Establishment of interleukin‐18 time‐resolved fluorescence immunoassay and its preliminary application in liver disease

ABSTRACT Background To establish a time‐resolved fluorescence immunoassay of interleukin (IL)‐18 (IL‐18‐TRFIA) and detect its concentration in different liver disease serum samples. Methods The IL‐18 coating antibody and the Eu3+‐labeled detection antibody were used for the IL‐18‐TRFIA to detect serum IL‐18 concentration in patients with liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, fatty liver disease, and healthy controls. The double‐antibody sandwich method was used and methodological evaluation was performed. Results The average intra‐ and inter‐assay coefficient of variation for IL‐18‐TRFIA was 4.80% and 5.90%, respectively. The average recovery rate was 106.19 ± 3.44%. The sensitivity (10.96 pg/mL) was higher than that obtained using the ELISA method (62.5 pg/mL). The detection range was 10.96–1000 pg/mL. IL‐6 and galectin‐3 did not cross‐react with IL‐18‐TRFIA. The serum concentration of IL‐18 was (776.99; 653.48–952.39 pg/mL) in hepatitis C, (911; 775.55–1130.03 pg/mL) in fatty liver, (1048.88; 730.04–1185.10 pg/mL) in liver cancer, and (949.12; 723.70–1160.28 pg/mL) in hepatitis B. Moreover, IL‐18 serum levels were significantly higher in patients than the healthy controls (483.09; 402.52–599.70/mL) (p < 0.0001). Autoimmune hepatitis with a serum IL‐18 concentration of 571.62; 502.47–730.31 pg/mL was not significantly different from the healthy controls (p > 0.05). Conclusion We established a highly sensitive IL‐18‐TRFIA method that successfully detected serum IL‐18 concentrations in different liver diseases. Furthermore, IL‐18 serum concentration was higher in patients with liver cancer, hepatitis C, hepatitis B, and fatty liver disease compared to healthy controls.


| INTRODUC TI ON
, also designated interferonγ (IFNγ) inducing factor, is a single nonglycosylated peptide chain with a molecular weight of 18 kDa that was first identified as a cytokine in 1996. 1   has been reported in most cell types, including endothelial cells, macrophages, dendritic cells, vascular smooth muscle cells, and Kupffer cells. 2,3 The cytoplasmic IL-18 precursor is an inactive 192 amino acid polypeptide with a molecular weight of 24 kDa, which becomes cleaved by group I or inflammatory caspases. 4 The active 18 kDa mature IL-18 then exerts proinflammatory and anti-inflammatory effects. 5 A comprehensive description of its roles in numerous diseases such as nephropathy, 6 leukemia, 7 atherosclerosis, 8 obesity, 9 diabetes, 10 and HIV 11 has been previously reported. According to the literature, the expression of IL-18 is elevated in the serum of patients with hepatitis B 12 and liver cancer 13 compared to healthy controls. Therefore, we aimed to establish a highly sensitive timeresolved fluorescence immunoassay (TRFIA) 14 and evaluate its application for detecting IL-18 in the serum of liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, and fatty liver disease patients.

| Serum samples
Blood samples were collected from healthy individuals and five different types of liver disease (liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, and fatty liver disease) from the Fifth People's Hospital of Wuxi (n = 186 samples). The samples were centrifuged at 1200 × g for 5 min to obtain the serum, which was then stored at −20°C. No hemolysis or lipid turbidity steps was included in the preparation of serum samples. The selection criteria for healthy subjects included: negative results for HBs antigens and hepatitis C virus (HCV) antibodies, and normal liver function. The research project was approved by the Ethics Committee of Wuxi Fifth People's Hospital (ethical code: 2020-023-1).

| Preparation of solid-phase coated antibodies
The coating antibody was diluted to 4 μg/mL with 50 mmol/L of carbonate buffer (pH 9.6), after which 100 μL of solution was added to each well of a 96-well microtiter plate and incubated overnight at 4℃. Next, we removed the coating antibody solution and washed the plates with DEM-3 plate washer. Next, we added 150 μL of sealing solution to each well, sealed the plates, and incubated them at 25℃ for 2 h. Lastly, the sealing solution was removed, the plate was washed, vacuum dried, vacuum packed in an aluminum foil bag, and stored at −20℃.

| Preparation and purification of Eu 3+ labeled antibody
A total of 0.3 mg of labeled antibody was added to the Millipore centrifuge tube with a filter membrane and centrifuged at 2862 × g for 8 min. Following centrifugation, the pellet was washed eight times with 300 μL of labeling buffer. Next, 50 μL of labeled antibody and 100 μg of europium labeling reagent were thoroughly mixed and incubated at 28°C overnight with constant shaking. We then used the SepHadex-G50 chromatography column to separate, purify, and elute the eluent, while simultaneously collecting the effluent (2 mL/ tube). Next, 5 μL of stock solution and 100 μL of enhancement solution were added to each tube to measure the fluorescence coefficient (counts per second, CPS). After combining the first peak tube, it was stored in the freezer at −20°C.

| TRFIA evaluation of IL-18
TRFIA detection of IL-18 was carried out using the two-step double-antibody sandwich method. First, we added a volume of 100 μL IL-18 antigen standard (62 pg/mL, 125 pg/mL, 250 pg/mL, 500 pg/mL, and 1000 pg/mL) or serum to each antibody-coated well. After a 1 h incubation period at 37℃, the plate was washed twice using a plate washer and patted dry. Next, we added 100 μL of diluted Eu 3+ detection antibody (1:400) to each well and incubated the plate at 37°C for 1 h. The plate was washed 6 times using the plate washer and patted dry. Next, we added 100 μL of enhancement solution to each well, and agitated the plate on a micro shaker for 5 min. The fluorescence coefficient (CPS) was subsequently measured. The method used for determining the concentration of IL-18 in serum samples was the same as that described for antigen standards.

