Development Of An Affimer-antibody Combined Chemiluminescence Immunoassay For Serum Glypican-3 In Hepatocellular Carcinoma

Glypican-3(GPC3)is a promising tumor marker for hepatocellular carcinoma(HCC).So far,there is no reliable serum kit for clinical diagnosis.Objective:The aim of this study is to develop a new analytical sandwich chemiluminescence immunoassay(CLIA)method for serum glypican-3(GPC3)detecting basing on the combination of a member of non-antibody binding protein(Affimer)and an antibody(Ab).The performance of the new proposed Affimer-mAb CLIA was assessed in the laboratory both with recombinant GPC3 protein and with clinical samples.And the value of Affimers as a substitution for antibody is discussed.Methods:1.The Affimers and mAbs prepared by our previous study were purified and identified.A pair of materials with the highest signal-to-noise ratio(SNR)was screened as coated and labeled reagents by a checkerboard method.2.A sandwich reaction based on the Affimer and mAb was applied for GPC3-CLIA development.The reaction parameters,including the concentration of Affimer coated magnetic beads,the ratio of antibody and NHS-LC-Biotin,the amount of acridine ester labeled streptavidin,etc.were optimized.The small-scale of GPC3 CLIA kits were prepared.3.All the typical index for analytic performance of the proposed assay were assessed,including detection limitation,specificity and recovery rate,etc.The comparation to the dual-antibody kit was also undertaken.4.GPC3 levels were detected in clinical serum samples(from patients with HCC,hepatic cirrhosis,intrahepatic cholangiocarcinoma,chronic hepatitis B,chronic hepatitis C,gastrointestinal cancer,lung cancer,and healthy individuals)by the proposed assay and three other dual-antibody kits.GPC3 in tissues from HCC patients were also detected by IHC.Area under the curve(AUC)analysis was performed for determining the diagnostic value for HCC.Linear regression and Spearman's correlation were performed to analyze the results of quantitative data between two methods,and for Kappa value results of qualitative data.Significant difference was set at P<0.05.Results:1.All purified materials met the experimental requirements.Affimer-GPC3-22 as capture reagent and mAb-7D11 as label reagent were prepared for the subsequent experiments in CLIA kit development.2.The key optimized parameters for Affimer-Ab combined CLIA were as follows:The model of instrument run was two-step method.The concentration of GPC3-22 coated magnetic beads was 1:80(W/W).The ratio of 7D11 to NHS-LC-Biotin was 1:10(W/W).The amount of AE-labeled SA used was 1:50(W/W).The pH of the dilution buffer was 7.80,The Volume of reference calibration was 60 ?L.Perfect concentration of coated magnetic beads,biotinylated antibody and acridine ester labeled streptavidin were 800?g/mL,1:250 and 1:500,respectively.Under those optimized conditions,a calibration curve for the proposed CLIA was developed,with the working curve equation as y=3.95+1.03x,and the linear correlation coefficient of 0.9999.3.Analytic performance of the proposed CLIA assay demonstrated a high detection limitation(0.03 ng/mL)and specificity(0-0.002%),a wide linear range(0.03-600 ng/mL)with a good linear correlation coefficient(0.9999).The recovery rates were at the range of 91.88%-104.53%,and the inter-and intra-assay CVs ranged between 6.06%to 8.98%.No interferences were detected with bilirubin,hemoglobin and triglycerides.The sealed kits were stable for no less than 12 months,and the rest reagents should be discarded 14 days after use.Compared to the dual-antibody CLIA kit,the proposed CLIA assay had a better detection limitation,higher specificity,wider linear range.4.The normal reference range of the proposed assay was set at 0-1.1 ng/mL.The mean level of serum GPC3 was obviously higher in HCC compared to the other groups(>16 folds,P<0.001).The decreasing of GPC3 were obtained in post-operative serum at Day 1 and Day 7,with a gradual decline trend.A poor correlation(correlation coefficients ranged from-0.286 to 0.478)was observed through pairwise comparison of different kits.However,only the newly developed CLIA kit showed high specificity and correlated well with the "gold standard" GPC3-immunohistochemistry.Conclusions:This study indicates that Affimer-antibody CLIA can be used to generate a sensitive immunodiagnostic kit,which offers the potential for a highly specific clinically-relevant detection system.