HBsAg Test: Hepatitis B Surface Antigen Confirmatory EIA

The Alinity s HBsAg Confirmatory assay is used to confirm the presence of hepatitis B surface antigen (HBsAg) in human serum and plasma by means of specific antibody neutralization on the Alinity s System. The assay is intended to be used for confirmation of samples found to be repeatedly reactive by the Alinity s HBsAg assay.

About Hepatitis B Surface Antigen

Hepatitis B virus (HBV) is the causative agent of hepatitis B. An estimated 257 million individuals are living with hepatitis B virus infection. More than 887 000 people die annually of HBV-related liver disease. Globally, chronic hepatitis B is a major cause of liver cirrhosis and hepatocellular carcinoma.^{1, 2}

HBV belongs to the hepadnavirus family and is a partially double-stranded DNA virus. It consists of a central core nucleocapsid containing viral DNA, DNA polymerase, and a surrounding envelope consisting of HBsAg, which is expressed during HBV infection. Additionally, HBV-infected cells produce spherical or long filamentous particles that consist of excess HBsAg.³

The virus is divided into multiple major serotypes (e.g., adr, adw, ayr, ayw) based on antigenic determinants present on the envelope proteins, and into at least 8 genotypes (A–H) according to overall nucleotide sequence variation of the genome. Differences among genotypes can affect the disease severity, course and likelihood of complications, response to treatment, and possibly vaccine protection.^2-5 HBV, unlike other DNA viruses, replicates through a reverse transcription step. The reverse transcription process lacks proofreading capability; therefore, HBV is subject to a mutation rate more than 10 times higher than the mutation rate of other DNA viruses. Surface antigen gene mutations may cause changes in the antigenic structure of HBsAg, resulting in reduced recognition by

some antibodies to HBsAg.^6-11 HBV is transmitted through sexual, parenteral, and perinatal routes. Transmission may also occur through transfusion of HBV contaminated blood and blood products. After infection with HBV, HBsAg is the first antigenic marker, appearing 1 to 12 weeks after exposure and 2 to 6 weeks before the onset of clinical symptoms. HBsAg persists during this acute phase and clears late in the convalescence period. Failure to clear HBsAg within 6 months indicates a chronic hepatitis B infection.^{2, 3} HBsAg assays are used to screen blood and blood products for the presence of HBsAg to prevent transmission of HBV infection to recipients of blood or blood products. HBsAg assays are also used to screen organ and tissue donors. In addition, HBsAg assays are used to identify persons infected with HBV and to monitor the status of infected individuals in combination with other hepatitis B serological markers. Testing for HBsAg as part of an antenatal screening program may identify HBV infected mothers and allow for appropriate immune prophylaxis of the newborn. Specific antibody neutralization assays are used to confirm the presence of HBsAg.^12-18

This assay is used to confirm the presence of hepatitis B surface antigen (HBsAg) in human serum and plasma, using chemiluminescent microparticle immunoassay (CMIA) technology. This assay consists of two single tests that are both pre-treatment immunoassays.

Sample and Pre-Treatment 1 are combined and incubated. When HBsAg is present in the sample, it is neutralized by the antibody (anti-HBs) in Pre-Treatment 1. The pretreated sample, anti-HBs coated paramagnetic microparticles, and anti-HBs acridinium labeled conjugate are combined to create a reaction mixture and incubated. Any non-neutralized HBsAg present in the sample binds to the anti-HBs coated microparticles and to the anti-HBs acridinium labeled conjugate. The neutralized HBsAg is blocked from forming a sandwich with anti-HBs coated microparticles and acridinium labeled anti-HBs conjugate. The mixture is washed. Ancillary wash buffer is added and incubated. Following a wash cycle, Pre-Trigger and Trigger Solutions are added. This sequence is repeated for the sample and Pre-Treatment 2, except Pre-Treatment 2 does not

contain anti-HBs and will not neutralize HBsAg in the sample. The resulting chemiluminescent reaction is measured as relative light units (RLU). There is a direct relationship between

the amount of HBsAg in the sample and the RLU detected by the system optics.

The Alinity s HBsAg Confirmatory sample to cutoff (S/CO) result is determined by comparing the chemiluminescent RLU in the reaction to the cutoff RLU determined from an active calibration. If the non-neutralized sample (incubated with Pre-Treatment 2) result is greater than or equal to the cutoff of 0.70 S/CO and the RLU of the neutralized sample (incubated with Pre-Treatment 1) is reduced by at least 50% compared to the non-neutralized sample, the sample is considered confirmed positive for HBsAg.

Test performed by Eurofins DPT, 6933 S. Revere Parkway, Centennial, CO 80112.

This test has been cleared or approved for diagnostic use by the U.S. Food and Drug Administration. See package insert for more information.