EVALUATION OF A COMPUTER-ASSISTED, KINETICS-BASED ENZYME-LINKED IMMUNOSORBENT-ASSAY FOR DETECTION OF CORONAVIRUS ANTIBODIES IN CATS
EVALUATION OF A COMPUTER-ASSISTED, KINETICS-BASED ENZYME-LINKED IMMUNOSORBENT-ASSAY FOR DETECTION OF CORONAVIRUS ANTIBODIES IN CATS
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DOI：
10.1128/jcm.17.2.202-217.1983
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发表时间：
1983-01-01
影响因子：
9.4
通讯作者：
SCOTT, FW
中科院分区：
文献类型：
--
作者：
BARLOUGH, JE;JACOBSON, RH;SCOTT, FW
采用计算机辅助、基于动力学的酶联免疫吸附测定（ELISA）来检测猫血清中的冠状病毒抗体。初始实验中使用的碱性抗原稀释剂（碳酸盐-碳酸氢盐缓冲液，pH 9.6）在病毒和对照抗原比色皿中产生了弥漫性非特异性颜色反应，这些反应与测试血清中的冠状病毒抗体水平存在矛盾的相关性。通过使用较低 pH 值的抗原稀释剂（例如水和磷酸盐缓冲盐水）可最大限度地减少这些干扰反应。然后可以在大量血清中检测到针对抗原组织培养液中非冠状病毒成分的基于背景动力学的 ELISA 反应性，特别是在滴度较低的样品中。大部分这种反应性被证明与细胞培养液中的牛γ-球蛋白有关。它不具有血清批次或物种特异性；多种牛血清批次以及来自其他哺乳动物和禽类（鸡）物种的个别批次血清也发生了反应。当用于抗原制备的细胞在不含γ-球蛋白的牛血清中生长时，反应性显着降低。基于动力学的 ELISA 生成校正斜率值，可以校正单个测试血清中残留的背景反应性，从而消除假阳性反应的潜在主要来源。这些研究表明，控制猫冠状病毒血清学中的非特异性反应对于获得特异性抗体反应的有用估计至关重要。
A computer-assisted, kinetics-based enzyme-linked immunosorbent assay (ELISA) was adapted for detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based ELISA reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then be detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine .gamma.-globulins in cell culture fluid. It was not serum lot or species specific; a variety of bovine serum lots and individual lots of serum from other mammalian and avian (chicken) species also reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in .gamma.-globulin-free bovine serum. Generation of corrected slope values from the kinetics-based ELISA made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. These studies indicated that the control of nonspecific reactivity in feline coronavirus serology is essential to obtain useful estimates of specific antibody responses.