Ordronneau P, Abdullah L H, Petrusz P
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
J Immunol Methods. 1991 Sep 13;142(2):169-76. doi: 10.1016/0022-1759(91)90103-m.
In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.
为了包被用于酶免疫测定（EIA）的微量滴定板，氨基酸和其他半抗原通常与较大的蛋白质分子偶联。此类缀合物的形成并非总是可重复的。这可能导致半抗原 - 蛋白质化学计量比不一致、半抗原在蛋白质上的取向不利以及/或者可用半抗原浓度在孔间存在差异。在此所述的测定中，兴奋性氨基酸（EAA）谷氨酸（Glu）通过戊二醛（GA）直接偶联到聚苯乙烯微量滴定孔上。对测定的每一步都进行了最大效率测试。以Glu作为竞争物所得的EIA具有出色的重现性（变异系数 = 5.87%），半数有效浓度（EC50）为2.02×10⁻⁵ M，检测限为1.26×10⁻⁶ M。这种EIA方法通常适用于多种针对氨基酸和小肽的抗血清以及广泛的竞争物质。它可用于表征抗原结合的构象要求、测定谷氨酸或鉴定未知溶液中具有谷氨酸样结构的化合物。
