Method for preparing pyrethroid pesticide polyclonal antibody
A pyrethroid, polyclonal antibody technology, applied in the direction of serum immunoglobulins, etc., can solve the problems of incompetence, low detection limit and high sensitivity of rapid preliminary screening of large-scale samples
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Embodiment 1
[0018] Synthesis of artificial antigens:
[0019] Synthetic active ester method of immunogen: Dissolve 0.0196 g PBA in 2 ml DMF, add equimolar amounts of DCC and NHS, and stir overnight at room temperature. Slowly add the above mixture to 10 ml buffer containing 0.0900 g BSA (PBS:CBS=4:1), stir magnetically at room temperature for 30 min, then react at 4°C for 6 h, and the reaction solution is added to 1L phosphate buffer at 4°C (pH 7.2) dialyze overnight (change medium 4 times). The dialyzed solution was scanned with BECKMAN DU640 UV scanner, and the binding ratio was calculated.
[0020] Synthesis of Coating Progen:
[0021] The active ester method was used, and the specific preparation method was similar to that of the immunogen synthesis. The dosages were 0.0301 g of PBA, 0.0722 g of DCC, 0.0347 g of NHS, and 0.0805 g of OVA. The dialyzed solution was scanned with BECKMAN DU640 UV scanner to calculate the binding ratio.
[0022] Preparation and purification of antibodi...
Embodiment 2
[0026] The synthesis of the artificial antigen and the preparation and purification of the antibody were carried out according to the steps in Example 1.
[0027] The synthesis of the original coating adopts the following method:
[0028] Dissolve 0.0693 g of 6-aminocaproic acid, 0.0796 g of EDC, and 0.2072 g of OVA in water, adjust the pH to 7.5 with dilute NaOH, react in the dark at room temperature for 18 h, purify with Sephadex G-10, freeze, and store in dryness. Dissolve the above sample in water containing 20% DMF, otherwise known as PBA 0.1090 g, dissolve it in DMF, slowly add it dropwise to the above sample, and then add DCC (0.0932 g) dissolved in DMF, so that the final concentration of DMF is 30 % (v / v) was reacted in the dark at room temperature for 18 h, the mixture was filtered to remove precipitates, and dialyzed. The dialyzed reaction solution was scanned with BECKMAN DU640 UV scanner to calculate the binding ratio.
Embodiment 3
[0030] Establishment of standard inhibition curve
[0031] According to the principle of square matrix titration, determine the optimal working concentration of antigen and antibody. Under the optimal working conditions, the inhibition rate (I%) of different concentrations of PBA on the binding reaction between the two coating agents and the antibody was determined, I=100[(OD 对照 —OD 药剂 ) / OD 对照 ]. Draw the standard curve with the inhibition rate as the ordinate and the standard sample concentration (C) as the abscissa. Through regression analysis, the regression equation I=B lgC+A is established, and the concentration I in the inhibitory calculation is calculated. 50 and the lower limit of detection I 10 .
[0032] specificity analysis
[0033] Respectively measure the I of permethrin, cypermethrin, deltamethrin, cyhalothrin, fenpropathrin, fenvalerate and other pyrethrin pesticides to the antibody reaction 50 value, I of PBA 50 Other pesticides with pyrethroids I ...
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