不同稀释液对混合酶标抗体稳定性的影响

Effect of different diluents on stability of mixed enzyme-labeled antibody

目的 探讨不同稀释液对混合酶标抗体稳定性的影响,筛选出一种适用于混合酶标抗体的稀释液,可同时保护辣根过氧化物酶(horseradish peroxidase,HRP)偶联二抗和碱性磷酸酶(alkaline phosphatase,AP)偶联二抗鸡尾酒混合物的稳定性。方法 以Tris-HCl缓冲盐溶液为基础溶液,设置4种不同的稀释液配方(A、B、C、D),每种配方包含不同类型的稳定剂,如蛋白、金属离子、表面活性剂、抑菌剂等,采用不同的稀释液配置混合酶标抗体,在冷藏(2~8℃)和37℃条件下存储,通过ELISA、酶活测定及免疫组织化学(immunohistochemistry,IHC)染色法检测其稳定性。结果 D液配方[Tris(50 mmol/L)+牛血清白蛋白(1.5%,g/100mL)+Tween-20(0.3%)+Proclin-950(0.1%,g/100 mL)+氯化钠(150 mmol/L)+L-Ascorbic Acid(1 mmol/L)+L-methionine(5 mmol/L)+L-histidine(5 mmol/L)+酪蛋白酸钠(1%,g/100 mL)+氯化锌(0.1 mmol/L)+海藻糖(8%,g/100 mL)]保存试剂的稳定性最好,在37℃条件下保存10周,HRP偶联二抗和AP偶联二抗的工作液效价最高,HRP和AP的活性剩余比率分别为93.46%和96.07%,IHC结果 无明显差异。结论 该配方稀释液可用于混合酶标抗体工作液的配置。

Objective To investigate the effect of different diluents on the stability of the mixed enzyme-labeled antibody,and screen a suitable diluent for the mixed enzyme-labeled antibody,which can protect the stability of the cocktail mixture of horseradish peroxidase(HRP) conjugated secondary antibody and alkaline phosphatase(AP) conjugated secondary antibody.Methods Using Tris-HCl buffer as the base solution,four different diluent formulations(A,B,C,D) were prepared with different kinds of stabilizers contained in each formula,such as protein,metalion,surfactant and bacteriostatic agent.Mixed enzyme-labeled antibodies were prepared with different diluents and stored at 2~8 ℃ and 37 ℃,which were detected for the stability by ELISA,enzyme activity assay and immunohistochemistry(IHC) staining.Results D solution [Tris(50 mmol/L)+BSA(1.5%,g/100 mL)+Tween-20(0.3%)+Proclin-950(0.1%,g/100 mL)+sodium chloride(150 mmol/L)+L-Ascorbic Acid(1 mmol/L)+L-methionine(5 mmol/L)+L-histidine(5 mmol/L)+sodium caseinate(1%,g/100 mL)+zinc chloride(0.1 mmol/L)+trehalose(8%,g/100 mL)] was of the optimal protective effect.When stored at 37 ℃ for 10 weeks,the working solution of HRP conjugated secondary antibodies and AP conjugated secondary antibodies showed the highest titer,the activity residual ratios of HRP and AP were 93.46% and96.07%,respectively,and there was no significant difference in IHC results.Conclusion This formula diluent can be used for the preparation of mixed enzyme-labeled antibody working solution.