Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone.
High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r=0.99, n=109). Fab was good in all aspects of immunoassay--capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.
Brockmann
Eeva-Christine
EC
Department of Biotechnology, University of Turku, FI-20520 Turku, Finland. eechbr@utu.fi
Vehniäinen
Markus
M
Pettersson
Kim
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods; D011994:Recombinant Proteins / Q000276:immunology; D013972:Thyrotropin / Q000032:analysis* / Q000276:immunology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D013972:Thyrotropin