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

Abstract

Abstract:Introduction: 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. Methods: LOS was extracted by a modified hot phenol method from the aqueous and phenol phase and its concentration and purity assayed. Protein contaminations of the samples were determined spectrophotometrically and their endotoxin contents were assayed by the Limulus amebocyte lysate (LAL). Result: 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).

Development and Optimization of a Homemade ELISA Kit for Detection of Antibodies Against Haemophilus influenzae Type b

“…The NMR and FTIR analyses were performed on vials of lyophilized PRP, containing 9.8 g powder of PRP. Data indicate > 90% similarity between purified PRP and standard PRP obtained from previous studies ( 25 ).…”

“…Resuspension of the precipitate was performed in 0.3 M sodium chloride. Orcinol was added to the pellet for assessing ribose ( 11 , 14 , 25 , 26 ). The ribose concentration was determined by measuring the absorbance of the solution at 670 nm and comparing it to a standard curve prepared by assaying pure ribose.…”