α1-酸性糖蛋白单克隆抗体的制备及检测方法的建立

Preparation of Monoclonal Antibody and Development of An ELISA for α1-Acid Glycoprotein

目的制备α1-酸性糖蛋白（α1-Acid glycoprotein，α1-AGP）单克隆抗体（McAb），建立α1-AGP的ELISA检测方法，用于检测人体血液、尿液及各种组织液中α1-AGP的水平。方法用高氯酸沉淀人血清中α1-AGP，经离子交换层析和高压液相层析纯化后，免疫BALB／c小鼠，建立特异性抗α1-AGP单克隆抗体杂交瘤细胞株，制备并鉴定McAbs，分析各株分泌抗体的效价、特异性、位阻群、类及亚类。制备针对α1-AGP的双抗体夹心ELISA检测试剂盒，分析其稳定性、灵敏度和特异性，并进行临床评价。结果建立了9株特异性抗α1-AGP单克隆抗体杂交瘤细胞株，均分泌IgG抗体，所有抗体的轻链均为κ链。根据位阻分析结果，将9个细胞株分泌的抗体分为5个位阻群，腹水效价在1×10-4 - 1×10^-7之间，且均有较好特异性。制备的α1-AGP双抗体夹心ELISA试剂盒灵敏度达2ng/ml，且检测结果与α1-AGP含量呈现良好的线性关系（r=0．9916），并与其他血清蛋白无交叉反应，试剂盒置37℃放置3d及4℃放置2个月，稳定性良好。用该试剂盒检测96份正常人血样，其α1-AGP平均值为0．43—1．32mg／ml；105份Ⅱ型糖尿病患者血样，其平均值为0．88—2．35mg／ml，经统计学分析，两者差异有显著意义。结论已成功制备出α1-AGPMcAb，并建立了针对α1-AGP的双抗体夹心ELISA，制备的试剂盒可用于临床检测。

Objective To prepare monoclonal antibody（McAb） and develop an ELISA for α1-acid glycoprotein（ α1-AGP） in blood, urine and tissue fluid of humans. Methods Precipitate the α1-AGP in human sera with perehloric acid and purify by ion exchange chromatigraphy and HPLC. Immunize BALB/c mice with the purified α1--AGP to establish the hybridome cell strains secreting specific McAb to α1-AGP. The specific McAbs were prepared and identified, then analyzed for titer, specificity, steric groups, class and subclass. Select the McAb with high sensitivity and good specificity as a candidate antibody to develop an ELISA kit. The developed kit was analyzed for stability, sensitivity and specificity, and evaluated in clinic. Results Nine hybridoma cell strains secreting specific McAbs to α1-AGP were established. All the secreted McAbs were IgG,and their light chains were of x type.The McAbs were divided into 5 steric groups,all of which showed good specificity and titers of 1×10-4 - 1×10^-7 in ascites. The developed ELISA kit reached a sensitivity of 2 ng/ml and showed no cross reaction with other serum protein,and good linear relationship was observed between detection result and α1--AGP content （ r = 0.991 6）. The kit showed good stability after storage at 37℃ for 3 d or at 4℃ for 2 months.The mean al-AGP levels in blood samples from 96 healthy persons and 105 patients with diabetes type Ⅱ determined by the kit were 0.43 - 1.32 mg/ml and 0.88 -2.35 mg/ml respectively, which showed significant difference. Condusion The specific McAb to α1--AGP was successfully prepared, and an ELISA kit for αl-AGP was developed, which might be used for the assistant diagnosis fo diabetes in clinic.