Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies
Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies
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DOI：
10.1128/cvi.00112-06
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发表时间：
2006-09-01
影响因子：
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通讯作者：
Nahm, Moon H.
中科院分区：
文献类型：
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作者：
Burton, Robert L.;Nahm, Moon H.
吞噬细胞杀伤试验是发展和改进肺炎球菌疫苗的重要手段。需要一种用于肺炎球菌抗体的高通量、可靠、标准化和完全特征化的OPA。为了满足这一需要，我们开发并鉴定了一种针对肺炎球菌13个血清型(1、3、4、5、6A、613、7F、9V、14、18C、19A、19F和23F)的四重复合OPA(MOPA4)。13个目标细菌只对下列抗生素中的一种产生抗药性：光霉素、链霉素、壮观霉素和甲氧苄啶。在对检测条件进行优化后，通过对30份老年人血清进行MOPA4和单血清型检测，确定了MOPA4的准确性。两种分析方法得到的优化滴度符合很好(r(2)>；0.95)。尽管22个(满分390分；类似于6%)结果差异超过两倍，但差异是不可重现的。该方法是特异的：含有同源多糖(PS)的预吸附试验血清可完全消除调理活性，但不相关的PS(各5微克/毫升)池则不起作用。批内和批间变异系数分别为10%和22%。MOPA4结果不受每个化验组中不同的目标肺炎球菌血清型的影响。此外，HL60细胞与细菌的比例可以变化两倍，而不会影响结果。我们的结论是，MOPA4具有足够的敏感性、准确性、特异性、精确性和健壮性，足以进行大规模的临床研究。此外，MOPA4应允许使用通常可从幼儿获得的有限数量的血清对多价肺炎球菌疫苗进行评估。
Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 613, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well (r(2) > 0.95). Although 22 (out of 390; similar to 6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 mu g/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.