Method Description

Specimens are first screened by the Genetic Systems hepatitis B surface antigen (HBsAg) 3.0 EIA. All reactive results are confirmed by the Genetic Systems HBsAg Confirmatory Assay 3.0 (HBsAg Neutralization) at an additional charge.

HBsAg:

The Bio-Rad GS HBsAg EIA 3.0 is a qualitative third-generation enzyme immunoassay which uses mouse monoclonal antibodies to detect anti-HBsAg in human serum or cadaveric serum specimens. Wells of a microwell strip plate are coated with mouse monoclonal antibody to HBsAg (anti-HBs). Patient serum and appropriate controls are added to the wells and incubated with bound antibody. If HBsAg is present, it will bind to the antibody and not be removed by washing. The strips are washed to remove any unbound material. Washing is followed by the addition of conjugate solution (peroxidase-conjugated mouse monoclonal antibodies directed against HBsAg). The conjugate solution will bind to the antibody-HBsAg complex, if present. Unbound conjugate is removed by a wash step. Next, working 3,3',5,5'-tetramethylbenzidine (TMB) solution is added to the plate and allowed to incubate. A blue or blue-green color develops in proportion to the amount of HBsAg present in the sample. The enzyme reaction is stopped by the addition of acid, which changes the blue-green color to yellow. The absorbance values of controls and specimens are determined using a spectrophotometer with wavelength set at 450 nm.(Package insert: Genetic Systems HBsAg 3.0 EIA. Bio-Rad Laboratories; 02/2019)

HBsAg Confirmation:

The repeatedly reactive specimen is incubated with HBsAg confirmatory reagent (human antibody to HBsAg). If HBsAg is present in the specimen, it will be neutralized by the HBsAg confirmatory reagent. The treated specimen is re-assayed using the Genetic Systems HBsAg 3.0 EIA assay. The neutralized HBsAg is prevented from binding to the HBsAg antibody-coated microwells, which results in a reduction of signal. A non-neutralized control of the specimen (treated with HBsAg-negative control [human] in place of the HBsAg confirmatory reagent) is tested in parallel to the neutralized specimen for comparison of signal. Genetic Systems HBsAg EIA 3.0 repeatedly reactive specimens are confirmed as positive by the Genetic Systems HBsAg Confirmatory Assay 3.0 if the reduction in signal of the neutralized specimen is greater than or equal to 50% of the corresponding non-neutralized specimen, and the non-neutralized specimen signal is greater than or equal to the assay cutoff.(Package insert: Genetic Systems HBsAg Confirmatory Assay 3.0. Bio-Rad Laboratories; 01/2009)