A human liver alcohol dehydrogenase enzyme-linked immunosorbent assay method specific for class I, II, and III isozymes.
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.
Montavon
P
P
Division d'Endocrinologie et Biochimie Clinique, C.H.U.V., Lausanne, Switzerland.
Felber
J P
JP
Holmquist
B
B
Vallee
B L
BL
D000426:Alcohol Dehydrogenase / Q000032:analysis*; D000906:Antibodies / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007527:Isoenzymes / Q000032:analysis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007527:Isoenzymes; D000426:Alcohol Dehydrogenase