Development of immunoenzymatic technique for detection of hepatitis B surface antigen using streptavidin-biotin

Abstract

Enzyme-linked immunosorbent assay (ELISA) is one of the versatile technique used in research study and diagnosis. Nowadays there is a discovery of a specific reaction between avidin protein or streptavidin and vitamin biotin forming a strong binding which could be used to develop the ELISA technique for the detection of Hepatitis B surface antigen by using 2 clones of hybridoma cells numbered as 129 and 1C5 for the production of the monoclonal antibody. These two monoclonal antibodies were used to label with biotin and alkaline phosphatase enzyme. Followed by the coating of streptavidin at various concentrations on microtiter plates and the prepared reagents were tested for optimal conditions for the detection of HBsAg. It was found that the monoclonal antibody produced from the hybridoma cell no. 129 was suitable to label with biotin and the hybridoma cell no. 1C5 was suitable to label with alkaline phosphatase enzyme with the concentration of streptavidin at 1 ug/mL was suitable for coating the microtiter plate. By comparing the efficiency of the ELISA incorporating streptavidin and biotin to the conventional ELISA, it was found that the reagent of the streptavidin-biotin ELISA could detect Hepatitis B surface antigen (HBsAg) at the minimum concentration of 12 ng/mL while the conventional ELISA could detect at 25 ng/mL. In addition, the comparison of the efficiency in detection of HBsAg utilizing the Streptavidin-Biotin ELISA to ELISA commercial reagent kit obtained from Abbott company in 213 specimens, it was found that the sensitivity, specificity and efficiency were 100%, 98.29% and 99.06% respectively. Therefore, the efficiency of the ELISA technique for the detection of the Hepatitis B surface antigen could be improved by using streptavidin and biotin