Quantitative Determination of a Peanut Anionic Peroxidase by Specific Monoclonal Antibodies

A monoclonal antibody (46-12-C12) for use in a solid-phase enzyme-linkedimmunosorbent assay (ELISA) specific for an anionic peroxidase(APRX) from peanut (Arachis hypogaea L.) suspension cell mediumwas developed. The McAb (IgG₁) had a high affinity (2.77 ? 10¹¹)and specificity for APRX, and showed only weak interaction witha-amylase and virtually no reactivity with other enzymes, suchas MCPRX (peanut), minor CPRX (peanut), peroxidase (horseradish),RuBP case (spinach), -glucosidase (rice), ß-glucosidase(almonds), acid phosphatase (potato), catalase (bovine liver)and glucose-6-phosphatase (yeast). Sample dilution curves werefound to parallel the standard curve. The detection limit was0.002 ? 10^–12 mol APRX. The absorbance was linear at concentrationsbetween 0.004–24 ? 10^–12 mol APRX. Three hundredsamples could be analysed per day by one person, with a semi-automaticperformance. Using this assay, levels of APRX have been determinedin a number of biological extracts of different origin. Key words: Peanut, anionic peroxidase, monoclonal antibody, enzyme-linked immunosorbent assay (ELISA)     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Identification of an Antigenic Determinant on Anionic Peanut Peroxidase by Monoclonal Antibodies

Two monoclonal antibodies (46–12-C12 and 23–6-C12)raised against anionic peanut peroxidase were found to haveindependent epitope sites. These topographic sites were foundto be located within a tryptic glycopeptide (Atgp) from theanionic isozyme by both indirect and non-competive ELISA andWestern blotting. The Atgp has a M_r equal to 11 000 of which 70% is carbohydrateand the peptide is probably highly hydrophobic as determinedby its high R_F (0.83) value and the amino acid composition.McAb 23–6-C12 recognized a contiguous epitope which encompassedalso the sole N-linked oligosaccharide on the anionic isozyme.That the monoclonal antibody also recognized the oligosaccharideon the -amylase, ß-glucosidase, acid phosphatase,and horse-radish peroxidase may be related to similarities insugars. Sugar removal from the Atgp or from the cross-reactivepeptide of enzymes caused loss of antibody affinity. The monoclonal antibody 46–12-C12 recognized specificallya conformational epitope near the region of the cysteine, tryptophaneand methionine residue on Atgp. Digestion of the anionic isozymeby trypsin resulted in a 40-fold loss of affinity with thismonoclonal antibody. Moreover, treatment of the Atgp with performicacid or trifluoromethane sulphonic acid caused a loss of affinitybetween the treated Atgp and this monoclonal antibody. Key words: Monoclonal antibodies, peanut, anionic peroxidase, glycopeptide, trypsin digest     

Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B^-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B.     

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules

Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.     

Production and Characterization of Monoclonal Antibodies Against Myelin-Associated Glycoprotein

Monoclonal antibodies against myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen lymphocytes from BALB/c mice immunized with human myelin-associated glycoprotein purified from CNS myelin. Three groups of antibodies were identified: IgG antibodies recognizing the polypeptide moiety and IgG and IgM antibodies recognizing the carbohydrate moiety of the intact molecule. Properties of these antibodies were examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the immunostaining technique using human CNS and peripheral nerve myelin, and ganglioside fractions isolated from human brain and peripheral nerve, and with immunohistochemical staining of human peripheral nerves. Part of human peripheral blood mononuclear cells was stained with the antibodies against the carbohydrate moiety, but not with IgG antibodies recognizing the polypeptide moiety. Natural killer activity was partially reduced after treatment of human peripheral blood lymphocytes with an IgM antibody and complement in vitro. The possibility that anti-myelin-associated glycoprotein antibodies might play a role in the pathogenesis of demyelinating diseases through modification of natural killer activity is discussed.     

牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定

为制备牛病毒性腹泻病毒(BVDV)糖蛋白E2单克隆抗体(MAb),利用原核表达并且纯化的重组糖蛋白E2(rE2)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合.采用以BVDV为检测抗原的间接ELISA筛选阳性细胞克隆,经3次克隆纯化后获得2株稳定分泌抗E2特异性MAb的杂交瘤细胞株,分别命名为4E3与1G11.用4E3与1G11杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rE2及BVDV包被的ELISA测得的效价分别是6.21×106和6.83×105及6.83×105和7.5×104.间接ELISA、Western blot、IFA试验表明两株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性.经抗体亚类鉴定4E3与1G11均为IgM/K.特异性试验表明4E3与1G11这2株MAb均不与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛腺病毒3型反应;其中4E3不与猪瘟病毒反应,而1G11则可与猪瘟病毒发生交叉反应,这种反应特性可试用于BVDV与猪瘟病毒的鉴别诊断.所制备的4E3与1G11 MAb可以用于BVDV抗原的检测,为建立检测BVDV E2蛋白血清抗体的ELISA奠定了基础.     

Production and Characterization of Monoclonal Antibodies to Potato Virus A

Abstract Purified potato virus A (PVA) was used for immunization to produce monoclonal antibodies (MAb). The type of ELISA with purified PVA or non–purified PVA, played an essential role in selecting MAb with different specificity.
Two MAb's (MAb–1 and MAb–2) were selected, using indirect ELISA (I–ELISA) with purified PVA. Competition experiments suggested that MAb–1 and MAb–2 reacted with the same epitope on purified PVA (epitope 1). ELISA, IEM and SDS–PAGE–immunoblotting experiments showed that epitope–1 was only present on purified PVA but not on non–purified PVA, suggesting that this epitope was introduced during the purification. Assays at different steps during purification indicated that epitope–1 was only exposed after plant components and reducing agents were removed from the PVA extract.
Three MAb's (MAb–3, MAb–4 and MAb–5) were selected by indirect double antibody sandwich ELISA (IDAS–ELISA) with non–purified PVA. These MAb's reacted in I–ELISA or IDASELISA with purified PVA as well as with non–purified PVA and might be useful for routine diagnosis. MAb–3, 4 and 5 cross–reacted with some other potyviruses in I–ELISA and in IDAS–ELISA. MAb–1 cross–reacted with 5 out of 7 other potyviruses in I–ELISA, but not in IDAS–ELISA.     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P₂ isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N₂ fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P₂ but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P₁ isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P₁ was three orders of magnitude lower than for AAT-P₂. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P₂.     

Characterization of Monoclonal Antibodies against Etiological Agents of Weil's Disease

Monoclonal antibodies against etiological agents of Weil's disease were produced by cell fusion technology. Twenty hybridomas were produced through the fusion of P3×63Ag8.653 cells with spleen cells from BALB/c mice immunized against Leptospira interrogans serovar icterohaemorrhagiae RGA strain and serovar copenhageni Shiromizu and M20 strains. Reactivities of the antibodies produced by the hybridomas were determined by the microscopic agglutination test. Among the five hybridoma antibodies to the RGA strain, two reacted specifically to serovar icterohaemorrhagiae, two reacted to serovar icterohaemorrhagiae at a high titer and serovar copenhageni at a low titer, and one reacted to serovars icterohaemorrhagiae, copenhageni, pyrogenes, and canicola. Of the ten hybridoma antibodies to the Shiromizu strain, one reacted specifically to serovar copenhageni, seven reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, and two exhibited intermediate properties. Of the five hybridoma antibodies to the M20 strain, three reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, one reacted to serovar copenhageni at a low titer and serovar icterohaemorrhagiae at a high titer, and one reacted to serovars copenhageni, icterohaemorrhagiae, and pyrogenes. The results revealed that each serovar has its own antigen(s) and their common antigens. In addition, 20 strains of leptospires were recently isolated and tested with three monoclonal antibodies characterized by different reactivities. Twenty strains were clearly identified by their antibodies, i.e., 16 strains were identified as serovar icterohaemorrhagiae and three strains were identified as serovar copenhageni. The remaining strain, which was not agglutinated by three antibodies, was identified as serovar autumnalis by an agglutination test with immune rabbit sera.     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

