Development of a two-site immuno-PCR assay for hepatitis B surface antigen.
Hepatitis B virus (HBV) gene transcription may occur at very low levels resulting in HBsAg concentrations in serum and liver below the limit of detection by currently available immunoassays. An assay has been developed that combines the specificity of two high affinity anti-HBs monoclonal antibodies (MAb) directed against distinct and separate determinants in the 'a' domain of HBsAg with the highly sensitive polymerase chain reaction (PCR) detection method. Following capture of HBsAg present in serum samples, the second anti-HBs MAb, which is biotinylated, is added. Binding of the second antibody allows the subsequent specific binding of streptavidin and a biotinylated linear DNA molecule derived from a bluetongue virus (BTV) gene. Presence of this DNA is then detected by PCR using BTV-specific primers. The PCR product is quantified by a liquid-phase oligonucleotide enzymatic assay, which further increases the sensitivity of the technique. The use of a two-site MAb capture and PCR detection system for HBsAg was shown to greatly enhance the specificity and sensitivity of the assay and detect as little as 0.5 pg of HBsAg in serum samples. It is suggested that the principles of this technique could be applied to measure other low level viral antigens in serum and biological samples.
Maia
M
M
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown 02129, USA.
Takahashi
H
H
Adler
K
K
Garlick
R K
RK
Wands
J R
JR
D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D015139:Blotting, Southern; D001820:Bluetongue virus / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D006510:Hepatitis B Antibodies / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000097:blood* / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D001710:Biotin; D019809:Streptavidin