A vhh-elisa kit suitable for the analysis of triazophos residues
A kit, the technology of triazophos, applied in the field of ELISA kits, can solve the problems of difficult on-site detection, complicated pretreatment, high cost of analysis methods, etc., and achieve low cost of sample detection, less time-consuming, and simple pretreatment process Effect
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Embodiment 1 3
[0024] Example 1 Preparation of Triazophos-coated Antigen
[0025] A conjugated complex was prepared with hapten and bovine serum albumin as the coating antigen. The preparation method is as follows:
[0026] (1) Weigh 7.4mg O-ethyl-O-3-(1-phenyl-1,2,4-triazolyl)-N-(3-carboxymethyl)phosphorothioate (MW=370 ) (0.02mmol), 2.65mg of NHS (MW=115) (0.024mmol), 4.8mg of DCC (MW=206) (0.023mmol) were dissolved in 200 μL of anhydrous DMF, and stirred overnight at room temperature. Centrifuge the reaction solution (5000rpm, 10min), discard the precipitate, and the supernatant is the active ester.
[0027] (2) Dissolve 20mg of BSA (MW=67000) in 2mL of carbonate buffer solution (0.05mol / mL, pH9.5), add 150μL of active ester solution drop by drop under stirring, slowly, and finish adding in about 20min . Then the stirring reaction was continued at room temperature for 4 h.
[0028] (3) The reaction solution was put into a dialysis bag and dialyzed with PBS (0.01mol / L pH7.4). The liq...
Embodiment 2 3
[0029] Example 2 Construction of Triazophos Phage Display Nanobody Library
[0030] The active ester method was used to couple the hapten to keyhole limpet hemocyanin, and the specific method was as follows:
[0031](1) Weigh 59.7mg O-ethyl-O-3-(1-phenyl-1,2,4-triazolyl)-N-(5-carboxypentyl)phosphorothioate (MW=398 ) (0.15mmol), 17.825mg NHS (MW=115) (0.155mmol), 31.518mg DCC (MW=206) (0.153mmol) were dissolved in 1500 μL of anhydrous DMF, and stirred overnight at room temperature. Centrifuge the reaction solution (5000rpm, 10min), discard the precipitate, and the supernatant is the active ester.
[0032] (2) Take 6mL of KLH solution (6.8mg / mL), add 1200μL of active ester solution drop by drop under stirring, and add slowly in about 20 minutes. Then the stirring reaction was continued at room temperature for 4 h.
[0033] (3) The reaction solution was put into a dialysis bag and dialyzed with PBS (0.01mol / L pH7.4). The liquid was changed every 6 hours, and the liquid was ch...
Embodiment 3
[0060] Example 3 Screening of specific triazophos phage display nanobodies
[0061] Coat the first well of a 96-well ELISA plate with the coated antigen of Example 2, at a coating concentration of 100 μg / mL, overnight at 4°C; the next day, pour out the coating solution, wash 3 times with PBST, and the enzyme The first and second wells of the target plate were blocked with BSA, incubated at 37°C for 1 hour; poured out the blocking solution, and washed 3 times with PBST; added the phage antibody library of Example 2 to the first well, and reacted for 2 hours; poured out the liquid, Pat dry on clean absorbent paper, wash 5 times with PBST; add 100 μL triazophos standard to the first well, react for 1 h; suck out the liquid in the first well, add to the second well, react for 1 h, Phage bound to BSA were removed; the eluate was collected, 5 μL was used for titer determination, and the rest was used for amplification.
[0062] Add the phage eluate to fresh Escherichia coli ER2738 ...
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