Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A
Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A
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DOI：
10.1128/cvi.00371-13
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发表时间：
2013-10-01
影响因子：
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通讯作者：
Briles, David E.
中科院分区：
文献类型：
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作者：
Daniels, Calvin C.;Kim, Kyung-Hyo;Briles, David E.
对肺炎球菌荚膜多糖抗体的标准调理吞噬杀伤试验（OPKA）进行了修改，以评估肺炎球菌表面蛋白A（PspA）的保护介导抗体。我们发现，通过将与补体和吞噬细胞的孵育时间从45分钟增加到75分钟，很容易检测到保护活性。在另一个修改中，我们使用了表达PspA但不表达肺炎球菌表面蛋白C（PspC）的荚膜2型靶菌株。单独或组合使用这些修饰，针对PspA的重组α-螺旋或富含脯氨酸结构域的兔抗血清介导>50%的靶菌株杀伤。正常人血清介导该改良OPKA中肺炎球菌杀伤的能力与其抗PspA抗体水平及其保护小鼠免受3型菌株致命感染的能力相关。当正常人血清与重组PspA（rPspA）一起吸附在琼脂糖凝胶上时，小鼠对肺炎球菌的被动保护和在修饰的OPKA中的杀伤丧失，从而支持修饰的OPKA用于检测PspA的保护性抗体的潜在效用。在标准OPKA中，抗PspA的单克隆抗体在亚保护量的抗胶囊存在下具有强保护性。因此，目前建立的针对胶囊的抗体的高通量OPKA可以以两种方式之一进行修改，以允许评估针对PspA的抗体的调理功效。
The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant alpha-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.