IMPROVED ELISA CONDITIONS FOR DETECTION OF PLANT-VIRUSES
IMPROVED ELISA CONDITIONS FOR DETECTION OF PLANT-VIRUSES
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DOI：
10.1016/0166-0934(81)90018-5
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发表时间：
1981-01-01
影响因子：
3.1
通讯作者：
BAUM, RH
中科院分区：
文献类型：
--
作者：
MCLAUGHLIN, MR;BARNETT, OW;BAUM, RH
以三叶草黄脉病毒（CYVV）及其同源抗血清为材料，在聚苯乙烯板上研究了时间和温度对酶联免疫吸附试验（ELISA）的影响。重复的晶格正方形和约登正方形实验设计用于测量和解释与聚苯乙烯板内的样品位置相关的吸收值的变化。包被抗体对聚苯乙烯的吸附相对较快，在5 ℃下1小时内达到最佳测定效率。当以2.5 μ g/ml施用时，在ELISA过程中，抗原和酶联抗体（缀合物）在其各自步骤中的结合也是快速的。将抗原和缀合物各自温育2小时足以能够检测100 μ l样品中的20 ng CYVV，但任一反应物的更长温育改善了结果。在该病毒浓度下，抗原孵育时间减少1/2可通过使缀合物孵育时间加倍来补偿，反之亦然。在5 ℃下孵育缀合物。而不是30度。C使最终ELISA读数增加> 2倍。在室温下，底物水解遵循经典的一级动力学。通过比较家兔免疫应答过程中相对较早和较晚产生的抗血清，证明了ELISA中晚期抗血清的更高效率。以花生花叶病毒和花生矮化病毒及其同源抗血清为材料，研究了抗原和结合物孵育时间对最佳检测效率的影响。两种病毒的结果与CYVV相似。这些时间和温度对ELISA的影响应适用于大多数兔血清-病毒组合。
Clover yellow vein virus (CYVV) and homologous antisera were used to test effects of time and temperature on enzyme-linked immunosorbent assay (ELISA) in polystyrene substrate plates. Replicated lattice square and Youden square experimental designs were used to measure and account for variation in absorption values associated with sample position within polystyrene plates. Adsorption of coating antibody to polystyrene was relatively rapid, reaching optimum assay efficiency in 1 h at 5.degree. C when applied at 2.5 .mu.g/ml. Binding of antigen and enzyme-linked antibody (conjugate) in their respective steps during ELISA was also rapid. Incubation of antigen and conjugate for 2 h each was adequate to enable detection of 20 ng CYVV in a 100 .mu.l sample, but longer incubation of either reactant improved results. At this virus concentration, reduction in antigen incubation time by 1/2 could be compensated by doubling the conjugate incubation time and vice versa. Incubation of conjugates at 5.degree. C rather than 30.degree. C increased final ELISA readings > 2-fold. Substrate hydrolysis followed classic 1st order kinetics at room temperature. Greater efficiency of late antisera in ELISA was demonstrated by comparison of antisera produced relatively early and late during a rabbit''s immune response. Alfalfa mosaic virus and peanut stunt virus with their homologous antisera were used to test the effects of antigen and conjugate incubation times for optimum assay efficiency. The results with both viruses were similar to those obtained with CYVV. These time and temperature effects on ELISA should be applicable to most rabbit serum-virus combinations.