Development of competitive and noncompetitive immunoassays for clothianidin with high sensitivity and specificity using phage-displayed peptides.

Clothianidin is a widely used pesticide that has been banned from outdoor use by the European Union due to its toxicity. To improve the sensitivity and specificity of existing clothianidin immunoassays, we developed competitive and noncompetitive immunoassays for clothianidin based on phage-displayed peptides. Cyclic 8-, 9-, and 10-residue peptide libraries were constructed using an optimized phagemid pComb-pVIII to prevent the loss of theoretical library diversity. Twenty-eight peptidomimetics and two anti-immunocomplex peptides were isolated through a blended panning process and used to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), respectively. After optimization, the half inhibition concentration (IC50) and half saturation concentration (SC50) of competitive and noncompetitive P-ELISAs were 3.83 ± 0.23 and 0.45 ± 0.02 ng/mL, respectively. Competitive P-ELISA showed 2.6-18.2% cross-reactivity with imidaclothiz, nitenpyram and imidacloprid. Importantly, noncompetitive P-ELISA, which has the best specificity and great sensitivity for clothianidin, showed no cross-reactivity with the analogs. The average recoveries of competitive and noncompetitive P-ELISAs were 73.8-104.1% and 76.6-102.2%, respectively, while the relative standard deviations were ≤ 11.0%. In addition, the results of P-ELISAs in the analysis of blind samples were consistent with those of high-performance liquid chromatography.

Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

Sortase-Mediated Phage Decoration for Analytical Applications.

Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL-1, respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.