If an initial fourth generation screening test is positive, the test is repeated. If the two tests are positive, or if the initial result was positive and the repeat result using the same assay was negative, then a secondary assay may be necessary.
Western blot had been the gold standard for HIV confirmation for many years. In this technique, HIV viral antigens, derived from virus grown in culture, are separated by electrophoresis according to their molecular weights and transferred onto a nitrocellulose paper/strip. When a positive sample is added to the strip and developed using conjugate and substrate, bands develop on the strip in the areas of the antigens that correspond to the antibodies in the serum sample.The presence or absence of antibodies to HIV-1 in a sample and the identity of the antibodies that are present are determined by comparison of each nitrocellulose strip to the strips used for the non-reactive, weakly reactive, and strongly reactive controls tested along with the samples. Western blot testing only detects patient antibodies to virus; it does not detect viral antigens. Western blots need to be interpreted. They are read visually and criteria are applied such as "a band needs to be found for any two of the following antigens (p24, gp41, or gp12-/gp160). The gel may also have HIV-2 antigens as well (depending on the manufacturer). Western blot assays are not always easy to read. Bands can be faint or difficult to discern.
The Centers for Disease Control and Prevention (CDC) no longer recommends western blot for confirmation of reactive initial immunoassay results because western blot can produce false-negative results in late infection as antibody titers diminish or indeterminate results early in the course of HIV infection. An indeterminate result is frustrating to a patient and clinician as there is much anxiety around the confirmation of an HIV diagnosis. If western blot testing is inconclusive, repeat testing should take place two or more weeks later. At that time there may be higher titers of antibodies present in the patient and the western blot may be more clearly positive.