肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体，并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ，ELISA），用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周，获得高滴度的抗多糖血清，经过亲和层析纯化，获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体，加入多糖样品，再以生物素化的抗体作为检测抗体，建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围，并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32；该方法的线性检测范围为1．56～50 ng/mL；最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基，回收率分别为102％和108％；该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法，其特异性、准确性和精密度均良好，可以特异地检测肺炎球菌1型多糖浓度。

Preparation of polycional antibodies against pneumococcal serotype 1 capsular polysaccharide and development a sandwich ELISA in determination of concentration for type 1 capsular polysaccharide

Objective To prepare polyclonal antibodies against capsular polysaccharide of pneumococcal serotype 1, and use the antibodies to develop a sandwich ELISA in determination of concentration for pneumococcal capsular polysaccharide of serotype 1 in the process of fermentation and purification .Methods High titer serum of anti-capsular polysaccharide se-rotype 1 was obtained after immunizing rabbits by using of inactivated streptococcus pneumoniae cells of serotype 1 for 6 weeks.IgG was purified through affinity chromatography and used as a coating antibody .Polysaccharide samples , alone with in house polysaccharide standard , were then added followed by using biotinylated antibody as a signal antibody .The optimal linear range of the standard curve , specificity, accuracy and precision of the developed method were validated accordingly.Results The antibody titer of rabbit immune serum reached to 1 ∶32, the standard PS1 linear detection range was 1.56 ng/mL to 50 ng/mL, detection limit was 3.13 ng/mL.Standard polysaccharide was mixed with polysaccha-rides from different serotypes or culture media before asssay , the recovery ratio was 102%and 108%respectively .Intra-as-say precision and inter precision of the method were 6.08%and 7.01%.Conclusion High titer rabbit anti-serum against type 1 capsular polysaccharide of streptococcal pneumoniae was prepared .The developed sandwich ELISA method showed high specificity , good accuracy and precision .This method can be used in specific detection of concentration for pneumo-coccal polysaccharide serotype1 .