| Organophosphorus pesticides(OPs) are a class of cholinesterase-inhibiting insecticides typified by effectiveness, broad-spectrum insecticidal activity, and lower cost, and are hence widely applied in both agricultural and domestic applications. O,O-diethyl organophosphorus pesticides(DPPs), one of the principal sub-classes of the OPs, are widely used in agriculture. The sensitive detection of DPP residue in agricultural products and environmental samples is therefore critical.Recombinant antibodies mainly involve the use of the single-chain variable fragment(sc Fv) or antigen-binding fragment(Fab) of the parent antibody. Many sc Fvs have demonstrated reduced affinity towards the antigen and display relatively low stability in comparison with the Fab fragments. However, Fab expression in the periplasm of Escherichia coli is less efficient than for sc Fv. Therefore, both sc Fv and Fab recombinant antibodies face certain challenges in their application, which also pose major obstacles to their commercialization as diagnostic tools. Recently, a novel recombinant antibody format was reported, namely the single-chain Fab fragment(sc Fab), which integrates properties of both Fab and sc Fv and with similar antigen affinities. sc Fab also has the potential for increased expression yield of functional antibody fragments in both E. coli and Pichia pastoris expression systems.(1) Construction of the p Comb3XSS-sc Fabs vectorThe two genes encoding Fd and κ were amplified by PCR. The Fd gene was ~675 bp in length while the κ gene was ~645 bp as measured with agarose gel electrophoresis(Fig. 2, lanes 1–4). The Fd and κ fragments including the linker overhangs were joined in two different orientations(κ-linker-Fd and Fd-linker-κ) with splicing overlap extension PCR to generate two sc Fab genes with a size of ~1365 bp each.(2) Expression of the two κ/Fd sc FabsAfter induction, the periplasmic proteins were extracted with cold osmotic shock and confirmed with SDS-PAGE and western blotting. The molecular weight of the recombinant sc Fabs was predicted to be ~50 k Da according to their amino acid sequences, which agreed with the western blotting results. This band was not observed in the induced periplasmic protein extract from E. coli TOP10F’ cells containing non-recombined p Comb3 XSS plasmid only. These results demonstrated that the sc Fabs were successfully expressed.(3)Optimization of various κ/Fd sc Fab orientationsAn ic ELISA based on anti-Fab-HRP antibody was developed to compare the sensitivities of the two different Fd/κ orientations towards the O,O-diethyl OPs. The results suggested that the 50% inhibition of binding(IC50) values of the κ-linker-Fd sc Fab for coumaphos and parathion were determined by ic ELISA to be ~1.5 ng/m L and 3.1 ng/m L, respectively, clearly surpassing that of the Fd-linker-κ sc Fab(~3.7 μg /m L and 8.5 μg /m L, respectively). The IC50 values of the κ-linker-Fd sc Fab for other free DPPs were determined by ic ELISA to be shown. Other free DPPs cannot bind the Fd-linker-κ sc Fab, even when additional DPPs were present at high concentrations of 1 mg/m L.(4)Characterization of the sc FabThe expression, specificity, and stability of the optimal sc Fab orientation were compared with that of their homologous sc Fv and Fab.To study the expression of sc Fab, periplasmic protein extracts of E. coli-expressed sc Fab, sc Fv, and sc Fab were confirmed with western blotting and reactivity against coating antigen. No bands were apparent for Fab in the periplasmic extract. To confirm whether Fab was expressed, albeit at a low level, the periplasmic extract was concentrated 4 times and subjected again to western blotting. A distinct band of ~23 k Da was then visible. However, a band of ~25k Da was detected for sc Fv of the periplasmic extract and a band of ~50k Da was detected for sc Fab in the periplasmic extract. For the specificity test, the cross-reactivity and IC50 values of the κ-linker-Fd sc Fab for other O,O-diethyl OPs were determined, which showed that the IC50 and CR of the κ-linker-Fd sc Fab were similar to that of its homologous Fab derived from the same parent Mab, and were relatively low in comparison with sc Fv. To evaluate the stability of the sc Fab, each recombinant antibody was incubated at 37°C over 9 days. Binding activity against coated antigen and sensitivity determined once per day with ic ELISA. The sc Fab Amax and IC50 curves over time tended to be slightly lower compared with those for Fab, while sc Fv displayed even larger decreases over time in comparison with sc Fab and Fab. These results indicated that the stability of Fab and sc Fab was superior to that of sc Fv.(5)Analyses of spiked samplesTo evaluate the precision of the developed ic ELISA assay, a Qu ECh ERS approach was applied. The interference of the OP matrix in the standard curve became insignificant after all the extracts were diluted 1:2. The Qu ECh ERS treatment was also applied to the analysis of Chinese cabbage, cucumber, and lettuce samples spiked with three different concentrations of four different DPPs. The mean recovery values of the four selected DPPs were in the desirable range of 81.2~121.8% for Chinese cabbage, 90.8~116.1% for cucumber, and 82.7~123.4% for lettuce. Coefficients of variation(CV) ranging from 6.8 to 20.1% were obtained. The relationship between the data from ic ELISA and GC is shown Good correlation(R2 = 0.9887). Overall, the results indicate that the developed ic ELISA method can be used as a rapid screening tool for the analysis of DPPs in food samples. |
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