测定人红细胞嘧啶5′核苷酸酶含量的双抗体夹心ELISA方法

Establishment of Quantitative Assay for Human Erythrocyte Pyrimidine 5′-Nucleotidase Content by Double Antibody Sandwich ELISA

为了进一步探讨嘧啶 5′核苷酸酶 (P5′N)缺乏症的发病机制 ,我们利用P5′N的特异性底物尿苷一磷酸 (UMP)作为配基 ,制备尿苷一磷酸 己二酰肼 琼脂糖 4B(UMP ADH Sepharose 4B)亲和层析柱 ,结合硫酸铵分级分离和沉淀、离子层析及亲和层析等方法提纯人红细胞P5′N。将所得P5′N免疫家兔 ,获得兔抗人抗体。以兔抗人P5′N抗体为包被抗体 ,辣根过氧化物酶 (HRP) 兔抗人P5′N抗体为显示抗体 ,经方阵法、抗原阻断试验及抗原替代试验后 ,建立定量测定人红细胞的P5′N双抗体夹心酶联免疫吸附实验。实验结果显示 ,所得兔抗人红细胞P5′N抗体效价为 1∶4 ,所建双抗体夹心ELISA法灵敏度为 5ng ml,其阻断率大于 95 %,而取代率小于 30 %。同时 ,测定正常人红细胞P5′N含量为 (71.77± 10 .98)ng mgNHP。该法特异性强 ,敏感性高 ,可检测最低含量为 5 - 2 0ng ml的P5′N含量 ,在临床上能适应大样本测定。

For exploring pathogenesis of pyrimidine 5′-nucleotidase(P5′N) deficiency, a quantitative assay method for human erythrocyte pyrimidine 5′-nucleotidase was established. The specific substrate uridine monophosphate(UMP) of P5′N was used as ligand. The UPM-ADH-Sepharose 4B affinity column was prepared. P5′N of human erythrocyte was purified by ammonium sulfate fractionation and precipitation, ion chromatography and affinity chromatography. Rabbit anti-human P5′N antibody was acquired by immunizing rabbits with purified P5′N. Using rabbit anti-human antibody as the coated anti-body and HRP-rabbit anti-human antibody as demonstrated anti-body, the double antitody sandwich ELISA for quantitative assay of human erythrocyte P5′N was established after square rank trial, antigen blocked trial and antigen substituted test. Results showed that the titer of rabbit anti-human erythrocyte was 1∶4 and the sensitivity of double antibody sandwich ELISA was 5 ng/ml. Its blocking rate was more than 95% and the rate of substitution less than 30%. The content of P5′N was (71.77+10.98) ng/mg NHP in normal human erythrocyte. A new ELISA method for quantitative determination of human erythrocyte P5′N was first established. It not only had high specificity and sensitivity but also could assay the minimun content of P5′N as 5-20 ng/ml. It could be a suitable method for large sample in clinics.