抗金葡菌肠毒素C2和抗人甘露聚糖结合凝集素单克隆抗体的制备、鉴定及ELISA检测方法的建立和应用

发布时间：2018-09-08 08:35

【摘要】： 金葡菌肠毒素(Staphylococcal enterotoxins，SE)是一组由金黄色葡萄球菌分泌至胞外的水溶性蛋白，依据血清分型主要有肠毒素A、B、C、D、E等。其中肠毒素C又分为C1，C2，C3三个亚型。与普通抗原比，金葡菌肠毒素在很低浓度时就能活化的大量T细胞并产生强大免疫效应，因此被认为是超抗原。在肠毒素家族中，肠毒素A、B研究较多，肠毒素C则研究较少。本研究的前期工作已从一种由金葡菌培养产物制得的生物制品-金葡素注射液的原液中提取了肠毒素，经氨基酸序列测定和比较确定是肠毒素C2亚型，并通过药效学试验证实了SEC2是金葡素注射液有效成分。由上述实验可认为，SEC2含量是控制产品质量的可信指标。因此，建立一种简便可靠的定量检测SEC2的方法就十分必要。本课题通过制备抗SEC2的特异性单克隆抗体，建立了检测SEC2的双抗体夹心ELISA法。采用杂交瘤单克隆技术，运用已建立的间接ELISA法筛选，获得四株分泌抗SEC2单克隆抗体(monoclonal antibody，McAb)的杂交瘤细胞株。四个杂交瘤细胞株分泌单克隆抗体的Ig类别均为IgG_1(k)，效价达1∶10~5—1∶10~7。采用辛酸-硫酸铵沉淀法纯化各株杂交瘤细胞腹水，经SDS-PAGE电泳分析，纯化各株McAbs的纯度均在80％以上。各株抗SEC2 McAbs能特异性识别SEC2，与牛血清白蛋白，人血清白蛋白及SEA、SEB、SEC1、SED型肠毒素无交叉反应，仅McAb387与高剂量(＞1μg／ml)的SEC1有交叉反应。采用非竞争法测得4株McAbs亲和常数在1.1×10~9M~(-1)至2.46×10~(10)M~(-1)之间。应用常规方法标记纯化McAbs，并经阻断抑制试验筛选McAbs识别位点，其中只有McAb 4F7和McAb 4D7之间识别抗原位点完全相关，其余McAbs之间识别抗原位点完全不相关。选择二株识别不同抗原位点的抗SEC2McAbs，运用于双抗体夹心抗原的ELISA方法，，建立定量检测SEC2的ELISA方法，灵敏度为0.5ng／L，批内变异系数为3.71％至6.93％，批间平均数变异系数1.02％，回收率为97.8％至101％，回收率实验变异系数为2.2％至5.2％。应用建立的单克隆抗体ELISA法测定了40批厂家送检的金葡素注射液样品中SEC2含量，同时与多克隆抗体ELISA法测定结果比较，结果显示两方法测定的SEC2含量无显著差异，单克隆抗体法结果略低于多克隆抗体法。50批金葡素注射液的SEC2含量在5-16ng／ml之间，变异系数为16.8％。本研究成功的制备了抗SEC2 McAbs，建立了特异、准确的定量检测ELISA方法，并用于金葡素注射液制品的SEC2含量检测，为超抗原研究和金葡素注射液质量控制提供了有力的手段。甘露(聚)糖结合凝集素(mannose-binding lectin or mannan-binding lectin；MBL)s是由肝脏分泌的具有胶原样结构的Ca~(2+)依赖的血浆C型凝集素，是先天免疫的重要组成部分。通过其糖基识别域MBL可识别并结合至很多微生物病原体的表面，随后激活补体系统发挥中和和／或调理作用。作固有免疫成分和急性期蛋白，特别是在后天免疫系统成熟前阶段和在感染后IgM出现之前，MBL都有重要意义。血循环中MBL含量水平主要由基因型决定，但许多非基因因素也对它有明显影响。MBL不足或缺失与反复感染、反复流产、自身免疫病以及某些肿瘤等许多疾病相关。定量检测MBL的试剂盒是进行相关研究的必要条件，但目前MBL国内没有这种试剂盒，进行MBL相关研究工作主要依赖进口试剂盒，其价格昂贵，影响了国内的研究发展。本课题通过采用杂交瘤技术制备了两株抗人MBL单克隆抗体(monoclonalantibody，McAb)并建立了MBL的定量ELISA检测方法。研制了MBL检测试剂盒，并初步应用于检测不同人群的MBL含量。以健康人血浆纯化的MBL为抗原免疫小鼠，运用已建立的间接ELISA法筛选，获得两株分泌抗人MBL单克隆抗体的杂交瘤细胞株。