国际生物制品学杂志
國際生物製品學雜誌
국제생물제품학잡지
INTERNATIONAL JOURNAL OF BIOLOGICALS
2012年
6期
281-285
,共5页
潘殊男%肖詹蓉%王宇星%张霖阳%史雨舟%王玲%张萍
潘殊男%肖詹蓉%王宇星%張霖暘%史雨舟%王玲%張萍
반수남%초첨용%왕우성%장림양%사우주%왕령%장평
酶联免疫吸附测定%脑膜炎双球菌菌苗%多糖类,细菌%方法
酶聯免疫吸附測定%腦膜炎雙毬菌菌苗%多糖類,細菌%方法
매련면역흡부측정%뇌막염쌍구균균묘%다당류,세균%방법
Enzyme-linked immunosorbent assay%Meningococcal vaccines%Polysaccharides,bacterial%Methods
目的 建立测定A、C、Y、W135群脑膜炎球菌多糖疫苗(groups A,C,Y,W135 meningococcal polysaccharide vaccine,MPV4)C群多糖含量的双抗体夹心ELISA法.方法 制备抗C群多糖多克隆抗体,所得的多克隆抗体经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法对其标记辣根过氧化物酶.分别将抗C群多糖多克隆抗体作为包被抗体和酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对C群多糖进行特异性定量测定.结果 建立的双抗体夹心ELISA法特异性较好,未检出与A、Y、W135群多糖的交叉反应.C群多糖在2.5～20.0 ng/ml质量浓度范围的剂量反应曲线线性最佳,相关系数大于0.99.该法的准确度和精密度均较好,试验内和试验间变异系数和多糖回收率分别为0.6％ ～9.1％和87.5％ ～ 100.0％,定量限度为4.0 ng/ml.采用该法测定3批MPV4显示,C群多糖含量及多糖分子大小KD值和回收率的测定结果均与先前的测定结果一致,均符合暂行质量标准.结论 建立的双抗体夹心ELISA法可用于MPV4 C群多糖的关键质量指标测定.
目的 建立測定A、C、Y、W135群腦膜炎毬菌多糖疫苗(groups A,C,Y,W135 meningococcal polysaccharide vaccine,MPV4)C群多糖含量的雙抗體夾心ELISA法.方法 製備抗C群多糖多剋隆抗體,所得的多剋隆抗體經辛痠-硫痠銨沉澱法純化後,用過碘痠鈉法對其標記辣根過氧化物酶.分彆將抗C群多糖多剋隆抗體作為包被抗體和酶標二抗,建立雙抗體夾心ELISA法,優化反應條件,對C群多糖進行特異性定量測定.結果 建立的雙抗體夾心ELISA法特異性較好,未檢齣與A、Y、W135群多糖的交扠反應.C群多糖在2.5～20.0 ng/ml質量濃度範圍的劑量反應麯線線性最佳,相關繫數大于0.99.該法的準確度和精密度均較好,試驗內和試驗間變異繫數和多糖迴收率分彆為0.6％ ～9.1％和87.5％ ～ 100.0％,定量限度為4.0 ng/ml.採用該法測定3批MPV4顯示,C群多糖含量及多糖分子大小KD值和迴收率的測定結果均與先前的測定結果一緻,均符閤暫行質量標準.結論 建立的雙抗體夾心ELISA法可用于MPV4 C群多糖的關鍵質量指標測定.
목적 건립측정A、C、Y、W135군뇌막염구균다당역묘(groups A,C,Y,W135 meningococcal polysaccharide vaccine,MPV4)C군다당함량적쌍항체협심ELISA법.방법 제비항C군다당다극륭항체,소득적다극륭항체경신산-류산안침정법순화후,용과전산납법대기표기랄근과양화물매.분별장항C군다당다극륭항체작위포피항체화매표이항,건립쌍항체협심ELISA법,우화반응조건,대C군다당진행특이성정량측정.결과 건립적쌍항체협심ELISA법특이성교호,미검출여A、Y、W135군다당적교차반응.C군다당재2.5～20.0 ng/ml질량농도범위적제량반응곡선선성최가,상관계수대우0.99.해법적준학도화정밀도균교호,시험내화시험간변이계수화다당회수솔분별위0.6％ ～9.1％화87.5％ ～ 100.0％,정량한도위4.0 ng/ml.채용해법측정3비MPV4현시,C군다당함량급다당분자대소KD치화회수솔적측정결과균여선전적측정결과일치,균부합잠행질량표준.결론 건립적쌍항체협심ELISA법가용우MPV4 C군다당적관건질량지표측정.
Objective To establish a double antibody sandwich ELISA for determination of group C polysaccharide in groups A,C,Y,W135 meningococcal polysaccharide vaccine (MPV4).Methods The prepared polyclonal antibodies against group C polysaccharide were purified by octanoic acid-ammonium sulfate precipitation method,and then labeled with horseradish peroxidase by sodium periodate method.The polyclonal antibodies against group C polysaccharide were used as coated antibodies and the second enzyme-labled antibodies to establish a double antibody sandwich ELISA.The reaction conditions of the established ELISA were optimized,and specific quantitative determination of group C polysaccharide was carried out by the established ELISA.Results The specificity of the double antibody sandwich ELISA for determination of group C polysaccharide was high,and no cross reactions with groups A,Y and W135 were detected.The best linearity in dose-response curve of group C polysaccharide was found in a range of 2.5-20.0 ng/ml (r ＞ 0.99).The precision and accuracy of the established ELISA were good.Coefficients of variation and recovery rates of intraand inter-assay were 0.6％-9.1％ and 87.5％-100.0％,respectively.Quantitation limit was identified as 4.0 ng/ml.Group C polysaccharide contents,molecular dimension KDvalue and recovery rates of three batches of MPV4 detected by the established ELISA were in accordance with those of previous determination and temporary quality control standards.Conclusion The double antibody sandwich ELISA can be applied to detecting the key quality indexes of group C polysaccharide in MPV4.
