Optimization of Rough Lipopolysaccharide Extraction from Brucella abortus by Phenol – Chloroform - Petroleum ether

Brucellosis is an important zoonotic disease which threatens public health and livestock industry. The aim of this study was to optimize extraction of rough lipopolysaccharide from Brucella abortus which can be used as the antigen for the development of serological tests to diagnose vaccinated animals. This applied study was performed in the Brucellosis Research and Production Laboratory. For this purpose, the rough strain was cultured in Brucella agar specific medium and final diagnosis was performed by PCR method. The rough lipopolysaccharide of Brucella abortus was extracted by phenol-chloroform-petroleum ether method and the results of rough LPS extraction were approved by LAL and SDS - PAGE methods. Extraction and precipitation of Lipopolysaccharide with cold methanol and sodium acetate was performed by using Phenol – Chloroform - Petroleum ether method. Then Lipopolysaccharide was identified by Limulus Amebocyte Lysate (LAL) test and formation of clots indicated presence of Lipopolysaccharide.  Also SDS – PAGE (14 % Polyacrylamide gel) followed by silver nitrate staining showed a 12 KDa band which indicates Rough Lipopolysaccharide. According to the results of this study, it seems that Phenol – Chloroform - Petroleum ether method is an excellent method for extraction of rough LPS of antigen for the development of serological tests and ELISA Kit to diagnose vaccinated animals.