[Design of enzyme-linked immunosorbent assay method for detection of anti-streptolysin-O antibodies on base of recombinant streptolysin-O]

ABSTRACT

Group A, beta hemolytic streptococci are among the major causative agents of otorhinolaryngology infections. Inadequate treatment of disease may lead to serious disorders such as acute rheumatic fever and glomerulonephritis. Anti-streptolysin-O [ASO] is commonly used as a marker in the diagnosis of infection. Purification of native streptolysin-O has several difficulties and its industrial production process is time consuming with very low yield and the risk of biological contamination. In this study, we used a recombinant streptolysin-O protein as an antigen to detect ASO antibodies in enzymes-linked immunosorbent assay [ELISA]. We amplified streptolysin-O gene by polymerase chain reaction [PCR] method and subcloned in prokaryotic expression vector PET28a. E.coli. BL21-DE3-plySs strain was transformed with PET28a-streptolysin-O and gene expression was induced by IPTG. ELISA microplates were coated with different concentration of streptolysin-O protein. Level of ASO antibodies were detected by ELISA method. The results obtained from ELISA method were compared with inhibition of hemolysis assay as a standard method. The results showed that there is a positive significant correlation between the ELISA and inhibition of hemolysis method[r=0.97, p=0.0001]. The sensitivity and specificity of ELISA for detection of ASO anti bodies were 100% and 83%, respectively. ELISA developed with recombinant streptolysine O showed a good sensitivity for detection of ASO antibodies. It is suggested that this method could be suitable for immunoassays