vhh-elisa kit for analysis of cyantraniliprole and chlorantraniliprole residues and its application
A technology of chlorantraniliprole and cyantraniliprole, which is applied in the field of ELISA kits for analyzing cyantraniliprole and chlorantraniliprole residues, can solve the problems of low stability, achieve less time-consuming, pre-treatment The effect of simple process
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Embodiment 1
[0053] The preparation of embodiment 1 cyantraniliprole-coated antigen
[0054] Take the hapten CNAP-4C: (2-(3-bromo-1-(3-chloropyridine-2-pyridyl)-1H-pyrazole-5-carboxamido)-5-cyano-3-methyl A conjugated complex was prepared from benzamido)valeric acid and bovine serum albumin as the coating antigen (CNAP-4C-BSA). The preparation method is as follows:
[0055] (1) 5.66 mg of (2-(3-bromo-1-(3-chloropyridine-2-pyridyl)-1H-pyrazole-5-carboxamido)-5-cyano-3-methylbenzene Formamido)valeric acid, 1.725mg N-hydroxysuccinimide and 2.678mg N,N'-dicyclohexylcarbodiimide were dissolved in 200μL anhydrous dimethylformamide, and the reaction was stirred overnight at room temperature; The reaction solution was centrifuged, the precipitate was discarded, and the supernatant was collected;
[0056] (2) Dissolve 20 mg of bovine serum albumin in 2 mL of 0.05M, pH 9.6 carbonate buffer, add dropwise to the supernatant under stirring, and continue stirring for 4h at room temperature after the ...
Embodiment 2
[0058] Example 2 Construction of cyantraniliprole and chlorantraniliprole nanobody library
[0059] The hapten of embodiment 1 is coupled with keyhole limpet hemocyanin using the active ester method, and the specific method is as follows:
[0060] An equimolar amount of the hapten CNAP-3C: (2-(3-bromo-1-(3-chloropyridine-2-pyridyl)-1H-pyrazole-5-carboxamido)-5-cyano- 3-methylbenzamido)butyric acid, NHS and DCC were dissolved in DMF and the reaction was stirred overnight at room temperature. The reaction solution was centrifuged to discard the precipitate, and the supernatant was the active ester. The supernatant was added to the keyhole limpet hemocyanin solution under stirring, and the stirring reaction was continued for 4 h at room temperature. The reaction solution was put into a dialysis bag and dialyzed with PBS. After centrifugation, the supernatant was collected and freeze-dried to obtain the conjugate of the hapten CNAP-3C and keyhole limpet hemocyanin (CNAP-3C-KLH)...
Embodiment 3
[0086] Example 3 Screening of cyantraniliprole Nanobodies
[0087] The first well of a 96-well microtiter plate was coated with the coating antigen of Example 1 at a coating concentration of 50 ng / mL, overnight at 4°C; the next day, the coating solution was poured out, washed three times with PBST, and the The first and second wells of the microtiter plate were blocked with BSA and incubated at 37°C for 1 h; the blocking solution was poured out and washed three times with PBST; the phage antibody library of Example 3 was added to the first well and reacted for 2 h; the liquid was poured out , pat dry on clean absorbent paper, and wash 5 times with PBST; add 100 μL cyantraniliprole standard to the first well, and react for 1 h; aspirate the liquid in the first well, add it to the second well, and react After 1 h, the phage bound to BSA was removed; the eluate was collected, 5 μL was used for titer determination, and the rest was used for amplification.
[0088] The phage eluat...
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