Design and production of a chemiluminescent immunoassay for the early detection of HIV infection

Combined, simultaneous detection of anti-human immunodeficiency virus (anti-HIV) immunoglobulin and HIV core protein distinguishes fourth-generation (combination assay) HIV screening and diagnostic immunoassays from third-generation (double-antigen sandwich) antibody detection immunoassays. Prior to the introduction of fourth-generation assays, commercial immunoassays for blood screening and diagnosis of HIV infection were based either on detection of HIV core (p24) protein or on detection of HIV-specific antibodies, notably those antibodies directed against HIV transmembrane proteins (tmp). Antibodies against these proteins consistently appear during seroconversion of HIV-infected individuals and remain throughout the course of infection. Fourth-generation immunoassays have targeted reduction of the seronegative window period to achieve a continued decrease in the residual risk of transfusion-transmitted HIV infection. Combining antibody and antigen detection in a single immunoassay format achieves a reduction in the seroconversion window because HIV core protein (p24) appears transiently in the blood and has been used as a marker of antigenemia prior to a detectable humoral immune response to HIV infection. Antigen (p24) testing in a combined (fourth-generation) format has been estimated to reduce the seroconversion window by a few days to as much as about 2 weeks in comparison with third-generation single-format antibody detection assays. Despite enhanced seroconversion sensitivity, fourth-generation assays will be most valuable only if the specificity and sensitivity of individual antibody and antigen detection formats are not compromised when they are combined into a single immunoassay. Sensitive combination assays will need to detect antigen at levels equivalent to those of single-format antigen assays in order to be recommended as replacements for current antigen tests. This prerequisite, however, presents a considerable technical challenge not met by all combination assays. In addition, not all combined formats have achieved the low (non confirmed) repeat-reactive rates expected of blood donor screening and diagnostic assays that result in high specificities such as those typically displayed by third-generation single-format antibody assays. With this work we produced a fourth generation chemiluminescent immunoassay for the detection of HIV-1 and HIV-2 infections, achieving an antigen assay sensitivity approaching that of single-format antigen tests, a specificity equivalent to that of a third-generation single-format antibody assay, and a high degree of total precision.