Design of non-competitive flow injection enzyme immunoassays for determination of haptens. Application to digoxigenin

Abstract

A theoretical model immunoradiometric assay (IRMA) was adapted to provide a non-competitive flow injection enzyme immunoassay for haptens and used as a guide in studying the effects of different parameters on the sensitivity, precision and dynamic range of the assay. As well as the concentration of the antibody–enzyme conjugate, the affinity constant, the run time through the affinity column, the homogeneity of the antibody population and purity of the antibody–enzyme conjugate were all shown to be important parameters in the optimisation of the assay. The findings were used to design an optimised enzyme flow injection immunoassay for the model compound digoxigenin in standard solutions. A linear calibration curve was established in the range 0.38–7.7 fmol of digoxigenin, resulting in a precision of 14.8% RSD at 1 fmol and 3.7% RSD at 7.7 fmol. Antibody fragments reacting with digoxigenin and labelled with alkaline phosphatase, (Fab–AP) were used to convert 4-methyl umbelliferyl phosphate to a fluorescent product measured downstream. The sample throughput was 15 h −1 and over 60 injections were possible before regenerating the affinity column.