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 characterization of monoclonal antibodies against adenylate kinase

Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAMD8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 × 108 M-1 and 9.6 × 108 M-1, while their interactions to AK in solution are much weaker, with affinity constants of 7.0 × 104 M-1 and 3.9×106M-1, respectively. Thus, McAb3D3 and McAMD8 react preferentially to the immobilized AKs. Since pro-teins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAMD8 are directed against non-native AK.     

高特异性抗桔霉素单克隆抗体的制备及鉴定

以1, 4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白偶联抗原CIT-BSA,将偶联抗原免疫BALB/C小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株稳定分泌抗桔霉素抗体的杂交瘤细胞株H2-F8．该单克隆抗体经过初步鉴定,抗体类型为IgM类,抗体的相对亲和力为4.17×10⁸ L/mol,单抗与黄曲霉毒素B1、赭曲霉毒素A、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等毒素的交叉反应率均低于0.1%,与红曲色素中的橙色素和红色素的交叉反应率均低于0.01%．在此基础上建立了间接竞争ELISA检测方法,线性范围为0.05～1.0 μg/L,IC₅₀值为0.3 μg/L．结果为快速检测桔霉素的酶联免疫检测方法的建立和检测试剂盒的研制提供技术依据．     

抗重金属汞离子抗体的制备及鉴定

汞、镉、铅等重金属引起的环境污染已在世界范围内造成危害。快速、廉价地监测生境中重金属是减小其对人类及动物危害的先决条件。传统检测方法无法满足高通量的现场检测,建立更快速、更经济的免疫分析法检测汞离子是生产及经济发展的需要。本研究中,报道了汞特异性单克隆抗体的制备与筛选方法和结果。因Hg2+太小以至于不能引起免疫反应,所以用螯合剂(二乙烯三胺五乙酸,DTPA)将金属离子与载体蛋白(匙孔血蓝蛋白,KLH)连接起来。成功合成、鉴定汞复合物抗原后,免疫BALB/c小鼠,通过细胞融合获得了稳定分泌抗体的杂交瘤细胞。用极限稀释法亚克隆,通过ELISA筛选,获得了2株稳定分泌抗汞离子抗体的细胞株(H2H5,H1H8)。小鼠腹腔注射1×107H2H5、H1H8细胞株制备腹水,腹水抗体效价都在1∶51200以上。经鉴定两株杂交瘤均为IgG1亚类,轻链为kappa型且分泌抗体稳定性较好。实验结果为汞离子残留免疫学检测方法的建立提供了技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。     

鼠冠状病毒核衣壳蛋白的抗体制备与抗原决定簇分析

核衣壳(nucleocapsid,N)蛋白有稳定病毒基因组、调控病毒复制及细胞状态的特殊作用。鼠肝炎病毒(murine hepatitis virus,MHV)为乙型冠状病毒属的原型病毒,是研究冠状病毒N蛋白功能的经典模型。本研究用去污剂处理鼠冠状病毒粒子暴露N蛋白,另用原核表达纯化的重组N蛋白分别免疫小鼠,制备多克隆及单克隆抗体。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹分析结果显示两类抗体均具有高灵敏度和特异度,与甲型冠状病毒猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)的N蛋白无交叉反应。原核表达缺失突变的N蛋白分析结果显示,多克隆抗体与单克隆抗体2E6识别的鼠冠状病毒N蛋白抗原决定簇完全一致,位于N端结构域(N-terminal domain,NTD)C端与SR之间的58个氨基酸残基内。此外,基于单克隆抗体2E6的ELISA及免疫荧光法能检测到感染细胞中和培养上清液中的N蛋白组分,且其含量与病毒复制的滴度一致。这些结果表明,鼠冠状病毒复制过程中粒子与细胞中的N蛋白可能维持相似的结构,使NTD与SR之间的部分氨基酸残基一直暴露在表面,从而形成了优势抗原决定簇。     

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 epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed in E. 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 monoelonal 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 lueiferase, 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 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 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.     

Structural analysis of the xyloglucan from Phaseolus coccineus cell-walls using cellulase-derived oligosaccharides

Oligosaccharides, obtained by digestion of a xyloglucan from the cell walls of Phaseolus coccineus with cellulase, have been isolated by gel filtration. Oligosaccharides containing 2–6 residues accounted for ～57% of the hydrolysate, with larger oligosaccharides (d.p. ～10) and partially degraded xyloglucan accounting for ～32% of the polymer. The major glycosidic linkages were determined by methylation analysis. Methylated penta- and hexa-saccharide alditols were isolated by reverse-phase h.p.l.c. and characterised by e.i.-m.s. and f.a.b.-m.s. Methylated derivatives of the di-, tri-, and tetra-saccharide alditols were examined by g.l.c.-m.s. in the e.i. and c.i. modes. A structure based on these results is proposed.     

Preparation and characterization of monolconal antibodies against the human thrombin-antithrombin III complex

Monoclonal antibodies were raised against human thrombin-antithrombin III complex by a hybridoma technique. Among them, five monoclonal antibodies, designated as JITAT-4, -14, -16, -17 and -19, were found to react with thrombin-antithrombin III, but not with its nascent components, α-thrombin or antithrombin III. Their respective immunoglobulin classes are IgG₁ for JITAT-16 and -19, and IgG_2a for JITAT-4, -14 and -17. Besides the thrombin-antithrombin III complex, they all bound to the Factor Xa-antithrombin III complex and the active-site-cleaved two-chain antithrombin III as well. Moreover, the reactivity of these two antibodies to the neoantigens was not affected by heparin, suggesting that their epitopes are independent of heparin-induced conformational changes of antithrombin III. Two of them, JITAT-16 and -17, were categorized as high-affinity antibodies to thrombin-antithrombin III complex, the dissociation constants being 6.7 nM and 4.8 nM, respectively. However, they do not share antigenic determinants. These monoclonal antibodies may allow us to explore more precisely the reaction between antithrombin III and thrombin or its related enzymes.     

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.     

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.     

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。     

A multiple-column approach to the methylation analysis of plant cell-walls

A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.     

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.