微生物学免疫学进展
微生物學免疫學進展
미생물학면역학진전
PROGRESS IN MICROBIOLOGY AND IMMUNOLOGY
2013年
5期
1-5
,共5页
刘威%廖红梅%谢谦%黄放%邹莎莎%马诚%江山%崔长法
劉威%廖紅梅%謝謙%黃放%鄒莎莎%馬誠%江山%崔長法
류위%료홍매%사겸%황방%추사사%마성%강산%최장법
肺炎球菌1型多糖%免疫学检测法%夹心ELISA%多糖浓度
肺炎毬菌1型多糖%免疫學檢測法%夾心ELISA%多糖濃度
폐염구균1형다당%면역학검측법%협심ELISA%다당농도
Pneumococcal polysaccharide serotype 1%Immunoassay%Sandwich enzyme-linked immunosorbent assay ( S-ELISA)%Polysaccharide concentration
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。
目的:利用肺炎毬菌1型全菌體製備多剋隆抗體,併且利用該抗體建立肺炎1型莢膜多糖夾心酶聯免疫吸附分析法( Enzyme-linked immunosorbent assay ,ELISA),用于檢測髮酵和純化過程中的多糖濃度。方法用滅活的1型肺炎鏈毬菌免疫傢兔6週,穫得高滴度的抗多糖血清,經過親和層析純化,穫得高純度的兔抗肺炎1型多糖抗體IgG。以純化IgG作為包被抗體,加入多糖樣品,再以生物素化的抗體作為檢測抗體,建立夾心ELISA法檢測肺炎1型多糖濃度。確定標準麯線的最佳線性範圍,併對該方法進行特異性、準確性和精密度驗證。結果兔免疫血清經過雙嚮免疫擴散檢測抗體滴度可達1∶32;該方法的線性檢測範圍為1.56~50 ng/mL;最低檢測限為3.13 ng/mL。在標準品中混入其他型彆多糖或培養基,迴收率分彆為102%和108%;該方法批內精密度和批間精密度分彆為6.08%和7.01%。結論建立的夾心ELISA方法,其特異性、準確性和精密度均良好,可以特異地檢測肺炎毬菌1型多糖濃度。
목적:이용폐염구균1형전균체제비다극륭항체,병차이용해항체건립폐염1형협막다당협심매련면역흡부분석법( Enzyme-linked immunosorbent assay ,ELISA),용우검측발효화순화과정중적다당농도。방법용멸활적1형폐염련구균면역가토6주,획득고적도적항다당혈청,경과친화층석순화,획득고순도적토항폐염1형다당항체IgG。이순화IgG작위포피항체,가입다당양품,재이생물소화적항체작위검측항체,건립협심ELISA법검측폐염1형다당농도。학정표준곡선적최가선성범위,병대해방법진행특이성、준학성화정밀도험증。결과토면역혈청경과쌍향면역확산검측항체적도가체1∶32;해방법적선성검측범위위1.56~50 ng/mL;최저검측한위3.13 ng/mL。재표준품중혼입기타형별다당혹배양기,회수솔분별위102%화108%;해방법비내정밀도화비간정밀도분별위6.08%화7.01%。결론건립적협심ELISA방법,기특이성、준학성화정밀도균량호,가이특이지검측폐염구균1형다당농도。
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 .