Antibody preparation method capable of detecting multiple organophosphorus pesticide residuals
A technology for the preparation of organophosphorus pesticides and antibodies, which is applied in the fields of peptide preparation methods, organic chemistry, chemical instruments and methods, etc., and can solve practical problems such as strong specificity and limited application of immunoassay methods
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Embodiment 1
[0031] Such as figure 1 Shown, present embodiment synthesis comprises three steps:
[0032] In the first step, O, O-diethylphosphorothioate was reacted with 4-aminobutyric acid to synthesize a general hapten containing the common structure of various organophosphorus pesticides - O, O-diethylphosphorothioate O,O-diethyl-N-(3-carboxypropyl)thiophosphoramidate (DENP). details as follows:
[0033] Weigh 1.0 g, O, O-diethyl thiophosphoryl chloride dissolved in 5 mL of methanol, placed in an ice bath.
[0034] Weigh 0.69g of KOH and 0.67g of 4-aminobutyric acid, and dissolve them in 5mL of methanol.
[0035] While stirring, add the mixed solution of GABA and KOH dropwise into the O, O-diethylphosphorothioate solution. React in an ice bath for 2 h, filter the reaction mixture, concentrate under reduced pressure, then use 20 ml of NaHCO solution with a concentration of 50 mg / mL to transfer the concentrated solution to a separatory funnel, extract with 10 mL of n-hexane, discard t...
Embodiment 2
[0051] After the antibody was obtained in the same manner as in Example 1, when establishing the method for analyzing the organophosphorus pesticide, except that PBS with 10% methanol was used when diluting the organophosphorus pesticide, the rest was the same as in Example 1. The inhibitory effects of various organophosphorus pesticides on the acquisition of antibodies are shown in Table 3,
[0052] Table 3. IC of several organophosphorus pesticides against the obtained antibodies 50 and IC 20
[0053] Pesticide name
Embodiment 3
[0055] After the antibody was obtained in the same manner as in Example 1, when establishing the method for analyzing the organophosphorus pesticide, except that PBS with 20% methanol was used when diluting the organophosphorus pesticide, the rest was the same as in Example 1. The inhibitory effects of various organophosphorus pesticides on the acquisition of antibodies are shown in Table 4,
[0056] Table 4. IC of several organophosphorus pesticides against the antibodies obtained 50 and IC 20
[0057] Pesticide name
[0058] In summary, it can be seen that the obtained antibody has a strong affinity with the hapten DENP, and also has cross-reactivity with phorate, demeton and diphorate, these three organophosphorus pesticides; Organophosphorus pesticides such as thion and quinthion also had a certain affinity with the obtained antibodies; the antibodies had poor affinity with dimethoate, diazinon and chlorpyrifos.
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