Establishment And Application Of IL-18 Time-resolved Fluorescence Immunoassay

Purpose and background:There are a large number of people suffering from hepatitis and liver cancer in our country,and the high incidence and high mortality caused by them have brought great suffering to people.There are many types of hepatitis,including:hepatitis B,liver cancer,non-alcoholic fatty liver and so on.However,the detection indicators of each liver disease are different and complex,which brings certain difficulties to the diagnosis of different liver diseases.Interleukin-18（IL-18）,as an inflammatory cytokine,has long been described as having a pro-inflammatory effect.Its pro-inflammatory effect is mainly caused by IL-18 induced interferon-γ（Interferon-γ,IFN-γ）.IL-18 has been reported to increase its concentration in many diseases:such as liver disease,leukemia,kidney disease and diabetes.However,the existing data showed that there are few studies on the concentration changes of IL-18 in hepatitis and liver cancer.Therefore,it is necessary to detect the IL-18 content in the serum of patients and analyze it in combination with clinical data.Currently,the detection method for IL-18 in diseases is enzyme-linked immunosorbent assay（ELISA）,which has insufficient sensitivity and detection range.In this study,a time-resolved fluorescence immunoassay（TRFIA）with high sensitivity,wide linear range,and strong specificity was established.We aimed to establish a time-resolved fluorescence immunoassay（IL-18-TRFIA）for IL-18 and detect its concentration in the serum of five liver diseases:liver cancer,hepatitis B,hepatitis C,non-alcoholic fatty liver and autoimmune hepatitis,and used the healthy controls as a control to evaluate its clinical application value.Method:Fixed the IL-18 coated antibody M02 on a 96-well ELISA plate,and we used Eu³⁺to label IL-18 to detect the antibody M01.We used the double antibody sandwich method to establish IL-18-TRFIA and evaluated the sensitivity and specificity.The IL-18-TRFIA method detected the concentration of IL-18 in the serum of liver cancer,hepatitis B,hepatitis C,non-alcoholic fatty liver,autoimmune hepatitis,and the healthy controls.Then we analyzed them in combination with their clinical data.Results:The intra-assay average coefficient of variation（CV）of IL-18-TRFIA was 4.80%,and the inter-assay average CV was 5.90%.The average recovery rate was 106.19±3.44%,and the sensitivity（10.96 pg/m L）was better than the commercial ELISA kit（62.5 pg/m L）.The detection range was 10.96-2000 pg/m L.Interleukin 6（IL-6）,Galectin-3（Galectin-3）and Interleukin 33（IL-33）had no cross-reactivity with this method.The concentration of IL-18 in serum was hepatitis C（776.99;653.48-952.39 pg/m L）,non-alcoholic fatty liver（911;775.55-1130.03 pg/m L）,liver cancer（1048.88;730.04-1185.10 pg/m L）and hepatitis B（949.12;723.70-1160.28pg/m L）which were significantly higher than the healthy controls（483.09;402.52-599.70 pg/m L）（P<0.0001）.However,the concentration of IL-18 in the serum of autoimmune hepatitis patients was（571.62;502.47-730.31 pg/m L）,which was not significantly different from the healthy controls（P>0.05）.It can be seen that the detection index of IL-18 had certain effects on liver damage and pro-inflammatory effects in patients with hepatitis C,non-alcoholic fatty liver,liver cancer and hepatitis B diagnostic value.Conclusion:This study first established the IL-18-TRFIA method,which was more sensitive than the ELISA method.After linear fitting,the multiple correlation coefficient（R²）of the two methods was 0.9596,and the method was stable.Studies have found that the concentration of IL-18 in the serum of patients with liver cancer,hepatitis C,hepatitis B and non-alcoholic fatty liver was significantly increased.The change in the concentration of IL-18 may have a certain value for the damage and pro-inflammatory effects of hepatitis or liver cancer.