两杂交瘤细胞株分泌单克隆抗体的Ig类别均为IgG_1(K)，效价达1∶10~6—1∶10~7，识别不同抗原位点。两株McAbs能特异性识别MBL，与牛血清白蛋白，人血清白蛋白及由基因突变导致的MBL缺失的阴性血清无交叉反应。采用辛酸-硫酸铵沉淀法和A蛋白亲和层析法纯化各株杂交瘤细胞腹水，经SDS-PAGE电泳分析，纯化各株McAbs的纯度均在90％以上。应用常规方法标记经纯化的McAb-B3。运用两株McAb建立定量检测MBL的ELISA方法，最佳检测条件为8μg／ml浓度包被抗体B10，1∶500稀释酶标抗体B3。该方法特异、灵敏，有较好重复性。其准确定量的检测范围为1～100ng／ml。批内变异系数3.9％至5.2％，批间平均数变异系数为1.6％，均回收率为98.0％至105.3％，回收率实验变异系数为2.1％至4.3％。应用建立的单克隆抗体ELISA法测定了52例健康人血清MBL含量，与丹麦试剂盒测定结果比较，两方法测定的MBL含量有较好一致性。本研究的方法检测范围更宽。用自建的MBL单抗ELISA夹心法对277例侗壮两民族血清MBL含量水平进行调查。结果经统计学分析显示，MBL含量侗族低于壮族(p＜0.01)，不同性别间无差异，不同年龄的MBL含量，在青春期明显高于其它各时期(p＜0.01)，MBL缺失率在青春期明显低于其他各时期，MBL缺失率与MBL含量存在负相关。对MBL低下的样本进行基因突变检测，其中ExonⅠ54位密码子突变51例，57位密码子突变1例，没有52位密码子突变。所有突变的样本其MBL含量均低于1μg／ml，MBL含量低下与ExonⅠ密码子突变密切相关。另一调查检测了204例儿童血清MBL水平，103例感染患儿(肺炎、脑炎、腹泻、败血症)MBL水平明显高于101例正常健康儿童，表明感染会导致MBL升高，MBL低水平可能与易发感染相关。
[Abstract]:Staphylococcal enterotoxins (SE) are a group of water-soluble proteins secreted by Staphylococcus aureus and extracellular. According to the serotype, SE is mainly composed of enterotoxins A, B, C, D and E. Enterotoxin C is divided into three subtypes, C1, C2, C3. Compared with common antigens, SE can activate a large number of T cells at very low concentrations and can activate them. Enterotoxins A and B have been studied more and enterotoxin C has been studied less in the enterotoxin family.
Enterotoxin was extracted from the original solution of Staphylococcus aureus injection, a biological product made from Staphylococcus aureus culture products. It was identified as enterotoxin C2 subtype by amino acid sequencing and comparison. The pharmacodynamic test confirmed that SEC2 was an effective component of Staphylococcus aureus injection. Therefore, it is necessary to establish a simple and reliable method for quantitative detection of SEC2.
