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, 6B, 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; approximately 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 mug/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.

译文

调理吞噬细胞杀伤试验 (OPAs) 对于开发和改进肺炎球菌疫苗至关重要。需要用于肺炎球菌抗体的高通量,可靠,标准化和完全表征的OPA。为了满足需求,我们开发并表征了针对13种血清型 (1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F和23F) 肺炎球菌的四倍多重OPA (MOPA4)。仅使13种目标细菌对以下一种抗生素具有抗性: 光钦,链霉素,壮观霉素和甲氧苄啶。优化测定条件后,通过在MOPA4和单血清型测定中测试来自老年人的30份血清来确定MOPA4的准确性。用两种测定获得的调理滴度一致 (r(2)> 0.95)。尽管22个 (390个; 大约6% 个) 结果差异超过两倍,但差异是不可重现的。该测定具有特异性: 具有同源多糖 (PS) 的预吸收测试血清完全消除了调理活性,但不相关的PS池 (每个5g/ml) 没有作用。测定内和测定间变异系数分别为10和22%。MOPA4结果不受每个测定组中具有不同目标肺炎球菌血清型的影响。此外,HL60细胞与细菌的比率可以在不影响结果的情况下改变两倍。我们得出的结论是,对于大规模临床研究而言,MOPA4具有足够的敏感性,准确性,特异性,准确性和鲁棒性。此外,MOPA4应允许评估多价肺炎球菌疫苗,而通常可从幼儿获得的血清体积有限。

+1
+2
100研值 100研值 ¥99课程
检索文献一次
下载文献一次

去下载>

成功解锁2个技能,为你点赞

《SCI写作十大必备语法》
解决你的SCI语法难题!

技能熟练度+1

视频课《玩转文献检索》
让你成为检索达人!

恭喜完成新手挑战

手机微信扫一扫,添加好友领取

免费领《Endnote文献管理工具+教程》

微信扫码, 免费领取

手机登录

获取验证码
登录