Ⅰ型前胶原氨基端肽化学发光免疫分析检测方法的建立及评价

Development and evaluation of a chemiluminescence immunoassay for type Ⅰ procollagen N-terminal peptide

摘要:

为了建立定量检测人血清中Ⅰ型前胶原氨基端肽 (Type Ⅰ procollagen N-terminal peptide，PINP) 的化学发光免疫分析检测方法，首先在谷氨酸棒状杆菌中分泌表达了PINP-α1链重组蛋白，以其为免疫原制备单抗，获得了2B10、8C12和1F11共3株可稳定分泌抗PINP-α1链的单抗杂交瘤细胞株。进一步配对筛选后，以单抗8C12偶联生物素作为捕获抗体，单抗1F11标记辣根过氧化物酶作为检测抗体，二者与样本中的PINP结合形成夹心复合物，再与包被有链霉亲和素的磁微粒形成完整的检测系统，从而定量检测人血清中PINP的浓度。对该方法进行条件优化后，确定捕获抗体、检测抗体的最佳工作浓度均为3 μg/mL，孵育时间为30 min；本方法的最低检测限为1.22 ng/mL，批内、批间的变异系数均在10%以内，线性范围为5–1 100 ng/mL，回收率在93%–107%之间。通过对160份临床样本进行检测，本方法检测结果与罗氏诊断试剂盒检测结果的相关系数R2为0.906 2。开发的磁微粒化学发光免疫分析检测方法可定量检测人血清中PINP的含量，有望成为骨骼疾病检查的辅助手段。

Abstract:

To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5–1 100 ng/mL, with recovery rates between 93%–107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.