A sub-picogram sensitive rapid chemiluminescent immunoassay for the detection of human fetuin A

A sub-picogram sensitive chemiluminescent immunoassay (CIA) was developed for the detection of human fetuin A (HFA), a specific biomarker for hepatocellular carcinoma and atherosclerosis. The anti-HFA antibody was covalently bound to 3-aminopropyltriethoxysilane-functionalized 96-well chemiluminescent microtiter plates (MTP) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide. The developed CIA detected HFA in the range of 0.3 pg mL−1-20 ng mL−1 with an analytical sensitivity of 5 pg mL−1, which was 125-fold more sensitive than that of commercial enzyme linked immunosorbent assay (ELISA). It is the most sensitive immunoassay for HFA detection. The overall immunoassay duration was reduced significantly by more than 3-fold in comparison to that of commercial ELISA. The anti-HFA antibody-bound MTPs, stored at 4 °C in 0.1 M PBS, were highly stable and did not show any significant decrease in functional activity for six months. The perfect correlation between the developed CIA and commercial ELISA for the detection of various concentrations of HFA spiked in diluted human plasma further demonstrates the potential utility of the developed HFA CIA for clinical diagnosis.