混合酸酐法合成雌二醇酶标抗原偶联工艺影响因素探讨

Synthesis of enzyme labeled estradiol antigen by mixed anhydride

目的研究混合酸酐法合成雌二醇酶标抗原偶联工艺影响因素。方法主要研究有机溶剂种类(二甲基亚砜、N-N二甲基甲酰胺、二氧六环)及有机溶剂添加量、雌二醇衍生物成酐温度及时间、反应摩尔比等偶联效果影响因素。结果有机溶剂选择N-N二甲基甲酰胺,添加量为50%,雌二醇衍生物成酐温度为冰盐浴,成酐时间为30 min,反应摩尔比为10∶1,偶联反应结束后G52层析纯化,冻干浓缩用PBS复溶后加入等体积甘油,得到的酶结合物效价高于1×10~4。结论通过偶联工艺优化获得高效价雌二醇酶标物。

Objective This paper studied the influence factors when synthesis of enzyme labeled estradiol antigen by Mixed anhydride. Methods Those factors include species of organic solvents（ dimethyl sulfoxide,N-dimethylformamide,dioxane） and the amount of organic solvent. The temperature and time of anhydride formation of estradiol derivatives,molar ratio of estradiol and HRP. Results The results showed that the organic solvents was N-dimethylformamide and its amount in horseradish peroxidase solution was 50%,the temperature of the anhydride of the estradiol derivative was 30 min,the molar ratio of estradiol and HRP was 10∶1. When the synthesis reaction was finished,choice G52 chromatography purification those sample,then lyophilized concentrate was reconstituted with PBS and an added equal volume of glycerol,the enzyme labeled estradiol antigen titer higher than 1 × 10~4. Conclusion Objective to obtain high efficient estradiol enzyme target by the hinge process optimization