Development Of Enzyme-linked Immunosorbent Assay For Daizein And Detection Of Daidzein Concentration In Sera Of Rats, Egg And Diet

1. The development of ELISA for DaidzeinThe antigen (OA-Da,BSA-Da)was made by the reaction of Da with ovi-albumin and bovi-albumin respectively .The poly-cloned antibody was prepared by immunizing New Zealand rabbits with ovi-albumin-Da(OA-Da)and was tested by double immunodiffusion against bovi-albumin-da(BSA-Da).The highest titer was 1:16.The ELISA result showed :the hightest value of anti-Da sera was 1:7000,the antibody has immuno-across reaction with HK99J-OH isoflavone and genistein which recovery was 5%,15%andlO% respectively.In the inhibitory competitive ELISA ,the concentration coating antigen and antibody were determined by chess-board experiment ,the regression equation was Logit Y=-1.26LgC+4.37(R2=0.98), inter-well variation were 1.30%(n=8). inter-plate variation were 4.30%(n=4),minimum detectable concentration was 0.lug/ml, the standard curve was liner within the range from 0. 10 ug/ml to 4.00 ug/ml ,the stability test showed CV=0.70%(n=8).2. Effect of daidzein and enzyme preparation supplemented to basal diet on the performances and Da concentration in sera and diet of ratsForty-eight growing rats including male and female were randomized into four groups respectively: basal diet ,basal diet plus 3mg/kg Da (Da),basal diet plus 0.01%enzyme preparation (E),basal diet plus 3mg/kg Da and 0.01%enzyme preparation (Da+E). Daidzein and enzyme preparation supplementation increased rats body weight gains ,feed conversion rate ,and daidzein concentration of sera and diet.In female rats compared with control ,Da, E and Da plus E groups respectively increased weight gains by 34.45%(p<0.01),25.50% and 31.79%,increased feed conversion rate by 17.45%,6.83%and 10.62%,increased Da concentration in sera by 16.53%,32.00%and 57.60%(p<0.01).In male rats ,comparing with control ,Da, E and Da plus E groups respectively increased weight gains by 15.32%,11.97%and 7.5%,and increased feed conversion rate by 6.30%,0.79%and6.59%, furthermore ,Da concentration in sera increased significantly (p<0.01).Daidzein in diets pretreated with 3 -glucuronidase were extracted with 95%ethanol. Compared to control , Da, E and Da plus E increased Da concentration by 23.06%,104.93%and 140.22% respectively.In conclusion ,Da and enzyme preparation increased rats performances and Da concentration in the sera of rats .To a certain degree, 3 -glucuronidase and enzyme preparation can speed Da released from diets and improve Da' s absorptivity . 3. Extraction and detection daidzein in eggs20 laying hens were randomly allotted to 2 groups which were feed basal diet and basal diet plus 6mg/kg Da. The daidzein in eggs which were collected in sequence three days were extracted with three methods and measured by ELISA. The test result showed extraction Da with P -glucuronidase was better than the other two method .comparing with eggs white ,Da concentration in eggs yolk increased by 5.49%.