Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.