程序性细胞死亡因子5夹心ELISA检测方法的建立及初步应用

Development of Sandwich ELISA for Detecting Serum Programmed Cell Death 5 and Its Clinical Application

建立一种快速检测人血清中程序性细胞死亡因子5(PDCD 5)的酶联免疫吸附试验(ELISA)方法,初步研究PDCD 5蛋白在血清中含量变化与疾病的相关性。用抗PDCD 5多克隆抗体包被酶标板,高碘酸钠法标记PDCD 5单克隆抗体作为酶标抗体,以PDCD 5重组蛋白为标准蛋白绘制标准曲线,以特异性、稳定性和灵敏度对ELISA方法进行评价。检测81份癌症患者及21份类风湿性关节炎患者血清,探讨血清PDCD 5蛋白与癌症、免疫缺陷病的相关性。经双扩散试验检测标记后的抗体和酶都具有生物学活性,初步建立了检测人血清PDCD 5蛋白的定量ELISA方法,该方法具有良好的特异性、稳定性,灵敏度达0.5ng/mL。临床检测应用表明,类风湿性关节炎患者血清PDCD 5含量明显高于正常人血清,癌症患者血清PDCD 5蛋白水平明显低于正常人含量(P<0.05),各种癌症患者之间PDCD 5含量差异不显著(P>0.05)。成功建立了一种检测人血清PDCD5的定量ELISA方法,为疾病的诊断提供临床参考。

To establish an enzyme-linked immunosorbent assay（ELISA）method to detect PDCD5 in human serum and evaluate the relitationships between patients and their PDCD5 quantity in blood,the microplate was precoated with anti-PDCD5 PcAb,and PDCD5 McAb linked with enzyme HRP to catalize substrates.The recombinant PDCD5 protein was used as standard,and the standard curve was established.Thus the sandwich ELISA method was developed for detecting PDCD5 in human serum quantitatively.After evaluating ELISA method in specificity,sensitivity and stability,the method was applied in detecting serum PDCD5 in 81 patients of cancer,and 21 patients of rheumatoid arthritis,then the relationship between serum PDCD5 levels and cancer,rheumatoid arthritis was evaluated.The results showedthe double diffusion assay confirmed the enzyme and antibody had the biological activity.And a quantitative ELISA method for detecting PDCD5 in human serum was successfully set up.The sensitivity of this medthod reached 0.5 ng/mL,the accuracy and reproducibility were good.Our results showed the concentration of serum PDCD5 was significantly higher in immunocompromised patients serum than in normal serum（P〈0.01）;The concentration of serum PDCD5 was significantly lower in cancer patients serum than in normal serum（P〈0.05）.There was no significant difference among several cancer patients.A rapid and simple quantitative ELISA method for detecting PDCD5 in human serum was established.And Provide reference value for clinical diagnosis of disease.