基于SPR技术定量检测肾透明细胞癌组织中NHERF3蛋白水平

Quantification of NHERF3 protein level in clear cell renal cell carcinoma by SPR technology

目的建立基于表面等离子共振(SPR)技术定量检测肾透明细胞癌组织中钠氢交换调控因子NHERF3蛋白水平的方法。方法将抗NHERF3抗体固定在CM5芯片,将重组NHERF3蛋白(0.78-50 nmol/L)和牛血清白蛋白BSA(3.125-100μmol/L)依次注入芯片表面,通过Biacore分析软件计算NHERF3与其抗体的亲和力。将NHERF3蛋白(0.0625-8μg/ml)依次注入高水平固定抗NHERF3抗体的芯片表面,用结合反应值通过非线性回归法建立NHERF3的标准曲线。将NHERF3蛋白稀释至8μg/ml,重复进样50次,检测方法的可重复性。将20对肾癌和癌旁组织蛋白提取液作为分析物,根据反应值计算NHERF3的浓度并比较肾癌和癌旁组织之间的差异。结果抗NHERF3抗体与NHERF3之间可以发生特异性强相互作用(K D=19.7 nmol/L)。该NHERF3定量检测方法的检测限(LOD)和定量限(LOQ)分别为8.12 ng/ml和27.07 ng/ml;50次重复检测的变异系数(CV)为4.25%。肾透明细胞癌组织中NHERF3蛋白的表达水平明显低于癌旁组织[(36.4±54.38)ng/μl vs(155.1±60.47)ng/μl,P<0.001]。结论基于SPR的定量检测方法适用于高通量检测肾透明细胞癌组织中NHERF3的蛋白表达水平。

Objective To establish a SPR method for quantification of NHERF3 protein level in clear cell renal cell carcinoma(ccRCC).Methods Anti-NHERF3 antibody was immobilized on a CM5 chip.Recombinant NHERF3 protein(0.78-50 nmol/L)and BSA(3.125-100μmol/L)were sequentially injected over the sensor surface.Affinity was analyzed with Biacore evaluation software.NHERF3(0.0625-8μg/ml)was injected over the sensor surface coated with anti-NHERF3 antibody at a high level.The NHERF3 standard curve was constructed by nonlinear regression of binding response values.NHERF3 was diluted at a concentration of 8μg/ml and was repeatedly injected for 50 times to assess the repeatability of method.The protein extracts from 20 pairs of ccRCC and adjacent normal renal tissues were served as analytes.The concentration of NHERF3 in tissues was determined by plotting the response value on standard curve and was compared between ccRCC and adjacent normal tissues.Results NHERF3 specifically interacted with anti-NHERF3 antibody with strong affinity(K D=19.7 nmol/L).The LOD and LOQ of the method were 8.12 ng/ml and 27.07 ng/ml,respectively.The signal across 50 cycles was stable with a CV of 4.25%.The expression level of NHERF3 was significantly lower in ccRCC than in adjacent normal tissues[(36.4±54.38)ng/μl vs(155.1±60.47)ng/μl,P<0.001].Conclusion SPR method can be used for high-throughput quantification of NHERF3 level in ccRCC.