A Simple Method for Extraction of Lipopolysaccharides from Brucella Melitensis

Abstract

Background: Brucellosis is the most common zoonotic bacterial disease around the world, and its causative agent is Brucella, a gram-negative bacterium. Lipopolysaccharide (LPS) molecules are the most significant surface antigen of Brucella bacteria. This antigen is used in a high number of research and diagnostic fields. The present study was designed in order to introduce a simple method to extract LPS from Brucella melitensis. Materials and Methods: Smooth strains of B. melitensis were cultured in large quantity, and LPS was extracted through changes in the hot-phenol method. The LPS concentration was measured by using the dimethyl-methylene blue method. The contamination rates of proteins and nucleic acids were determined using the bicinchoninic acid (BCA) method and absorbance measurement at 260 nm, respectively. Results: The amount of the extracted LPS was 1.1% of the wet weight of the bacteria. The rate of contamination with nucleic acid was measured to be 0.2% of the LPS. The protein contamination was not detectable through the BCA method. Conclusion: The advantages of our newly-introduced method are considerable decrease in the time required to extract LPS; independence from nucleic acid and protein digestive enzymes; an extraction rate of LPS equivalent to other applied methods; nucleic acid contamination equal or lower than other methods, and no protein contamination; and independence from equipment such as ultracentrifuge and chromatography. [GMJ.2021;10:e1944]