Purification of hapten-enzyme conjugates for enzymoimmunoassay

Chromatography on hydroxyapatite, including a potassium phosphate linear-gradient elution, was applied to the purification of hapten-enzyme conjugates to be used as immunoenzymatic tracers (namely, progesterone, phenobarbital, diphenylhydantoin coupled to glucose-6-phosphate dehydrogenase). The method effectively removed unreacted enzyme and separated the conjugate classes according to the number of substitutions between enzyme linking groups and hapten derivatives. The adequacy of the chromatographic procedures in improving the analytical performance of the enzymatic tracer appears to depend on direct interdependence between the degree of substitution and the immunological properties of the conjugates.