A Time-Resolved Fluorescence Immunoassay of Golgi Protein 73 and Its Application in Hepatitis B Virus Related Liver Cirrhosis

摘要

Chronic hepatitis B (CHB) is one of the world's most common liver diseases, causing huge health burden all over the world. Golgi protein 73 (GP73) is a novel biomarker for liver injuries that is also considered to be an indicator when the disease progresses in CHB patients. The aim of this study was to develop a time-resolved fluorescence immunoassay (TRFIA) assay for GP73 and primarily apply it in early diagnosis of disease progression in CHB patients. The sandwich immunoassay was used to detect the concentration of GP73 in both solutions and serum. GP73 was captured by anti-GP73 monoclonal antibody immobilized on micro titer wells and then banded together with another anti-GP73 antibody labeled with europium (Eu3+) chelates. Finally, the fluorescence was measured by using time-resolved fluorometry on a microplate reader. The performance of this TRFIA was evaluated using clinical serum samples. The limit of detection (LOD) for the GP73 was 1.75 ng/mL and the linear range was from 3.91 to 4000 ng/mL. When GP73 concentration was extended to more than 4000 ng/mL, Hook effect became quite obvious. The intra-assay CVs were between 4.70% and 9.95%, and the inter-assay CVs ranged from 7.50% to 14.90%. The recovery ranged from 80.83% to 114.57%. For interference test, though cross-reactivity between GP73 and three other types of chemicals were all very low, and hemoglobin appeared to have influence during the GP73 detection. The TRFIA assay was successfully developed and validated by 122 samples, including 48 liver cirrhosis (LC), 44 CHB and 30 normal individuals. Serum GP73 (sGP73) levels in the LC group were higher than those in the CHB and healthy groups. The developed TRFIA assay is of high sensitivity, specificity and accuracy. When this assay was applied to clinical samples, the results showed that the sGP73 levels in the LC patients were significantly higher than those of CHB patients and healthy controls, indicating that the TRFIA assay could be an optimal l tool for evaluating the sGP73 levels in patients with hepatitis B virus related liver cirrhosis. All the results laid the solid foundation for subsequent application of this novel assay in clinical use.