Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology

Aim of the study: Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood.
Material and methods: Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits.
Results: The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 μg/l and GP73 0.482 μg/l. The functional sensitivity was AFP 0.010 μg/l and GP73 0.640 μg/l. The range of detection was AFP 0.01-50 μg/l and GP73 0.64-100 μg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) μg/l and GP73 was 42.60 (39.10, 57.40) μg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) μg/l and GP73 was 136.10 (92.10, 261.70) μg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) μg/l and GP73 was 186.25 (96.73, 262.03) μg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001).
Conclusions: The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B.