Preparation and clinical application study of serum AFP-L3 protein immunoradiometric assay and chemiluminescent immunoassay kits

Objective To prepare serum alpha-fetoprotein isoform L3 (AFP-L3) immunoradiometric assay (IRMA) and chemiluminescence immunoassay (CLIA) kits, systematically evaluate their analytical performance, and explore their application value in the clinical diagnosis of hepatocellular carcinoma (HCC).

Methods One AFP-L3 monoclonal antibody was immobilized onto carboxyl-modified magnetic microparticles to serve as the solid-phase carrier, whereas another monoclonal antibody was labeled with ¹²⁵I for radioisotope detection and with acridinium ester for chemiluminescence detection for the preparation of AFP-L3 IRMA and AFP-L3 CLIA kits. Key performance indicators, such as the sensitivity, precision, and recovery rate of AFP-L3 detection, were compared and analyzed. Serum samples from the normal control group (n=50), liver cirrhosis group (n=54), and HCC group (n=83) were detected to evaluate the diagnostic efficacy of the two kits. For measurement data that did not follow a normal distribution, Mann–Whitney U test was used for comparison between two groups, and Kruskal–Wallis H test was used for comparison among multiple groups. The diagnostic efficacy of AFP-L3% (i.e., the proportion of AFP-L3 in total AFP) for HCC was evaluated using the receiver operating characteristic (ROC) curve.

Results The analytical sensitivity values of the prepared AFP-L3 IRMA and AFP-L3 CLIA for AFP-L3 were 0.17 and 0.13 ng/ml, respectively. The intra- and inter-assay variabilities were both less than 10%, and the mean recovery rate of samples was between 90% and 100%. The detection results of the two kits showed that the levels of AFP and AFP-L3 in the liver cirrhosis and HCC groups were higher than those in the normal control group, and the differences were statistically significant (Z=8.013–9.005, all P<0.05). The differences in serum AFP-L3% among the three groups were statistically significant (H=120.563, 120.732; both P<0.05). ROC curve analysis showed that the area under the curve (AUC) for the diagnosis of HCC by serum AFP was 0.787(95%CI: 0.722–0.852), with a sensitivity of 0.690 and a specificity of 0.780, the AUC for the diagnosis of HCC by serum AFP-L3% was 0.840 (95%CI: 0.777–0.903), with a sensitivity of 0.792 and a specificity of 0.943.

Conclusion The prepared AFP-L3 IRMA and AFP-L3 CLIA kits have good technical indicators, and they can be used for the early screening and clinical auxiliary diagnosis of HCC.