DESIGN OF NONCOMPETITIVE FLOW-INJECTION ENZYME IMMUNOASSAYS FOR DETERMINATION OF HAPTENS - APPLICATION TO DIGOXIGENIN

A theoretical model immunoradiometric assay (IRMA) was adapted to prov ide a non-competitive flow injection enzyme immunoassay for haptens an d used as a guide in studying the effects of different parameters on t he sensitivity, precision and dynamic range of the assay. As well as t he concentration of the antibody-enzyme conjugate, the affinity consta nt, the run time through the affinity column, the homogeneity of the a ntibody population and purity of the antibody-enzyme conjugate were al l shown to be important parameters in the optimisation of the assay. T he findings were used to design an optimised enzyme flow injection imm unoassay for the model compound digoxigenin in standard solutions. A l inear 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 labe lled 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 possibl e before regenerating the affinity column. (C) 1997 Elsevier Science B .V.