The Isolation Of Ag60 From Bcg Vaccine And It's Comefirmation With Immonuoassay Procedures

Document Type : Original Article


Center of Biological Cellular and Molecular, Shaheed Beheshti Medical Sciences University, Tehran, Iran


Introduction: Tuberculosis remains as an important socioeconomical and medical problem throughout the world and especially in Iran. Early and timely diagnosis of pulmonary and extrapulmonary tuberculosis is very vital to initiate prompt treatment. Current diagnostic methods are either slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. Their identification is important for both diagnosis and protection against mycobacteria. Antigen 60 (A60) is a thermostable antigen found in the cytosol of M.bovis and M.tuberculosis. Performing ELISA test using A60 for diagnosis of tuberculosis has shown goos results . In previous researches A60 also showed a protective effect against experimental infections and useful immuno therapeutic effects in prevention and treatment of cancer.
Materials and Methods: A60 was purified by size exclusion chromatography using sephrose 4B. A60 was recognized by bidimentional immunoelectrophoresis with anti BCG and Anti A60 antiserum. Double diffusion – precipitation test was performed using purified antigen and anti BCG antiserum. Purified A60 was fractionated in SDS – PAGE and transferred to nitrocellulose membrane and western blotting was performed using anti A60 antiserum as primary antibody and anti human immunoglobulin as secondary antibody.
Results: A60 appeared as the less mobile component in Crossed Immuno Electrophoresis. In agarose electrophoresis, A60 formed only one band and in immunodiffusion A60 formed two immunopercipitinogen line with anti BCG. In analyzing with dot blotting, both cytoplasm and cell wall of BCG showed positive reaction with anti A60 anti serum. When A60 was fractionated by SDS – PAGE and transferred to nitrocellulose membrane and analyzed in western blotting with anti A60, proteins of 65, 46, 40, 38 and 35 KD were identified.
Conclusion: It is concluded that A60 is a macro molecular antigen of BCG with molecular weight of 106 – 107 Da and has several proteins. A60 can easily be purified from BCG vaccine which is produced in Iran, by size exclusionchromatography using sepharose 4B.