,为进一步完善肠毒素SEC3的临床检测奠定了基础。     

Immunofluorescent staining method for use with monoclonal antibodies

This note describes an immunofluorescent staining method for cells in the S-phase which have been allowed to take up bromodeoxyuridine into their DNA in place of thymine. The technique involves the use of fluorescinated monoclonal antibodies against bromodeoxyuridine and allows rapid and accurate estimation of cells in the S-phase, the technique does not require interpretation by skilled technicians.     

Specific detection of the Andean strain of potato virus S monoclonal antibodies

Four mouse monoclonal antibodies (MAbs) specific for the Andean strain of potato virus S (PVS^A) were produced. The MAbs reacted with four isolates of PVS^Abut did not react with four isolates of ordinary strain of PVS (PVS^O). The MAbs did not react with six other members of the Carlavirus group including potato virus M. A MAb-based ELISA, using MAbs (IEB-1 and IEB-4-AP), was devised and shown to specifically detect PVS^A.     

Production and partial characterization of monoclonal antibodies for detection of entomopoxvirus from Melanoplus sanguinipes

Entomopoxviruses (EPV) are currently being considered as candidate grasshopper (Orthoptera: Acrididae) microbial control agents. Classical techniques for diagnosing infections in grasshoppers are laborious, time consuming, and sometimes inaccurate. Specific murine monoclonal antibodies were developed against an EPV from Melanoplus sanguinipes (Fab) for use in a nitrocellulose-based enzyme-linked immunoassay (dot-blot). An IgG_2b monoclonal antibody was used to diagnose infections in grasshoppers 13 days following injection with virions. Of 25 grasshoppers that had patent infections microscopically, 22 produced positive results on the dot-blot. In a second test, 39 patently infected grasshoppers all produced positive results. Seven additional grasshoppers in the first test and 2 in the second test gave positive reactions in the dot-blot method but virus was not detected upon microscopic examination. The monoclonal antibody did not cross-react with other commonly occurring grasshopper pathogens. The dot-blot method detected as few as 2.5×10⁶ purified EPV virions. The improvement over existing detection techniques should facilitate evaluation of EPV for field use.

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Prodution et caractérisation partielle d'anticorps monoclonaux pour la détection d'entomopoxyvirus de Melanoplus sanguinipes

Résumé Les criquets sont très nuisibles aux pâturages de l'Ouest des USA et du Canada. Les méthodes classiques de protection sont basées sur les traitements chimiques lors des pullulations. Un entomopoxvirus (EPV) extrait de M. sanguinipes est généralement considéré comme un outil, pour le contrôle des populations sur une longue période, en vue de la suppression des criquets. Les méthodes actuelles d'isolement de EPV sont longues, pénibles et peu fiables. Les tests d'adsorption des antigènes sur l'anticorps fixé et le dosage per l'anticorps enzymatiquement marqué sont efficaces, sûrs et donnent à temps des résultats pour déceler des entomopathogènes. Nous avons produit des anticorps monoclonaux de souris contre EPV de M. sanguinipes, et les avons utilisés dans des dosages immunoenzymatiques d'extraits protéiques adsorbés sur nitrocellulose. Les EPV sont recherchés sur des criquets injectés de virions 13 jours avant. Sur 25 criquets qui présentaient des infections nettes lors d'examens microscopiques, 22 ont donné des résultats positifs par sérologie. Dans un second test, sur 38 criquets nettement contaminés, tous ont donné des résultats positifs; 7 criquets suppl'ementaires mentaires dans le premier test et 2 dans le second test ont donné des résultats positifs en sérologie alors que l'examen microsopique n]avait pas test ont donné des résultats positifs en sérologie alors que l'examen microsopique n'avait pas révélé de virus. Ceci montre que ce type de détection est plus sûr que la méthode ordinaire. Les anticorps monoclonaux ne donnent pas de réactions avec les autres pathogènes courants des criquets. La méthode sérologique a permis de détecter même une concentration de 2,5×10⁶ virions EPV.

     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Probing eukaryotic RNA polymerases B with monoclonal antibodies

Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora comata were selected on the basis of different subunits recognition and inhibitory effect on enzyme activity. A library of 10 antibodies biased toward B180, B145, B39, B23,5 and B11 subunits was constructed. Most of these antibodies also recognize yeast, wheat germ and calf thymus RNA polymerase B. Subunits bearing antigenic determinants are not always homologous in Podospora and yeast enzyme. As some of these antibodies strongly inhibit enzyme activity they constitute potent probes for functional studies of corresponding subunits.     

Preparation monoclonal β-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique

Abstract

Immunoassay has been widely used in the screening of mycotoxins, which may be hazardous to the operator or the environment. This study was to develop a green way to measure zearalenone (ZEN) with a monoclonal β-type anti-idiotype antibody (Ab2β) against ZEN in place of ZEN standard. Six monoclonal β-type anti-idiotype antibodies were prepared. The 50% inhibitory concentration (IC₅₀) value to ZEN of the six antibodies was between 34.45?±?1.12–182.12?±?15.40?nM. A green ELISA was then developed and validated. The quantitative conversion formula between ZEN and the monoclonal Ab2β against ZEN was y?=?0.092x^0.722, R² = 0.990. The working range was 2.63–100.64?ng ml^?1. The recovery rate in spiked feed samples was from 82.15% to 102.79%, and the within-assay and between-assay coefficient variation (CV) level were less than 10.00%. A good correlation was obtained by high-performance liquid chromatography method (HPLC) to validate the developed method.     

Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA.

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

Preparation and characterization of monoclonal antibodies to human hexokinase type I

1. Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I. None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis. 2. All seven monoclonals recognize the two different subtypes of human hexokinase I equally well. Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides. 3. Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

Preparation and preliminary application of monoclonal antibodies against Trichokirin-S1, a small ribosome-inactivating peptide from the seeds of Trichosanthes kirilowii

Trichokirin-S1,a small ribosome-inactivating peptide recently purified from the seeds ofTrichosanthes kirilowii,has potential clinical applications because of its small molecular mass.Two stablestrains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) againstTrichokirin-S1 have been developed using the hybridoma technique.The isotypes of these two mAbs,1F11and 2A5,were determined to be IgG_(2a) and IgG_1,respectively.The affinity constants,which were measuredby non-competitive ELISA,were found to be 2.3×10~8 M~(-1) and 2.8×10~8 M~(-1),respectively.An immunoaffinitymethod using 2A5-coupled Sepharose 4B was successfully developed to purify Trichokirin-S1.These twoantibodies have also been used to detect Trichokirin-S1 in Western blot.     

Mouse monoclonal antibodies to swine blood group antigens

Eight mouse hybridomas with haemagglutination capacity to swine blood group antigens were obtained, three of them producing antibodies capable of being used as blood group reagents. Two detected the Ba factor and another the Fa factor. The others gave non-specific and weak reactions or cross-reaction with antigens present in more than one system. We conclude that mouse monoclonal antibodies are also suitable for use in swine as a complement of polyclonal reagents.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Linking radiosilver to monoclonal antibodies reduced by ascorbic acia

Radiosilver-111 and Radiogold-199 were proposed by us (1) as suitable isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111T _1/2=7.45 d and Au-199T _1/2=3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dialysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1∶1, 1∶2, 1∶5, 1∶10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver efflux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The comparative results with the two techniques are described.