Extraction and Purification of Haemophilus influenzae Type b Lipooligosac charide by Modified Phenol Method

Haemophilus influenzae type b (Hib) is a Gram negative bacterium and one of the causative agents of acute bacterial meningitis, especially in infants and children less than 5 years old. Lipooligosaccharide (LOS), one of the virulence factors which plays an important role in pathogenesis of Hib, has multiple applications in diagnosis and conjugate vaccines. In this study, LOS extracted from two Hib standard strains (ATCC 39930 and ATCC 10214) were compared. LOS was extracted by a modified hot phenol method from the aqueous and phenol phase and its concentration and purity assayed. Protein c ontaminations of the samples were determined spectrophotometrically and their endotoxin contents were assayed by the Limulus amebocyte lysate (LAL). The yield of the extracted LOS from strain ATCC10214 was about 475 μg/ml and the protein contaminations of the samples were approximately 0.07 mg/ml, whereas strain ATCC39930 yielded 520 μg/ml of LOS with protein contamination of 0.08 mg/ml. The results showed that the production of LOS by both strains was similar and the variation observed was not statistically significant (p < 0.001).