人心肌肌钙蛋白Ⅰ鲁米诺化学发光酶免疫分析法的建立

Establishment of chemiluminescence enzyme immunoassay for detecting human cardiac troponin Ⅰ

目的:制备两株抗人心肌肌钙蛋白Ⅰ(human cardiac troponinⅠ,cTnⅠ)单克隆抗体,建立鲁米诺化学发光酶免疫分析法。方法:制备腹水型单抗、饱和硫酸铵沉淀法和亲和层析柱Protein G纯化抗体,并通过SDS-PAGE和ELISA对抗体特异性进行鉴定,建立鲁米诺化学发光酶免疫分析法,并对30份临床血清样本进行检测。结果:本实验制备的两株单抗(分别命名为Ab1、Ab3;亚型都是Ig G1)能够识别以cTnⅠ单体、cTnⅠ-C复合物和cTnⅠ-T-C复合物不同形式存在的cTnⅠ。Ab1、Ab3的效价分别为1∶80万和1∶40万,亲和力常数分别为1.62×109L/mol、2.60×108L/mol。利用2株单抗建立的鲁米诺化学发光酶免疫分析法检测范围为:6.25~400 ng/ml。对30份临床样本进行检测,阳性检出率为77.3%,阴性检出率为100%。结论:建立了可以检测以cTnⅠ单体、cTnⅠ-C和cTnⅠ-T-C复合物形式存在的cTnⅠ的鲁米诺化学发光酶免疫分析法,具有更高的实用性。

Objective： Two anti-human cardiac troponin I（ cTnⅠ） monoclonal antibodies were prepared to establish Luminol chemiluminescence enzyme immunoassay. Methods： Obtaining ascites monoclonal antibody was purified by ammonium sulfate fractionation and protein G affinity chromatography column,and their specificity was identified by SDS-PAGE and ELISA methods. Luminol chemiluminescence enzyme immunoassay was successfully established,which was used to detect 30 clinical serum samples. Results：Two antibodies named Ab1 and Ab3 could recognize different forms of cTnⅠ including cTnⅠ monomers,cTnⅠ-C complexes and cTnⅠ-T-C complexes. Both Ab1 and Ab3 are Ig G1. The titers of the Ab1 and the Ab3 were up to 1 ∶800 000 and 1 ∶400 000,respectively. The affinity constants of Ab1 and Ab3 were 1. 62 × 109 L/mol and 2. 60 × 108 L/mol,respectively. The detection range of Lumino chemiluminescence enzyme immunoassay established by two monoclonal antibodies was 6. 25 ng/ml to 400 ng/ml. Detecting 30 clinical samples attained the positive detection rate of 77. 3% and the negative detection rate of 100%. Conclusion： The chemilu minescence enzyme immunoassay has been established to detect different forms of cTnⅠ including cTnⅠ monomer,cTnⅠ-C complexes and cTnⅠ-T-C complexes,which has better practicality.