双抗夹心法酶联免疫吸附试验影响因素探析

Exploration and analysis on influence factors in double antibody sandwich enzyme-linked immunosorbent assay

目的探析双抗夹心法酶联免疫吸附试验中牛血清清蛋白封闭,吐温20及浓度,样品与检测抗体的加样方式及孵育温度对酶联免疫吸附试验检测结果的影响。方法双抗夹心法酶联免疫吸附试验检测人白细胞介素(IL)-1Ra细胞因子作为模型,检测封闭条件,吐温20及其不同浓度,样品与检测抗体分开孵育及共同孵育,酶联孵育温度对标准品浓度与OD值关系的影响。结果酶联免疫吸附试验不封闭、洗液吐温浓度为0.05%且孵育温度为37℃的R^2=0.995 7;BSA封闭的R^2=0.981 1;洗液中吐温浓度0.005%时R^2=0.991 5,0.5%时R^2=0.9868;样品和检测抗体一同孵育1h或2h的R^2分别为0.964 5和0.874 2;孵育温度为25℃的R^2=0.996 1。结论BSA封闭步骤对低浓度的样品检测灵敏度不强;样品和检测抗体及酶稀释液中必须含有吐温20且浓度在0.05%条件下最合适;样品和检测抗体共同孵育会影响对高浓度样品的检测;37℃和25℃的孵育温度条件下的酶联免疫吸附试验检测灵敏度没有差异,但25℃孵育可以降低OD值背景。

Objective To explore and analyze the influence of blocking of bovine serum albumin,tween 20 and its concentration,sample adding mode of sample and detecting antibody and incubating temperature on the detection results of the double antibody sandwich enzyme-linked immunosorbent assay.Methods Detecting human IL-1Ra cytokines by the double antibody sandwich enzyme-linked immunosorbent assay was used as a model to measure the influence of the closure condition,tween 20 and its different concentrations,separated incubation and co-incubation of sample and detecting antibody,incubation temperature on the relation between standard substance concentration and ODvalue.Results R2 was 0.995 7when the enzyme-link was not closed,the concentration of tween 20 was 0.05%and incubating temperature was 37℃;R2 by blocking BSA was 0.981 1;when the concentration of tween 20 was0.005% or 0.5%,R2 was 0.991 5and 0.986 8respectively;R2 when the sample and the detection antibody were incubated together for 1hor 2hwas 0.964 5and 0.874 2respectively;R2 was 0.996 1when incubating at 25℃.Conclusion The BSA blocking step does not improve the detection sensitivity for low concentration samples;the sample,detection antibody and enzyme diluent must contain Tween 20 and the concentration less than 0.05%is most appropriate;the co-incubation of sample and detection antibody could affect the detection of the high concentration of sample;the enzyme-linked detection sensitivity has no difference under the conditions of incubation temperature of 37 ℃and 25 ℃,but the background of ODvalue could be reduced in incubation at 25 ℃.