苗鑫;王欣茹;赵志强;刘佳;刘月萍;刘方蕾;李燕婷;戈钰雯;谢贵林;谭小梅;

• 1:兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心

摘要(Abstract)：

目的建立间接ELISA方法检测小鼠血清中a型流感嗜血杆菌(Haemophilus influenzae type a,Hia)荚膜多糖(capsular polysaccharide,CPS)特异IgG含量,并对该方法进行验证。方法用1-氰基-4-二甲基氨基吡啶-四氟硼酸盐(1-cyano-4-dimethylaminopyridinium tetrafluoroborate,CDAP)活化Hia CPS制备酪胺化荚膜多糖(capsular polysaccharide-tyramined,CPS-Tyr),经Sepharose CL-4B层析柱分析其与Hia CPS的相对分子质量分布范围;对包被条件、封闭条件及显色时间进行优化,以碱性磷酸酶标记的羊抗鼠Ig G作为二抗,4-硝基苯磷酸二钠盐作为显色剂,建立小鼠血清中Hia CPS抗体的间接ELISA检测方法,并对该方法的特异性、线性及精密度进行验证。结果 CPS-Tyr与CPS相比,相对分子质量分布未发生明显变化。优化后的检测条件:包被抗原为CPS-Tyr,包被浓度为5μg/ml;以Costar42592为检测酶标板;含1%(bovine serum albumin,BSA)的封闭液37℃恒温封闭2 h;显色2 h。Hia阳性血清经不同浓度的Hia CPS和CPS-Tyr吸收后,抗原吸收的抑制率与浓度依赖性明显,抑制率在0¹00%之间。CPS-Tyr与CPS相比,抗原性保持一致,并且相同浓度的b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)荚膜多糖抗原对阳性血清的抑制率为0;血清稀释倍数与抗体效价之间呈负相关,斜率为-1.094,R2为0.998;本方法的实验内和实验间变异系数分别为8.8%和10.9%。结论建立的间接ELISA方法特异性、线性及精密度均符合要求,适用于检测小鼠血清中抗Hia CPS特异IgG的含量。

关键词(KeyWords)： a型流感嗜血杆菌;荚膜多糖;免疫球蛋白G;酶联免疫吸附试验

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 苗鑫;王欣茹;赵志强;刘佳;刘月萍;刘方蕾;李燕婷;戈钰雯;谢贵林;谭小梅;

DOI: 10.13200/j.cnki.cjb.001509

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