Clinical Chemistry, Vol 24, 1381-1385, Copyright © 1978 by American Association for Clinical Chemistry

Improved approach to sequential addition immunoassay

FD Lasky, J Al Razi and A Karmen

In the usual sequential addition enzyme immunoassays for drugs, the activity of the drug-labeled enzyme decreases continuously with time as more of it is bound to antibody. Sensitivity also decreases; the activity immediately after mixing is the most sensitive indicator of drug concentration. The reaction of enzyme-drug with antibody can be stopped by saturating the antibody with a larger quantity of unlabeled drug, which reacts with the antibody faster than does the enzyme- labeled drug. When drug is added soon after the reaction starts, the enzyme activity is stabilized and the sensitivity to small quantities of antigen is increased. This approach, with modification, should be applicable to sequential immunoassays in which other kinds of labels are used. The enzyme activity can be measured for a longer time, with the predictable increase in precision, as well as the ability to detect smaller quantities, to use less reagent, and to use end-point rather than kinetic assays.