مجله دانشکده پزشکی اصفهان (May 2016)
Enzyme-linked Immunosorbent Assay (ELISA) for Measurement of Antibody against Polyribosylribitol Phosphate Polysaccharide Capsule Extracted from Haemophilus Influenza Type-B Strain-Atf2
Abstract
Background: Haemophilus influenzae type b (Hib) is an encapsulated bacterium cause meningitis in infants worldwide. The capsular Polysaccharide antigen of this organism is a polymer made of ribosylribitol phosphate and is the most important virulence factor and the causative agent of many infections in children under 2 up to 5 years of age. The capsular Polysaccharide conjugated to a carrier protein is effective in the prevention of such infections. Methods: In this study Hib strain Atf2which was isolated and identified from a child with meningitis (previous studies), was cultured in a bioreactor (13-L Bio flo 2000(New Bruns Wick Scientific Co. USA)) containing Giolitti-Cantoni broth (GC broth). The culture was inactivated and polyribosylribitol phosphate (PRP) was extracted by various methods from the bacterial pellet and ultimately filtered through 0.25 µm filter. The filtrate was conjugated with tetanus toxoid (TT) as protein carrier and injected in to sepharos CL-4B gel. Fractions between 11 to 19 was pooled and used as a conjugate product (PRP-TT). Findings: The amount of 402 µg PRP was extracted from 109cfu/ml of bacteria. The amount of protein and nucleic acid was under 1% which is the amount recommended by World Health Organization (WHO) .The PRP recovery after conjugation which was measured by sodium deoxycholate (DOC) was 58%. The antibody response against PRP-TT raised in infant rats showed the highest titer against itself compare to extracted PRP in our own lab and the PRP purchased from National Institute for Biological Standards and Control (NIBSC). The similarity between standard PRP and extracted PRP, was shown by antibody titer in 1/200 dilution. Conclusion: The amount of 402 µg PRP was extracted from 109cfu/ml of bacteria. The amount of protein and nucleic acid was under 1% which is the amount recommended by World Health Organization (WHO). The PRP recovery after conjugation which was measured by DOC was 58%. The antibody response against PRP-TT raised in infant rats showed the highest titer against itself compare to extracted PRP in our own lab and the PRP purchased from NIBSC. The similarity between standard PRP and extracted PRP, was shown by antibody titer in 1/200 dilution.
