| Asialoglycoprotein receptor?ASGPR?is a liver-specific glycoprotein receptor,which not only exists on the surface of liver cell membrane,but also secretes into serum in a soluble form.In order to study the relationship between human ASGPR and the severity of liver injury,an ELISA method for quantitative detection of human ASGPR was established.Through the detection and statistical analysis of a large number of clinical serum samples,the correlation between sASGPR content in serum and liver injury was established,providing certain clinical value for the diagnosis of liver injury.?1?Establishment and optimization of ELISA method:The standard sASGPR used in the ELISA assay was purified by affinity chromatography from HepG2 cell culture supernatant.Western Blot analysis results showed that the purified sASGPR can be specifically recognized by ASGPR antibody.The concentration of standard preparation was determined to be 0.13mg?mL-1 by BCA method.GSA as the specific ligand of ASGPR,was synthesized by the reaction of HSA with aminogalactose,and the concentration of GSA was determined to be 6.6mg?mL-1.An ELISA method for quantitative detection of human ASGPR was established,and a mouse anti-human ASGPR1/2?E-1??Santa Cruz Biotechnology,SC-166633?antibody and goat anti mouse IgG-HRP were selected.The coating buffer was 0.05 M CBS buffer and the optimal coating concentration of GSA was 20?g?mL-1.The whole coating process took 24hours at 4?.Using 1%skim milk as the blocking solution,the bloking needed 2 h and was at37?.The incubation time of the sample was 2 h at 37°C.The first antibody was used in 1:50dilutions while the HRP-labeled antibody was used in 1:2000 dilutions.The TMB development time was 15 min.?2?Methodology validation:The minimum detection limit was 3.80?g?L-1,and the minimum quantitation limit was 5?g?L-1.The inter-assay coefficient of variation was 2.56%-5.64%.The intra-assay coefficient of variation was 2.14%-5.91%.The detection linear range was 4?g?L-1-100?g?L-1 with linear correlation coefficient r?29?0.990.The sample recovery rate was between 91.40%and 104.60%?permissible range:100%?10%?.The coated ELISA plate can be stored for 10 days at 37°C.The interference limit to hemoglobin was 2 g?L-1,the interference limit to triglyceride was 20 g?L-1,and the interference limit to bilirubin was 0.4g?L-1.?3?Application:?I?The sASGPR in the culture supernatant of HepG2 cells and Huh7 cells can be detected by this ELISA method.?II?A large number of clinical serum samples?healthy group and liver injury group?were detected by the ELISA method.Statistical analysis showed that sASGPR level in 18-60 age group and?29?60 age group were 91.60±61.01?g?L-11 and63.10±48.67?g?L-1,respectively.The difference between these two groups was statistically significant?P?27?0.001?.According to statistical analysis,gender was not a factor that would result in sASGPR content difference.Statistical analysis showed that sASGPR level in healthy group and liver injury group were 85.94±59.69?g?L-11 and 20.41±10.59?g?L-1,respectively.There was also a significant statistical difference between these two groups?P?27?0.001?.Drew ROC curve and the area under the ROC curve?AUC?was 0.852.The maximum of“Youden's index”?sensitivity+specificity-1?was 0.810,and the sensitivity and specificity were 88.34%and95.45%,respectively.Total compliance rate was 88.72%,cut-off value was 30.67?g?L-1,and kappa value was 0.426.The results indicated that the level of sASGPR in serum of patients with liver injury was lower than that of healthy serum.Therefore,sASGPR,as a new potential serum marker of liver injury,would provide certain clinical diagnostic value for the determination of liver injury.Besides,the ELISA method which could detect ASGPR quantitatively would achieve auxiliary effect in the diagnosis of liver injury. |
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