抗磺胺对甲氧嘧啶单克隆抗体的研制及其特性分析

目的制备针对磺胺对甲氧嘧啶的单克隆抗体,建立对该物质的免疫学检测方法。方法以BSA-磺胺对甲氧嘧啶为免疫原,免疫BALB／c小鼠,取脾细胞与小鼠Sp-2／0骨髓瘤细胞融合后,经筛选和亚克隆,建立杂交瘤细胞株。结果获得2株能稳定分泌抗磺胺对甲氧嘧啶抗体的细胞株。对抗体进行了特性分析,抗体的效价分别为1：400000和1：1630000,抗体类型及亚类都为IgGl。其中,单克隆抗体1H10的亲和力为1．4×109L／mol,利用该抗体采用竞争间接ELISA法检测磺胺对甲氧嘧啶的范围是1025—16μg/mL,最低检测浓度是8μg/mL。单抗1H10与其他6种磺胺药（SMM、SMZ、SM2、SD、SulfaquinoxalineSodium、Sulfametetyrazine）无交叉反应。结论单克隆抗体1H10可用于研制免疫学方法检测磺胺对甲氧嘧啶残留的产品。     

Preparation and characterization of monoclonal antibodies against human myeloperoxidase

We established 11 hybridomas producing monoclonal antibodies (MoAbs), designated AM, against human myeloperoxidase (MPO), by immunizing mice with the three forms of MPO (I, II, and III) purified from healthy human polymorphonuclear leukocytes (PMN) and characterized the specificity of the AM MoAbs. Ten of the AM MoAbs reacted similarly to each of the three forms using an enzyme-linked immunosorbent assay. When a cetyltrimethylammonium bromide (CETAB) extract of human PMN was electrophoresed in a CETAB polyacrylamide gel and transferred to a nitrocellulose filter, IgG1 class AM MoAbs immunostained only the MPO band of the proteins of the extract. In addition, the AM MoAbs reacted to two radioactive bands of 94 and 92 kDa in a HL-60 cell lysate labeled with [35S]methionine for 1 h. After a chase period of 24 h, these bands were replaced by four radioactive bands of 64.5, 43, 16.7, and 13.4 kDa, demonstrating that the MoAbs recognize not only mature MPO but also the MPO precursors of 94 and 92 kDa. The data also indicated that the two major bands of 64.5 and 13.4 kDa corresponded to heavy and light chains of mature MPO, respectively, and the additive intermediate bands of 43 and 16.7 kDa were MPO-related proteins. Moreover, AM MoAbs reacted to a similar extent to the deglycosylated form of MPO III with endo-beta-N-acetylglucosaminidase H (Endo-H). Thus, IgG1 class AM MoAbs recognized MPO with high specificity and reacted to the structure which is commonly conserved in the three mature forms of MPO (I, II, and III), MPO precursors, and deglycosylated MPO with Endo-H. AM MoAbs also specifically reacted to PMN and/or monocytes but did not react to lymphocytes when the cell staining method was used.     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed inE. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoclonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native luciferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

Preparation and characterization of polyclonal antibodies against human chaperonin 10

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.     

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.     

Preparation and characterization of monoclonal antibodies against soybean seed lipoxygenase isoenzymes

Sets of monoclonal antibodies have been prepared using two soybean seed lipoxygenase isoenzymes as the antigens. The antibodies were characterized by ELISA, Western blot analysis, immunoprecipitation, and in kinetic assays. Several antibodies displaying selectivity for the two closely related polypeptides were obtained, while the majority of the antibodies generated were crossreactive. Antibodies specific to the native and denatured forms of the two proteins were also obtained. Two of the monospecific antibodies were shown to immunoprecipitate the appropriate isoenzyme selectively from a mixture. When these antibodies were immobilized on agarose, they were successful in the immunoaffinity purification of the individual isoenzymes. In kinetic experiments certain antibodies were found to influence catalysis upon incubation with lipoxygenase. Antibodies which both inhibited and stimulated catalysis were identified.     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

Preparation and characterization of anti-paroxetine antibodies

6-nitroparoxetine was synthesized and reduced to 6-aminoparoxetine. After coupling to glutaraldehyde at the 6-position and to bovine serum albumin, the resulting Schiff's base was further reduced into an amino-derivative which served as the antigen. Anti-paroxetine antibodies were raised against this antigen in rabbits and the anti-paroxetine IgG purified by Protein A affinity chromatography. The anti-paroxetine IgG demonstrated high specificity towards paroxetine and 6-nitroparoxetine without significant cross-reactivity with other commonly used antidepressant and neuroleptic drugs. These antibodies may be useful for both plasma paroxetine level assays and uptake inhibitor binding site studies.     

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha)

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.     

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.     

Production and characterization of antibodies against microcystins

Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively.     

Development and characterization of antibodies against aflatoxins

A panel of ten monoclonal antibodies against aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant regions of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab-fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.     

Monoclonal antibodies against the plant cytokinin isopentenyl adenosine

Sixteen monoclonal antibodies against isopentenyl adenosine (iPA) were developed and characterized for reactivity towards this cytokinin and structurally related molecules by use of a competition fluorescence enzyme immunoassay. Antibodies with suitable affinity and specificity were used in an immunoassay to detect and quantify isopentenyl adenosine. Logit/log plots of fluorescence vs pmol of the cytokinin indicated that the assay had a measurement range of 0.03–256 pmol (10–8500 pg) with high linearity (r =–0.98). This competition fluorescence enzyme immunoassay showed that three antibodies cross-reacted with N-6-benzyladenine and its riboside: cross-reactivity with dihydrozeatin and its riboside, cis -zeatin, trans -zeatin riboside, adenine and adenosine was minor. When an immunoaffinity-HPLC technique was used to measure the cross-reactivity of two of the antibodies in the presence of known quantities of multiple cytokinins, iPA was bound in preference to the other cytokinins. One of the antibodies was used to quantify this cytokinin in developing wheat ( Triticum aestivum L. cv. Stephens) seeds and in wheat seeds spiked with known amounts of certain other cytokinins. The use of these antibodies in immunoassay in combination with HPCL for quantification of iPA in plant extracts is discussed.     

Production and characterization of antibodies against nivalenol tetraacetate

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.     

Preparation of monoclonal antibodies against a poly(U), poly(C) specific ribonuclease prepared from the insect Ceratitis capitata.

A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。