An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9 pg mL-1 to 20 ng mL-1 HFA, with a limit of detection of 7 pg mL-1. Furthermore, an analytical sensitivity of 10 pg mL-1 was achieved, representing a 51-fold increase in sensitivity over the commercial sandwich ELISA kit. The results obtained for HFA spiked in diluted human whole blood and plasma showed the same precision as the commercial kit. When stored at 4°C in 0.1 M phosphate-buffered saline (PBS, pH 7.4), the anti-HFA bound microtiter plates displayed no significant decrease in their functional activity after two months. The new ELISA procedure was extended for the detection of C-reactive protein, human albumin and human lipocalin-2 with excellent analytical performance. ; peer reviewed: yes ; NRC Pub: yes