Use of an In Vivo Biotinylated Single-Chain Antibody as Capture Reagent in an Immunometric Assay to Decrease the Incidence of Interference from Heterophilic Antibodies

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2005-05, Vol.51 (5), p.830-838
• Main Authors: Warren, David J, Bjerner, Johan, Paus, Elisabeth, Bormer, Ole P, Nustad, Kjell
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies, Heterophile - blood; Antibodies, Monoclonal; Biological and medical sciences; Biotinylation; Carcinoembryonic Antigen - blood; Carcinoembryonic Antigen - immunology; Cattle; Cell Line; Escherichia coli - metabolism; Europium; False Positive Reactions; Fluorescence Resonance Energy Transfer; Fluoroimmunoassay; Fundamental and applied biological sciences. Psychology; Humans; Hybridomas; Immunoglobulin Fab Fragments; Immunoglobulin Variable Region - genetics; Indicators and Reagents; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Reagents; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2004.046979

Heterophilic antibodies are a common source of interference in immunometric assays. We tested the hypothesis that the incidence of such interference could be decreased by use of a recombinant in vivo-biotinylated single-chain antibody (scFv) as the capture reagent. We established three assays for carcinoembryonic antigen (CEA) with the capture antibody either chemically biotinylated whole monoclonal T84.66 immunoglobulin, a corresponding F(ab')2 fragment, or a site-specifically biotinylated T84.66-derived single-chain antibody (scFv). Antibodies were attached to streptavidin-coated microplates. A common europium-labeled anti-CEA tracer monoclonal antibody was used. The F(ab')2 assay used a buffer that contained bovine immunoglobulin and aggregated irrelevant monoclonal antibody MAK33 as blocking agents. The whole T84.66 immunoglobulin and scFv assays were performed without addition of blocking agents. From a previous study of 11 261 sera, we tested 390 samples that had displayed heterophilic antibody interference and 179 samples that had not. After correction for bias and analytical variation [2.56 x SD (from the precision profile)], 383 samples displayed significantly different values (>1 microg/L) in the whole T84.66-based assay and the F(ab')2 assay. In contrast, only nine samples showed falsely high CEA concentrations in the scFv assay. After blocking agents were added to the assay buffer, eight of the nine samples displayed results equivalent to those of the F(ab')2 assay, and sample dilution produced equivalent results for the remaining sample. Their ability to be site-specifically biotinylated and their relative resistance to heterophilic antibody interference indicate that single-chain antibodies may be useful solid-phase reagents in immunometric assays.