The clinical sensitivity of the current anti-HIV assays is based for an important part on their reactivity with seroconversion panels. The most sensitive assay closes the seroconversion window as much as possible, thereby reducing the risk of transmitting false negative donations obtained from individuals infected recently. Because of the absence of anti-HIV antibodies during the early phase of infection, the seroconversion window can be narrowed partially by detection of HIV p24 Ag. To achieve this, the highest affinity anti-p24 binding antibodies were selected with BlAcore and applied in a direct assay format. To achieve optimal conditions for the anti-HIV part of the assay the HIV specific antigens viral HIV-1 gp160, HIV-2 gp36 and HIV-1 group O gp41 peptides were used. These antigens and antibodies were applied for microELISA coating as well as in the conjugate pearl, which is present in the well of the microELISA plate. The (analytical) anti-HIV-1/-2 and anti-HIV-1 group O sensitivity of this new assay, Vironostika HIV Uni-Form II Ag/Ab, is at least at the level of the current Vironostika HIV Uni-Form II plus O. When compared to the Vironostika HIV Uni-Form II plus O, the seroconversion window is narrowed by 1-2 weeks due to the incorporation of HIV p24 Ag detection. The level of reactivity of the anti-HIV and HIV Ag detection part can be improved by about a factor 2 by applying continuous shaking during sample incubation. Initial studies suggested that the specificity of the assay is identical to that of the Vironostika HIV Uni-Form II plus O, namely > 99.9%. Monitoring of proper execution of the assay handling steps was facilitated by implementing a purple dye in the conjugate pearl. Colourless specimen diluent changes into a green fluid upon dissolving of the conjugate pearl and turns subsequently into blue/purple upon sample addition. These visual changes can also be determined by spectrophotometric measurement at 620 nm.