Abstract: A repetitive immunoassay analytical method for determination of a free analyte is carried out by loading an affinity column of covalently bound analyte with tagged antibody, passing a continuous aqueous stream of carrier liquid over the column, introducing an aliquot of a sample to be analyzed for free analyte into the carrier stream upstream of the column, and monitoring the eluting carrier stream for a signal spike resulting from the presence of tagged antibody material released from the column by the application of free analyte in the analytical sample. Many samples may be analyzed by this method before the antibody-loaded affinity column needs to be regenerated. It is also disclosed that substrate-analyte conjugates of superior stability are produced by linking a substrate to a hydroxyalkyl analyte via an amino, hydrazide, or sulfide linking group replacing a hydroxy group.