Enzyme-linked Immunosorbent Assay (ELISA) for Measurement of Antibody against Polyribosylribitol Phosphate Polysaccharide Capsule Extracted from Haemophilus Influenza Type-B Strain-Atf2

Document Type : Original Article (s)

Authors

1 Department of Microbiology, Islamic Azad University, Damghan Branch, Damghan, Iran

2 Associate Professor, Hib Project, Department of Human Vaccines, Razi institute of Vaccine and Serum, Hessarak, Karaj, Iran

3 Assistant Professor, Department of Biochemistery, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

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.

Keywords

References

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Journal of Isfahan Medical School
Volume 34, Issue 375 - Serial Number 375
January and February 2016
Pages 238-244

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History

  • Receive Date: 11 July 2015
  • Revise Date: 04 May 2024
  • Accept Date: 06 April 2022

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