Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

Abstract

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a doseresponse curve with an SC 50 (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC 50 of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection. KeywordsPhage anti-immunocomplex assay; Phage-displayed peptide; Phage ELISA; Noncompetitive immunoassay; BDE 47; Brominated flame retardant Antibody-driven specificity and affinity have made immunoassays widely accepted analytical tools for the detection of a variety of substances, including small-molecularweight analytes such as environmental contaminants, pesticides, pharmaceuticals, personal care products, toxins, and hormones [1][2][3]. Immunoassays are generally categorized into one of two functional formats: non-competitive sandwich type or competitive. Macromolecules with two or more nonoverlapping epitopes can be detected by a non-competitive immunoassay in which one antibody immobilized on the solid support captures the target molecule and a secondary antibody conjugated with signal-producing molecules detects the captured protein. In the case of small analytes, most of the antigen is buried in the antibody binding pocket after binding; therefore, the analyte cannot be simultaneously recognized by The use of an antibody capable of recognizing an analyte-bound antibody enhances the affinity and specificity of the primary antibody because of the formation of a ternary complex, which translates into an improved noncompetitive assay with enhanced sensitivity [4][5][6][7][8]. Although there have been efforts to produce these anti-immune complex antibodies by immunization with analyte-antibody complexes, the method has rarely been successful. In addition, polyclonal antibodies (PAbs) cannot be used as an immunogens for the antiimmune complex antibody because of their heterogeneous nature. An alternative method, called open sandwich assay, was recently introduced for the development of homogeneous noncompetitive a...

Recent Progress in Noncompetitive Hapten Immunoassays: A Review

“…However, once these specific antibodies have been prepared, these methods can easily be standardized. The recently developed phage display technology is a powerful and reliable technique for generating antibodies, and has successfully been applied to the preparation of anti-idiotype antibody (Goletz et al, 2002;Zhang et al, 2002;Raats et al, 2003;Coelho et al, 2004;Tometta et al, 2007) and anti-metatype antibody (Kim et al, 2009;Kim et al, 2010). Also, a phage display technology-based "split-Fv system" has recently been developed to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to open sandwich immunoassay (Aburatani et al, 2003).…”

Simultaneous development of both competitive and noncompetitive immunoassays for 2,2′,4,4′-tetrabromodiphenyl ether using phage-displayed peptides

“…The noncompetitive phage ELISA using the phage clone selected from plate #2 was performed according to the method reported by Kim et al [2]. Again, the half-maximum signal enhancement concentration (EC 50 ) and the LOD (EC 10 ) were obtained from the four-parameter logistic calibration curve of a noncompetitive assay.…”

“…2,2′,4,4′-Tetrabromodiphenyl ether (BDE47) is one of the most ubiquitous and toxic congeners in the environment and can be an indicator of exposure to lower brominated PBDEs, and so was selected as the target analyte for immunoassay development [1]. Kim et al [2] have recently reported the selection of a polyclonal antibody-BDE47 immunocomplex binding peptides and the development of a sensitive noncompetitive assay for BDE47.…”

“…In the development of a conventional immunoassay for small-molecule pollutants, great efforts are directed toward the preparation of the immunoreagents, e.g., haptens and antibodies, which is a challenging and timeconsuming task [1]. The phage-display technique offers an attractive means of developing an alternate immunoassay format that may enhance assay performance by increasing sensitivity and specificity [2].…”

Competitive and noncompetitive phage immunoassays for the determination of benzothiostrobin

“…The phage display technique offers attractive materials to develop alternate immunoassays that may enhance assay performance by increasing sensitivity and selectivity [8]. Phage-displayed peptide libraries have been a powerful tool for isolating peptides that specifically bind to analyte-free antibodies (peptidomimetics) or analyte-bound antibodies (anti-immunocomplex peptides) [9].…”