Immunoradiometric assay for quantitative determination of Hepatitis B surface Antigen (HBsAg) in human serum or plasma
In 1965, Dr. Blumberg who was studying hemophilia, found an antibody in two patients which reacted against an antigen from an Australian Aborigine. Later the antigen was found in patients with serum type hepatitis and was initially designated "Australian Antigen". Subsequent study has shown the Australian Antigen to be the hepatitis B surface antigen (HBsAg). Initially there appeared to be three particles associated with hepatitis B infection: a large "complete" particle called the "Dane particle", an oblong 42nm particle and a small circular 22nm particle. Further research identified the Dane particle as the hepatitis B virion and the other two particles as excess surface protein. This former terminology is no longer used and the virus is referred to according to its structure. HBsAg presents an antigenic heterogeneity. The principle determinant is called “a” and is common to all the different types of HBsAg. There are two other pairs of major determinants that is d/y (1y, 2y, 3y) and w/r which are mutually exclusive. Therefore the following combinations are possible adw, adr, ayw, ayr. Subsequent association with hepatitis B virus (HBV) led to the development of sensitive, specific markers of HBV infection. During acute and chronic HBV infection, HBsAg is produced in excess amounts, circulating in blood as both 22nm spherical and tubular particles. HBsAg can be identified in serum 30~60 days after exposure to HBV and persists for variable periods depending on the resolution of the infection. Antibody to HBsAg (anti-HBs) develops after a resolved infection and is responsible for long-term immunity. Anti-HBc develops in both resolved acute infections and chronic HBV infections and persists indefinitely. Immunoglobulin M (IgM) anti-HBc appears early in infection and persists for greater than or equal to 6 month. It is a reliable marker of acute HBV infection.
The RIAKEY HBsAg IRMA Tube is a non-competitive immunoradiometric (IRMA) method (“sandwich”). The method employs two highly specific monoclonal anti-HBs antibodies which recognize two different epitopes of the molecule. One antibody is coated on solid phase (coated tube), the other, specific for the HBsAg and labeled with Iodine-125, is used as a tracer. Antibody-coated polystyrene tubes serve as solid phase. The tracer antibody and the coated antibody react simultaneously with the HBsAg present in the standards, control serum and samples. Unbounded material is removed by a washing step. The amount of bound tracer will be directly proportional to the HBsAg concentration and the remaining radioactivity bound to the tubes is measured in a gamma scintillation counter.