Preparation and application of group-selective monoclonal antibodies against eight triazine pesticides
A triazine, pesticide multi-residue technology, applied in animal/human proteins, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of less research on triazine pesticides, and achieve high sensitivity and low detection cost. , the effect of simple operation
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Embodiment 1
[0046] Embodiment 1: the preparation of hapten
[0047] (1) Synthesis of immune hapten 4-(4-isopropylamino-6 methylthio-1,3,5-triazine-2-)aminobutyric acid
[0048]①In a 50mL three-neck flask, dissolve 2,4,6-trichloro-1,3,5-triazine (2g) and isopropylamine (644mg) in dry dichloromethane (20mL), and cool to -30°C under nitrogen protection. ℃, slowly drop 10mL of diisopropylethylamine (DIPEA) (2.1g) diluted with dichloromethane into the flask, react at -20℃~-10℃ for 2h, concentrate the reaction solution, spin dry, and disperse in 50mL Water, filtered, collected filter cake, and recrystallized with ethanol to obtain 2.1 g of intermediate a (4,6-dichloro-N-isopropyl-1,3,5-triazin-2-amine) as a white solid.
[0049] ②In a 225mL three-neck flask, dissolve the above-mentioned intermediate a (1g), 4-aminobutyric acid (400mg) and DIPEA (939mg) in 10mL of absolute ethanol, and react at 60°C for 12 hours under the protection of nitrogen. Concentrate the reaction solution and spin dry. ...
Embodiment 2
[0059] Embodiment 2: the preparation of artificial antigen
[0060] (1) Preparation of immune antigen
[0061] Weigh the immune hapten H10.015mmol, NHS 0.03mmol, DCC 0.03mmol, dissolve in 200μL dimethylformamide (DMF), seal the reaction at room temperature under magnetic stirring overnight, centrifuge at 10000rpm for 5 minutes, take the supernatant and slowly add it to 1mL In 10mg / mL OVA-PBS solution, stirred at 4°C for 12h. After the reaction was completed, the reaction solution was placed in a dialysis bag and dialyzed in 0.01 mol / L pH 7.4 PBS buffer solution at 4°C for 72 hours, and the dialysis solution was changed every 6 hours. After the dialysis, the white emulsion (H1-OVA) in the dialysis bag was taken out and stored in a refrigerator at -20°C.
[0062] (2) Preparation of coated antigen
[0063] The coating antigen is prepared by coupling the coating hapten H2 with BSA, and the method is the same as the coupling of the immune antigen.
[0064] (3) Artificial antige...
Embodiment 3
[0067] Example 3: Animal Immunization
[0068] H1-OVA was used as an immunogen to immunize 6-8 week-old Balb / c female mice. Each mouse was given 100 μg of immunogen subcutaneously in the abdominal cavity and back of each mouse, once every two weeks, and immunized four times in total. For the first immunization, an equal volume of immunogen was emulsified with Freund's complete adjuvant, and for subsequent immunizations, an equal volume of immunogen was emulsified with Freund's incomplete adjuvant. One week after the third immunization, blood was collected from the orbit of the mice, and the titer and specificity of the serum were detected by indirect ELISA.
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