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 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 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 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 microgram/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.
Eriksson
S
S
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland. susann.eriksson@utu.fi
Vehniäinen
M
M
Jansén
T
T
Meretoja
V
V
Saviranta
P
P
Pettersson
K
K
Lövgren
T
T
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002478:Cells, Cultured; D002614:Chelating Agents; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005470:Fluorometry; D006801:Humans; D006825:Hybridomas; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000096:biosynthesis; D007811:Lanthanum; D008297:Male; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007811:Lanthanum; D017430:Prostate-Specific Antigen