Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay

Abstract

A novel sandwich immunoassay based immunochromatographic test strip (ICTS) has been developed for simultaneously measuring both butyrylcholinesterase (BChE) activity and the total amount of BChE (including inhibited and active enzyme) from 70 μLpost-exposure human plasma sample. The principle of this method is based on the BChE monoclonal antibody (MAb) capable of acting as both capture antibody and detection antibody. The BChE MAb which was immobilized on the test line was able to recognize both organophosphorus BChE adducts (OP-BChE) and BChE and provided equal binding affinity, permitting detection of the total enzyme amount in post-exposure human plasma samples. The formed immunocomplexes on the test line can further be excised from the test-strip for subsequent off-line measurement of BChE activity using the Ellman assay. Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. The whole sandwich-immunoassay was performed on one ICTS, greatly reducing analytical time. The ICTS sensor showed excellent linear responses for assaying total amount of BChE and active BChE ranging from 0.22 to 3.58nM and 0.22-7.17nM, respectively. Both the signal detection limits are 0.10nM. We validated the practical application of the proposed method to measure 124 human plasma samples from orchard workers and cotton farmers with long-term exposure to organophosphorus pesticides (OPs). The results were in highly agreement with LC/MS/MS which verified our method is extremely accurate. Combining the portability and rapidity of test strip and the compatibility of BChE MAb as both capture antibody and detection antibody, the developed method provides a baseline-free, low-cost and rapid tool for in-field monitoring of OP exposures.

Keywords: BChE monoclonal antibody; Biomarkers; Enzyme inhibition; Immunochromatographic test strip; Pesticides exposure.

Figure 1

(A) ELISA of the binding affinity of BChE MAb to BChE and OP-BChE. (B) Results of different samples on test strip reader analysis. The inset shows the visual color results on the test strip.

Figure 2

Optimization of parameters of the test strip. (A) Effect of the pH of working solution on detection of total enzyme. (B) Effect of the volume of MAb-Au on detection of total enzyme. (C) Effect of immunoreaction time on the test strip.

Figure 3

(A) Detection of different concentrations of total amount BChE in PBST with ICTS. Concentrations of BChE from 1–8 were 14.34, 7.17, 3.58, 1.80, 0.90, 0.45, 0.22, 0nM. (B) The calibration curve of the total amount of enzyme recorded by strip reader. (C) The calibration curve of active enzyme recorded by colorimetric analysis.

Figure 4

Comparison of % phosphorylation with human plasma sample by ITCS and LC/MS/MS methods.

Scheme 1

Illustration of the principle of one-step ICTS