Development of competitive and noncompetitive lateral flow immunoassays for pendimethalin using synthetic peptides.

Peptidomimetic and anti-immunocomplex peptides can be easily isolated from phage display libraries, and can be used as alternatives to chemical competing haptens to improve the sensitivity of small molecule immunoassay. In this work, 16 peptidomimetics and 7 anti-immunocomplex peptides of pendimethalin (PND) were obtained from cyclic 7-, 8-, 9-, and 10-residue peptide phage libraries. Peptidomimetic EJ-2 (CMFTGTDFPC) with the highest sensitivity in competitive phage enzyme-linked immunosorbent assay (ELISA) and immunocomplex peptide EF-30 (CNPGWPPIPC) with the highest sensitivity in noncompetitive phage ELISA were selected to prepare phage-free peptides with GGGSSK-biotin at the C-terminus. Competitive and noncompetitive lateral flow immunoassays (CLFIA and NLFIA) were developed by using the phage-free peptides. After optimization, the CLFIA and NLFIA showed visual limit of detections (vLODs) of 5 ng/mL and 2.5 ng/mL, respectively, which were improved two- and fourfold compared with a LFIA based on chemical hapten. The NLFIA showed better sensitivity than CLFIA in the detection of spiked samples, and can meet the detection requirements for agro-products regulated by EU and China. The detection results of CLFIA and NLFIA for blind samples were consistent with that of ultra performance liquid chromatography/tandem mass spectrometry.

Development of Ic-ELISA and Colloidal Gold Lateral Flow Immunoassay for the Determination of Cypermethrin in Agricultural Samples.

Cypermethrin (CYP) is an insecticide in the pyrethroid family and is used widely in agriculture and for public health purposes. However, CYP has been shown to have negative impacts on reproduction, immunity and nerves in mammals. In this study, a monoclonal antibody (mAb) against CYP was prepared and used to establish an indirect competitive immunosorbent assay (ic-ELISA) and colloidal gold lateral flow immunoassay (LFIA) for the quantitative and qualitative determination of CYP residues in agricultural products. The half inhibition concentration of the ic-ELISA was 2.49 ng/mL, and the cut-off value and visual limit of detection of the LFIA were 0.6 and 0.3 µg/mL, respectively. The recovery rates of the ic-ELISA ranged from 78.8% to 87.6% in tomato, cabbage and romaine lettuce. The qualitative results of LFIA and quantitative results of ic-ELISA and HPLC were in good agreement in blind samples. Overall, the established ic-ELISA and LFIA proved to be accurate and rapid methods for the determination of CYP in agricultural products.

New Approach to Generate Ratiometric Signals on Immunochromatographic Strips for Small Molecules.

The self-calibration capability of ratiometric signals has been widely considered to enhance the accuracy, sensitivity, and anti-interference ability of immunoassays. Exploring a new approach to generate ratiometric signals can provide more options for various requirements. Herein, we integrated the negative-readout competitive and positive-readout noncompetitive immunoassays into a single assay by employing different color tracers, labeled peptidomimetic and anti-immunocomplex peptides, to create a new unconstrained ratiometric signal approach. Using an immunochromatographic strip (ICS) and a fungicide benzothiostrobin as the analytical platform and analyte, respectively, we showed that this approach can be extensively applied to fluorescence and colorimetry readouts, which have also been proven for strong anti-interference ability to an external light environment. Moreover, the enormous intuitional color changes of ratiometric fluorescent and colorimetric ICSs (RFICS and RCICS) enabled the formation of the color reference cards (like the pH paper) for visual judgment. After adaptation with a portable smartphone, the quantitative detection limits for RFICS and RCICS were 0.17 and 0.44 ng mL-1, respectively. In addition, the ICSs showed good accuracy for the detection of benzothiostrobin in spiked samples.

Development of immunoassay based on rational hapten design for sensitive detection of pendimethalin in environment.

Pendimethalin (PND) is one of the most widely used selective herbicides, but it is considered a potential human carcinogen and persistent bioaccumulative toxic chemical. Herein, five haptens with carboxylic groups were synthesized based on rational design and used to immunize mice, respectively. Then the antibodies obtained were evaluated systematically, and an indirect competitive ELISA (ic-ELISA) was developed based on an anti-PND monoclonal antibody. The 50% inhibition concentration and limit of detection of ic-ELISA were 0.53 ng/mL and 0.07 ng/mL, respectively. The cross-reactivities of ic-ELISA for the analogs of PND were ≤ 1.1%. The average recoveries of PND ranged from 79.5% to 107.4% in spiked samples. A good correlation was achieved between the ic-ELISA results and UPLC-MS/MS results in the analysis of blind samples. Thus, this assay provides a rapid and accurate tool for the determination of PND in the agro-products and agricultural producing environment.

"Ready-to-use" immunosensor for the detection of small molecules with fast readout.

Immunoassays are commonly used methods for detection of small molecules that typically require numerous steps of the labeling between immune-recognition reagents and tracers, immobilization and recurrent washing, making them time consuming and difficult to adapt into point of care formats. Here we describe a "ready-to-use" homogeneous competitive immunosensor with an assay time of 10 min that is based exclusively on recombinant reagents. The signal is produced when the split fragments of the nano luciferase (Nluc) are brought together by the interaction of a heavy chain only variable domain (VHH) with a peptidomimetic of the target small molecule. A VHH to 2,4-dichlorophenoxyacetic acid (2,4-D) was used to isolated the peptidomimetic (NGFFEPWQVVYV) from phage display libraries using six panning conditions. Then the peptidomimetic and VHH were fused with the larger (LgN) and smaller piece (SmN) of split fragments of Nluc, respectively. In order to optimize the signal and sensitivity of the immunosensor, we explored the effects of the spacer between the peptidomimetic and LgN, the copy number of peptidomimetics, and the spacer between SmN and VHH, generating 24 combinations that allowed to conclude on their respective roles. Eventually, the developed "ready-to-use" immunosensor performed excellent signal-to-noise ratio and sensitivity, and could be applied to the detection of 2,4-D in real samples. Meanwhile, the immunosensor totally realizes labeling-free, immobilization-free and washing-free, also can be produced in a highly cost effective way.

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.

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.

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.

Development of a multicolor upconversion fluorescence immunoassay for the simultaneous detection of thiamethoxam and dextran by magnetic separation.

The contents of both pesticide residues and dextran are important parameters for evaluating the quality of sugarcane. In this study, a multicolor upconversion fluorescence immunoassay for the simultaneous detection of thiamethoxam and dextran was established on the basis of magnetic separation. Antigens of thiamethoxam and dextran were coupled to magnetic nanoparticles as the separation elements. Monoclonal antibodies of thiamethoxam (6C7D12) and dextran (3C6F7) were conjugated with the upconversion nanoparticles of NaYF4:Yb,Er with an emission wavelength at 544 nm and NaYF4:Yb,Tm with an emission wavelength at 477 nm to prepare the signaling elements, respectively. Due to the difference in the emission wavelength, the signaling elements bound on the separation elements could be detected simultaneously after separation by an external magnetic field. After optimization, the half-maximal inhibitory concentration (IC50) values of the immunoassay for thiamethoxam and dextran were 0.46 and 49.33 ng mL-1, respectively. The assay showed no cross-reactivity with the analogs of thiamethoxam and dextran except for clothianidin (8.7%). The average recoveries of thiamethoxam and dextran in sugarcane juice were 82.9-93.3% and 87.5-97.2%, respectively. The results indicated that the immunoassay could meet the requirements for the simultaneous quantitative detection of thiamethoxam and dextran.