| Walnut is one kind of nut with both economic and highly nutritional values. It was reported that walnuts have high protein content (26.1g/100g of raw walnut). In recent years, a variety of walnut products such as walnut powder and walnut milk mixed with the cheaper peanut powder or soy flour were sold to obtain greater profits in the Chinese market. Thus, it is essential for the adulteration test of walnut products to find a suitable method.Two kinds of antiserum against walnut protein were produced by immunization of New-Zealand white rabbits and Guinea Pigs. And the titers of antiseras were determined by an indirect ELISA method. It indicated that the immunized rabbits and guinea pigs were more sensitivity to the walnut protein than to the others (peanut, soybean and sesame) with the higher titer for rabbit sera of108and guinea pig sera of106. Furthermore, the rabbit antibody was purified and labeled by HRP. The rabbit antibody labeled by HRP had a titer of104. Furthermore, the specificity of the antibodies were determined by testing how tree nuts ingredients were respond with them. The results indicated that the two kinds of complex antibodies of the assay exhibited no cross-reactivity among these food ingredients and high specificity with walnut. Therefore, both antibodies had the the basic conditions for the establishment of ELISA method.The indirect ELISA method was established by rabbit anti-walnut protein polyantibody and goat anti-rabbit IgG polyvalent horseradish peroxidase conjugate. The reaction condition was optimized at the basic of expanding the range of determination and lower OD (450nm) value of control. Finally, the optimal reaction conditions were as the follows:the walnut standard solutions and food samples were diluted with coating buffer (0.05M carbonate-bicarbonate buffer, pH9.60) and coated on microtiter plates for2h at37℃or overnight at4℃. The washing buffer was PBS with0.05%Tween-20, blocking and dilution buffer were PBS with5%defatted milk and0.05%Tween-20. After washing, the plates were blocked for2h at37℃. The rabbit anti-walnut antibody was diluted (1:1000) with PBS and incubated for1h at 37℃. The goat anti-rabbit IgG polyvalent horseradish peroxidase conjugate with PBS for1h at37℃. The results indicated that the linear range of detecting walnut protein was from800-25ng/mL (R2>0.99). The recoveries of walnut protein solution ranged from88.47to111.5%.The sandwich ELISA was established by anti-walnut protein guinea pig and rabbit polyclonal antibody. The reaction condition was optimized at the basic of expanding the range of determination and lower OD (450nm) value of control. The optimal reaction conditions were as the follows:the washing buffer was PBS with0.05%Tween-20. blocking and dilution buffer were PBS with5%defatted milk and0.05%Tween-20. The blocked (first) anti-walnut protein guinea pig polyclonal antibody was diluted to1:5000in0.05M carbonate-bicarbonate buffer (pH9.6)2h at37℃or overnight at4℃. After washing, the plates were blocked for2h at37℃. Then the capture (second) rabbit polyvalent antibody of anti-walnut protein was diluted to1:1000100μL/well for1h at37℃. The results indicated that the linear range of detecting walnut protein was from160-20ng/mL (R2>0.99). The recoveries of walnut protein solution ranged from84.25to99.84%.Based on the results, the two kinds methods for detecting walnut protein were compared with each other. The sandwich ELISA method was chosen as the optimal method. After assembling the sandwich ELISA kits, the walnut products came from the market were determined. The results showed that parts of the products in dairy market were seriously adulterated. So the walnut sandwich ELISA kits meets the needs of walnut protein determination and adulteration detection on the market. |
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