A sandwich ELISA method for detecting SEC2 was established by preparing specific monoclonal antibodies against SEC2. Four hybridoma cell lines secreting monoclonal antibody (McAb) against SEC2 were obtained by using hybridoma monoclonal technique and indirect ELISA method. The antibodies were all IgG_1 (k) with titer of 1:10~5-1:10~7. The ascites of hybridoma cells were purified by octanoic acid-ammonium sulfate precipitation method. The purity of McAbs was above 80% by SDS-PAGE electrophoresis analysis. The anti-SEC_2 McAbs could specifically recognize SEC_2, bovine serum albumin, human serum albumin, SEA, SEB, SEC_1, SED type. Enterotoxin did not cross-react, but only McAb 387 did cross-react with SEC 1 of high dose (>1 ug/ml). The affinity constants of four McAbs strains were measured by non-competitive method between 1.1 (-1) and 2.46 (10) M (-1). McAbs was labeled and purified by routine method, and the McAb 4F7 and McAb 4D7 were screened by blocking inhibition test. Two strains of anti-SEC2 McAbs were selected to identify different antigen loci and applied to the ELISA method of double antibody sandwich antigen. An ELISA method for quantitative detection of SEC2 was established. The sensitivity was 0.5ng/L, the coefficient of variation in batch was 3.71% to 6.93%, and the mean variation between batches was 3.71% to 6.93%. The coefficients were 1.02%, the recoveries were 97.8% to 101%, and the experimental variation coefficients were 2.2% to 5.2%. The SEC2 content in 40 batches of Staphylococcus aureus injection samples was determined by the established monoclonal antibody ELISA method. The results showed that there was no significant difference in SEC2 content between the two methods. The results of monoclonal antibody assay were slightly lower than those of polyclonal antibody assay. The SEC2 content of 50 batches of Staphylococcus aureus injection ranged from 5 ng/ml to 16.8%, and the coefficient of variation was 16.8%. And Staphylococcal Enterotoxin C Injection quality control provides a powerful means.
Mannose-binding lectin or mannan-binding lectin (MBL) is a collagen-like Ca~ (2+)-dependent plasma lectin secreted by the liver and an important component of innate immunity. MBL is recognized and bound to the surface of many microbial pathogens through its glycosyl recognition domain and then activated. The complement system plays a neutralizing/regulating role. MBL plays an important role as an innate immune component and an acute phase protein, especially in the pre-mature stage of the acquired immune system and before the appearance of IgM after infection. Lack of MBL is associated with repeated infection, recurrent abortion, autoimmune diseases and some tumors. Quantitative detection of MBL kits is a necessary condition for related research. However, there is no such kit in MBL at present. The research on MBL mainly relies on imported kits, which is expensive and affects the development of domestic research.
Two monoclonal antibodies (McAb) against human MBL were prepared by hybridoma technique and a quantitative ELISA method was established for the detection of MBL. The MBL detection kit was developed and applied to detect the content of MBL in different populations. The mice were immunized with purified MBL from healthy human plasma using the established indirect ELIS method. Two hybridoma cell lines secreting monoclonal antibodies against human MBL were screened by A method. The two hybridoma cell lines secreted monoclonal antibodies of IgG_1 (K) with titer of 1:10~6-1:10~7, and recognized different antigen sites. The two McAbs could specifically recognize MBL, BSA, HSA and MB caused by gene mutation. The ascites of hybridoma cells were purified by octanoic acid-ammonium sulfate precipitation and A protein affinity chromatography. The purity of McAbs was above 90% by SDS-PAGE electrophoresis analysis. The purified McAb-B3 was labeled by routine method. The quantitative detection of MBL by two McAb strains was optimized by ELISA. The method is specific, sensitive and reproducible. The accurate quantitative detection range is 1-100ng/ml. The intra-assay coefficient of variation is 3.9% to 5.2%, the average inter-assay coefficient of variation is 1.6%, and the average recovery is 98.0% to 105.3%. The established monoclonal antibody ELISA method was used to determine serum MBL levels in 52 healthy subjects. The results of the two methods were in good agreement with those of the Danish kit.
The serum levels of MBL in 277 cases of Dong and Zhuang nationalities were investigated by self-made MBL monoclonal antibody ELISA sandwich method.Results Statistical analysis showed that the MBL content in Dong nationality was lower than that in Zhuang nationality(p<0.01),and there was no difference between the sexes.The MBL content in different ages was significantly higher than that in other periods during puberty(p<0.01). The MBL deletion rate was negatively correlated with the MBL content at other stages. The low MBL content was closely related to the Exon I codon mutation. Another survey detected serum MBL levels in 204 children, 103 infected children (pneumonia, encephalitis, diarrhea, sepsis) MBL levels were significantly higher than 101 healthy children, indicating that infection will lead to MBL increased, low MBL levels may be associated with susceptibility to infection.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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