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目的建立A、C、W135和Y群脑膜炎奈瑟菌血清抗体特异性体外杀菌试验方法 (serum bactericidal assay,SBA),并进行验证。方法对SBA中活菌收获时间、靶菌的制备、反应时间及补体进行优化,并确定质控血清Men10的质控范围。对优化后的方法进行特异性、线性、精密度验证。结果确定A600值为0.5~0.8时为收获细菌最佳收获时间;冻存菌种可替代新鲜制备菌种作为靶菌;最佳杀菌反应时间为60 min;17830和84321N批补体均可用于试验。质控血清Men10的质控范围A群为521~4 689,C群为1 047~9 423,W135群为1 779~16 008,Y群为1 416~12 741。A、C、W135和Y同群多糖吸收血清样品后能够抑制≥80%的同群SBA滴度,而异群多糖则<20%;血清样品稀释倍数与对应的SBA滴度呈显著负相关,斜率在-0.90~-1.10之间,Pearson相关系数在0.90~1.0之间,P均<0.001;同一实验内相同样品检测结果 CV≤25%,实验间相同样品的95%的检测结果落在检测值中位数3倍(一个稀释度)的范围内,CV≤40%,不同实验室间相同样品的检测结果至少有70%落在美国CDC公布结果的3倍(一个稀释度)范围内。结论建立了标准化的具国际间可比性的A、C、W135和Y群脑膜炎奈瑟菌SBA,该方法特异性、线性和精密度良好。
Objective To establish a serum bactericidal assay (SBA) against serogroup A, C, W135 and Y meningitidis in vitro and to verify the results. Methods The harvest time of live bacteria, preparation of target bacteria, reaction time and complement were optimized in SBA, and the quality control range of quality control serum Men10 was determined. The optimized method for specificity, linearity, precision verification. The results showed that the best harvesting time was obtained when the A600 value was 0.5 ~ 0.8. The frozen bacteria could replace the freshly prepared bacteria as the target bacteria. The best bactericidal reaction time was 60 min. Both the 17830 and 84321N lots could be used for the experiment. Quality control serum Men10 quality control range A group 521 ~ 4 689, C group of 1 047 ~ 9 423, W135 group of 1 779 ~ 16 008, Y group of 1 416 ~ 12 741. A, C, W135 and Y polysaccharides absorbed serum samples can inhibit the same group SBA titers ≥ 80%, while the polysaccharide was <20%; serum sample dilution times and the corresponding SBA titer was significantly negatively correlated, Slope between -0.90 ~ -1.10, Pearson correlation coefficient between 0.90 ~ 1.0, P <0.001; the same test results within the same sample CV ≤ 25%, 95% of the same sample between the test results fall in the test CV <40% for a median of 3x (one dilution), with at least 70% of the same sample from different laboratories falling within 3x (one dilution) of the results published by the U.S. CDC. Conclusion A standardized and internationally comparable group A, C, W135 and Y meningitidis SBA was established. The method was specific, linear and precise.