抗HCMV Pp65蛋白单克隆抗体的制备及性质研究

人类巨细胞病毒（Human cytomegalovirus,HCMV）是一种机会性感染疱疹病毒,在人群中感染比较常见,但在免疫缺陷个体与新生儿中可引起严重的疾病。HCMV Pp65是HCMV活动感染的主要标志,也是临床检验检测HCMV感染的重要靶标。为研发抗HCMV Pp65蛋白单克隆抗体作为临床免疫检测HCMV感染的关键原料,本研究采用重组表达的HCMV Pp65蛋白免疫BALB/c小鼠,将免疫小鼠淋巴结细胞与sp2/0细胞融合,采用间接ELISA法筛选阳性克隆与效价测定,再用Western blotting进行抗体特异性鉴定,最后用免疫捕获PCR和免疫荧光法评价其应用前景。最终获得了1株能稳定分泌高效价的抗HCMV Pp65单克隆抗体的杂交瘤细胞株,命名为8D6。Western blotting及间接ELISA检测其效价分别达1∶4 000和1∶105;细胞免疫荧光与免疫捕获PCR实验结果说明,该杂交瘤细胞株分泌的单克隆抗体具有良好的亲和力和特异性,具有作为外周血细胞免疫荧光细胞化学和免疫捕获PCR检测HCMV临床感染的关键原料,也可作为双抗体夹心法检测HCMV临床感染的关键原料,为建立HCMV感染快速灵敏的临床诊断试剂打下了重要的基础。     

Production and Use of Monoclonal Antibodies against Rice Embryo Lipoxygenase-3

A DNA fragment coding for a part of a putative phosphatidylinositol 3 kinase was cloned from Schizosaccharomyces pombe by cross-hybridization with Saccharomyces cerevisiae VPS34 gene, a yeast homologue of mammalian PI-3 kinase. The clone contained an open reading frame of 797 amino acids but lacked the initiation codon, ATG. The predicted amino acid sequence was homologous to those of S. cerevisiae VPS34 and mammalian PI-3 kinase genes. Disruption of the gene resulted in extremely low levels of PI-3-P and higher levels of PI-4-P, supporting the idea that the gene codes for the PI-3 kinase of S. pombe. The disruptants harbored large vacuoles and were sensitive to stresses such as high temperature or high concentration of monovalent and divalent cations.     

A Retrospective Look at the Cationic Peanut Peroxidase Structure

ABSTRACT:?

The cationic peanut peroxidase has been studied in detail, not only with regard to its peptide structure, but also to the sites and role of the three moieties linked to it. Peanut peroxidase lends itself well to a close examination as a potential example for other plant peroxidase studies. It was the first plant peroxidase for which a 3-D structure was derived from crystals, with the glycans intact. Subsequent analysis of peroxidases structures from other plants have not shown great differences to that of the peanut peroxidase. As the period of proteomics follows on the era of genomics, the study of glycans has been brought back into focus. With the potential use of peroxidase as a polymerization agent for industry, there are some aspects of the overall structure that should be kept in mind for successful use of this enzyme. A variety of techniques are now available to assay for these structures/ moieties and their roles. Peanut peroxidase data are reviewed in that light, as well as defining some true terms for isozymes. Because a high return of the enzyme in a pure form has been obtained from cultured cells in suspension culture, a brief review of this is also offered.     

Production and Preliminary Application of Monoclonal Antibodies Against Paulownia Witches' Broom MLO

Monoclonal antibodies against mycoplasma-like organisms (MLO) associated with paulownia witches’broom (PWB) were produced by using partially purified preparations from diseased paulownia. Splenic cells from immunized mice were fused with sp2/0murine myeloma cells. Screened by indirect ELISA using partially purified PWB-MLO and healthy paulownia extracts as detecting antigens, two hybridoma clones that stably secreted specific antibodies against PWB-MLO were obtained from 459 clones of four successful fusions. The monoclonal antibodies were isotyped and determined to be immunoglubin classes IgG2a and IgG3. Antibody titers of ascitic fluids were both over 1.6 × 104 assayed by indirect ELISA. Priliminary application on several specimens proved that they were the monoclonal antibodies against PWB-MLO.     

环磷酸鸟苷(cGMP)单克隆抗体杂交瘤细胞系的建立及其特性的鉴定

<正> 自从1975年单克隆抗体技术建立以来,已被应用在各个生物学领域中,国内外都尚未建立抗cGMP单克隆抗体的杂交瘤细胞株。我们为了获得更纯,特异性更高的抗cGMP抗体,对此项工作进行了探索,已筛选出能分泌抗cGMP抗体的杂交瘤细胞株,并进行了初步鉴定;现将工作简报如下: 用琥珀酰环磷酸鸟苷(ScGMP)与牛血清白蛋白相连后的化合物(ScGMP-BSA)为抗原免疫BALB/c小鼠,每月一次,共四次,最后一次为尾静脉连续注射三天,第四天将免疫