摘要
目的建立双抗体夹心ELISA法,对A群流脑多糖抗原进行特异性定量测定。方法制备抗A群多糖的特异性多克隆抗体,所得抗血清经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法制备辣根过氧化物酶标记多克隆抗体。分别以抗A群多糖多克隆抗体作为包被抗体及酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对A群多糖抗原进行特异性定量测定。结果一系列验证试验表明,该法特异性较好,未检出与C、Y、W135群多糖的交叉反应;1.25~20 ng/mL多糖浓度范围的剂量反应曲线线性最佳,相关系数大于0.98,经实验内10次及不同试验间以16、84、ng/mL测定3次A群多糖中的含量,变异系数在6.3%~11.5%间,回收率在91.8%~105.9%之间,符合常规质控要求,检测限量为4 ng/mL。采用该法测定3批ACYW135群四价脑膜炎球菌多糖疫苗中A群多糖含量、分子大小及回收率的结果均符合规程草案质量标准。结论建立的双抗体夹心ELISA法可尝试用于ACYW135群脑膜炎球菌多糖疫苗中A群多糖的关键质量指标的检测。
Objective To develop a double antibody sandwich ELISA in determination content of group A meningoeoccal polysaccharides. Methods The polyclonal antisera against meningoeoceal group A polysaccharides was purified by capric acid and ammonium sulfate precipitation, then the purified antibody was coupled to horseradish peroxidase ( HRP ) by sodi- um periodate method. The ELISA was developed by using of purified antibody for group A polysaccharides as capture anti-body and signal antibody, which was optimized by adjusting reactive condition, such as concentration of capture antibody and signal antibody, finally, a specific quantitative approach in determination content of polysaccharide group A was set up. Results This method showed an excellent specificity in detection of group A polysaecharides, which was based on a series of tests for verification. There were no cross reactions with group Y, C and W135 polysaccharides. The best linearity of dose-response curve was found in a range of 1.25w20 ng/mL ( r〉0.98 ) for content of group A polysaccharides, The good precision for the ELISA was obtained as coefficient of variation of 6.3%-11.5% and the good accuracy with the re-covery of 91.8%-105.9% in detection the group A polysaccharides standards as 16 ng/mL, 8 ng/mL, 4 ng/mL by both the intra-and inter-assays. As the result, the ELISA was applied in detection content of group A polysaccharides in three lots of group ACYW135 meningococcal polysaccharide vaccine, the quantitative limitation is determinated as 4 ng/mL in accordance with the requirement for a drafted conventional method. Conclusion The developed method may be applied in attempt to detecting crucial quality index for the content of group A polysaccharides in group ACYW135 meningococcal polysaccharide vaccine.
出处
《微生物学免疫学进展》
2012年第4期29-33,共5页
Progress In Microbiology and Immunology