Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.