| Sensitivity and specificity
To identify the lowest concentration of the analyte to be reliably detected from the background noise, we took the mean and standard deviation (SD) of the values at zero-concentration points of the ten standard curves, obtaining the mean +2 × SD. 15 The sensitivity of the method was then calculated based on the standard curve. IL-6 and galectin-3 were used as interfering substances to measure the cross-reaction rate.

| Recovery rate
To evaluate the serum recovery rate, two serum samples with  The results were then compared to those obtained using the TRFIA method.

| Clinical application of the TRFIA method
We next aimed to evaluate the preliminary clinical application of the TRFIA method. To this end, we used serum samples from pa-

| IL-18 standard curve
Following the measurement of the IL-18 standards using the TRFIA method, we generated a standard curve fitted with a degree function. The IL-18 standard curve equation was as follows: y = 28.87x -471.50, p < 0.0001, and the multiple correlation coefficient (R 2 ) was 0.9986.

| Accuracy
We selected three IL-18 antigen standards with low, medium, and high concentration levels, and used the IL-18-TRFIA method to test each one ten times. The average intra-assay CV was 4.80% while the average CV between groups was 5.90%.

| Sensitivity, detection range, and specificity
The fluorescence value of mean +2 × SD at the zero-concentration point corresponded to a concentration of 10.96 pg/mL; thus, the detection range was between 10.96-1000 pg/mL. The measured values of IL-6 and galectin-3 were 0.15% and 0.48% of the theoretical values, respectively, indicating that there was no cross-reaction with either of these proteins.

| Recovery rate
High IL-18 concentration standard was added to a low IL-18 concentration serum. The average recovery rate of IL-18 was 106.19 ± 3.44%, which was controlled between 85% and 115%.

| Correlation between TRFIA and ELISA
There was strong correlation between the results obtained following TRFIA and ELISA as shown in Figure 1.

| Clinical application
The IL-18 serum concentrations of liver cancer, hepatitis C, hepatitis B, and fatty liver disease patients were significantly higher than that of healthy controls. The positive rate was calculated using the IL-18 data obtained from the 20 healthy control serum samples and a value of 775.66 pg/mL was defined as the cutoff (mean +2 × SD).
The average concentration of IL-18 in the serum of patients with liver cancer was the highest (Figure 2). The IL-18 concentration in the serum of patients with liver cancer, hepatitis C, hepatitis B, fatty liver disease, and autoimmune hepatitis was plotted as a ROC curve were analyzed (Table 2). We discovered that the IL-18 serum concentration was positively correlated with GT level (p < 0.05; r = 0.532) and inversely correlated with HDL levels (p < 0.01; r = −0.697).

F I G U R E 1 Correlation between IL-18 concentration in serum
of patients with liver disease and healthy controls measured using the TRFIA and ELISA methods (y = 0.8972x + 4.61, R 2 was 0.9596, p < 0.0001) F I G U R E 2 Comparison of the serum IL-18 concentrations between five groups of liver disease patients and healthy controls

| DISCUSS ION
The expression of cytokines is almost undetectable in a healthy liver; however, certain factors make the liver susceptible to cytokinemediated damage, such as chronic alcohol intake and obesity. 17 Inflammatory cytokines play a classical role in the regulation of immune responses. Indeed, increasing evidence indicates that pathological changes in the liver, as well as its regeneration after injury, are mediated by cytokines, such as IFNγ, which have been shown to play an important role in liver damage. 18  ALT, GT, LD, PA, and AFU (p < 0.05), indicating that the increase of IL-18 was related to liver injury. It was highly significantly positively correlated with P Ⅲ NP (p < 0.01), which is a peptide chain of type Ⅲ procollagen molecule extending at the amino terminus. It is cleaved from the amino terminus by endonucleases when procollagen is secreted into the extracellular space and enters the blood circulation. 25 Serum P Ⅲ NP levels increase with the synthesis of collagen fibers in the liver, and these levels were found to be significantly increased in patients with liver cancer, suggesting that IL-18 may be involved in the process of liver fibrosis. However, as a classic marker for the diagnosis of liver cancer-AFP, 26  Changes such as hepatocyte swelling and necrosis caused by liver diseases will increase the permeability of the liver, capillaries, and blood vessels, making it easy for indexes such as DBIL, AST, and ALT to flow back into the blood from the liver cells and bile ducts. 31 In patients with nonalcoholic fatty liver, the detection indexes of ALT, AST, and GT were found to be significantly higher than those of healthy controls (p < 0.01). 32 In our study, the clinical Abbreviations: AFP, Alpha-fetoprotein; AFU, a-fucosidase; ALT, alanine aminotransferase; C Ⅳ, type IV collagen; GT, glutamyl transpeptidase; HA, hyaluronic acid; LD, lactate dehydrogenase; LN, laminin; P Ⅲ N P, N-terminal type III procollagen; PA, prealbumin. To our knowledge, this is the first study to apply the TRFIA method for the detection of IL-18 concentration in the serum of patients with liver diseases. We found that the IL-18 serum concentration of patients with various liver diseases differed compared to the healthy controls. Furthermore, the TRFIA method used here is more sensitive than the traditional ELISA and also exhibits good specificity. This method is relatively simple to use, efficient, and cost-effective; thus, it might prove useful for both laboratory and clinical settings.

ACK N OWLED G EM ENTS
During the research, thanks to Editage organization for giving me help in language writing.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data that related to this study are available from the corresponding author upon reasonable request.