Dual-Label Time-resolved Immunofluorometric Assay of Free and Total Prostate-specific Antigen Based on Recombinant Fab Fragments

Abstract

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 fragments and compared it to the same assay with intact monoclonal antibodies. Methods: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with α1-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 only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved 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 μg/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2–11% and 4–10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression 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 increased with the monoclonal assay but was undetectable with the recombinant assay. Conclusions: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

“…The TRF assay described here differs from those reported earlier. It utilizes labels that provide optimal fluorescence without the need for a separate dissociation-based fluorescence enhancement of the DELFIA assays [ 2 , 4 , 5 ] or a non-dissociative signal development step of the LANFIA procedure [ 3 ] and permits measurement of the fluorescence directly from the dry surface of the microtiter wells.…”

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing

“…Anti-TSH monoclonal antibodies (clones anti-TSH 5404 SP-1 and anti-TSH 5409 SPTNE-5) were purchased from Medix Biochemica (Kauniainen, Finland). Antibody fragments (anti-prostate specific antigen (PSA) Fab 5A10, Fab anti-TSH 5409, and anti-TSH 5404) were produced at the Department of Biotechnology in the University of Turku [ 25 , 26 ]. Affinity constants for the antibodies were 9.3 × 10 8 L mol −1 and 2.2 × 10 10 L mol −1 for anti-TSH 5409 and anti-TSH 5404, respectively [ 27 ].…”

Microchip electrophoresis coupled with on‐line magnetic separation and chemiluminescence detection for multiplexed immunoassay

“…These methods usually require expensive array detectors for spatial resolution of different immunoreactions areas. The label resolution technique employs multiple labels such as fluorescent dyes [], metal ions [], enzymes [], and nanoparticles [] with radiometric detectors or potential, mass‐, and time‐resolved detector for multiplexed immunoassay. However, the labels available for simultaneous determination of multiple analytes are limited.…”