Dual-label time-resolved immunofluorometric assay of free and total prostate-specific antigen based on recombinant Fab fragments

Background: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragme nts and compared it to the same assay with intact monoclonal antibodies. Methods: The capture Fab fragment, which binds free prostate-specific antig en (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects on ly free PSA is site-specifically labeled with a fluorescent europium chelat e, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-re solved fluorescence is used to measure both europium and terbium signals in one well. Results: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 mu g/L, respectively, for free and total PSA. The with in-run and day-to-day CVs were 2-11% and 4-10%, respectively. Mean recoveri es were 93% and 98% in female and male sera, respectively. Compared with th e commercial ProStatus PSA Free/Total Assay, the intercepts of the regressi on equations (r >0.99) were not significantly different from zero, and the slopes were 0.95-1.01. In one female serum sample, PSA was falsely increase d with the monoclonal assay but was undetectable with the recombinant assay . Conclusions: The performance of this novel assay based on recombinant compo nents is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifi cally derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody ori entation, density, and capacity as well as uncompromised binding affinity a re required. (C) 2000 American Association for Clinical Chemistry.