Montavon P, Felber J P, Holmquist B, Vallee B L
Division d'Endocrinologie et Biochimie Clinique, C.H.U.V., Lausanne, Switzerland.
Anal Biochem. 1989 Jan;176(1):48-56. doi: 10.1016/0003-2697(89)90270-4.
A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes.
已设计出一种基于酶联免疫吸附测定法的灵敏且便捷的定量检测人乙醇脱氢酶（ADH）同工酶的方法。针对抗原包被密度、抗血清稀释度以及用针对β1β1-ADH的兔抗血清孵育时间进行了优化，以使该同工酶纯化时的灵敏度极限达到1 ng/ml。利用所建立的最佳条件，用针对这些单个同工酶的兔抗血清对αβ1、αγ1、β1γ1、π和χ-ADH进行了定量检测。在该方法中加入硫柳汞（乙基（4-巯基苯甲酸-S）汞）或其他巯基特异性试剂可提高所有ADH同工酶的可溶性抗血清亲和力，从而提高灵敏度。硫柳汞是χ-ADH抗原-抗体结合的绝对必需物。由三类ADH的单个同工酶引发的多克隆兔抗血清表现出高度的同工酶类特异性。评估了抗体与β1β1、αγ1、αγ2、αβ1、β1γ1、β1γ2、π和χ同工酶的交叉反应性。针对I类同工酶β1β1和β1γ1的抗血清与所有I类同工酶以及π-ADH发生交叉反应。针对π和χ-ADH的抗体仅对其各自的抗原具有选择性和特异性。两者均不与任何I类同工酶发生交叉反应。亚基相互作用产生的构象效应可能是密切同源的I类同工酶之间交叉免疫反应性差异的原因。
