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目的建立双抗体夹心ELISA方法,定量检测重组小鼠共抑制分子B7-H4(mB7-H4)-人IgGα1 Fc段融合蛋白(mB7-H4-hIg)的分泌型表达。方法将表达mB7-H4-hIg的真核表达载体pmB7-H4-Fc转染COS-7细胞,并将该载体注入小鼠体内,制备重组mB7-H4-hIg融合蛋白,以羊抗人IgG为包被抗体,HRP标记的羊抗人lgG为检测抗体,通过配对实验、方阵滴定实验及绘制hlgG浓度与A450的标准曲线,建立定量检测重组mB7-H4-hlg融合蛋白双抗体夹心ELISA法。结果建立了双抗体夹心ELISA方法,检测的线性范围为10～500ng/ml。标准曲线的回归方程为:y=0.0028x+0.0899,R2=0.9762,P<0.05.应用该方法可快速检测体外和体内表达的重组mB7-H4-hlg融合蛋白的分泌量。结论建立了一种可快速定量检测重组mB7-H4-hlg融合蛋白分泌表达的双抗体夹心ELISA法。
OBJECTIVE: To establish a double-antibody sandwich ELISA method for the quantitative detection of the secretory expression of recombinant mouse costimulatory molecule B7-H4 (mB7-H4) -human IgGα1 Fc fragment fusion protein (mB7-H4-hIg). Methods The mB7-H4-hIg eukaryotic expression vector pmB7-H4-Fc was transfected into COS-7 cells. The vector was injected into mice to prepare recombinant mB7-H4-hIg fusion protein. Coated antibody and HRP-labeled goat anti-human lgG as the detection antibody. Through the paired experiment, square titration experiment and the standard curve of hlgG concentration and A450, a sandwich ELISA method for the quantitative detection of recombinant mB7-H4-hlg fusion protein double antibody was established. Results A double antibody sandwich ELISA was established. The linear range was 10 ~ 500ng / ml. The regression equation of the standard curve was: y = 0.0028x + 0.0899, R2 = 0.9762, P <0.05. This method can be used to rapidly detect the secretion of recombinant mB7-H4-hlg fusion protein expressed in vitro and in vivo. Conclusion A double-antibody sandwich ELISA was developed to detect the secretion of recombinant mB7-H4-hlg fusion protein rapidly and quantitatively.