Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies
Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies
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DOI：
10.1128/cdli.10.4.616-621.2003
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发表时间：
2003-07-01
期刊：
影响因子：
--
通讯作者：
Nahm, MH
中科院分区：
文献类型：
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作者：
Kim, KH;Yu, JU;Nahm, MH
为了评价肺炎球菌疫苗，调理吞噬细胞杀伤试验（OPKA）可作为肺炎球菌抗体酶联免疫吸附试验（ELISA）的补充。然而，肺炎球菌疫苗的评价需要确定对7至11种血清型的抗体应答，并且OPKA进行起来繁琐，并且需要比ELISA更多的血清。因此，OPKA很少用于评价肺炎球菌疫苗。为了克服这些局限性，我们已经开发了一个简单的多重（双血清型）OPKA通过使用耐药肺炎球菌的9个血清型。使血清型6 B、9V、19 A和23 F菌株对链霉素耐药，使血清型4、6A、14、18 C和19 F菌株对optochin耐药。多重OPKA与单一血清型OPKA相同，除了两个变化。首先，靶细菌是一种链霉素抗性菌株和一种optochin抗性菌株的混合物。其次，通过在含有酵母提取物和含有适当抗生素和2，3，5-三苯基氯化四氮唑的琼脂覆盖物的Todd-Hewitt琼脂平板上铺板，对每种血清型的存活细菌进行计数。通过使用单一血清型OPKA和多重OPKA分析来自用23价多糖疫苗免疫的成人的28个血清样品来评价多重OPKA的性能。多重OPKA对所需血清型具有特异性。多重和常规OPKA具有相当的检测灵敏度，并且对于所有9种血清型产生高度相关的结果（r（2）值范围为0.92 - 0.98）。对传统OPKA进行简单的修改，产生了一种多重检测，大大减少了工作量、试剂和必要的血清量。
For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r(2) values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.