Journal of Isfahan Medical School

Journal of Isfahan Medical School

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
Shafagh Khademi 1
Farhad Esmaily 2
Mehdi Aminian 3
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
Haemophilus influenza type b
Polyribosylribitol phosphate
Conjugation
Enzyme-Linked-Immunosorbent Assay (ELISA)
  1. Brooks GF, Butel JS, Morse SA. Jawetz, Melnick and Adelberg's medical microbiology. 24th ed. New York, NY: McGraw-Hill Medical; 2007.
  2. Murray PR, Rosenthal KS, Pfaller MA. Medical microbiology. 6th ed. Philadelphia, PA: Saunders; 2005.
  3. Hilleman MR, Tai JY, Tolman RL, Vella PP. Coupled H. influenzae type b vaccine [US Patent 4,459,286]. 1984.
  4. Marburg S, Kniskern PJ, Tolman RL. Covalently-modified bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with big eneric spacers and methods of preparing such polysaccharides and conjugates and of confirming covalency. [US Patent: Registration No. 4 882 317]. 1989.
  5. Davey PG, Cruikshank JK, McManus IC, Mahood B, Snow MH, Geddes AM. Bacterial meningitis--ten years experience. J Hyg (Lond ) 1982; 88(3): 383-401.
  6. Barbour ML, Mayon-White RT, Coles C, Crook DW, Moxon ER. The impact of conjugate vaccine on carriage of Haemophilus influenzae type b. J Infect Dis 1995; 171(1): 93-8.
  7. Kelly DF, Moxon ER, Pollard AJ. Haemophilus influenzae type b conjugate vaccines. Immunology 2004; 113(2): 163-74.
  8. Granoff DM, Cates KL. Haemophilus influenzae type b polysaccharide vaccines. J Pediatr 1985; 107(3): 330-6.
  9. Eskola J, Ward J, Dagan R, Goldblatt D, Zepp F, Siegrist CA. Combined vaccination of Haemophilus influenzae type b conjugate and diphtheria-tetanus-pertussis containing acellular pertussis. Lancet 1999; 354(9195): 2063-8.
  10. Mawas F, Bolgiano B, Rigsby P, Crane D, Belgrave D, Corbel MJ. Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide. Biologicals 2007; 35(4): 235-45.
  11. Afshar M, Esmaily F, Aminian M, Asli E, Haadi E, Torabi M, et al. A study on Haemophilus influenzae type b growth rate and capsule production in different media. Archves of Razi Institute 2012; 67(1): 7-12.
  12. Esmaily F, Aminian M, Tavangar AR, Hadi A. Comparison of bacterial biomass and PRP production between different isolates of Haemophilus influenza type b (Hib) under different culture conditions. Archives of Razi Institute 2011; 66(1): 43-9.
  13. Takagi M, Lima RB, Albani SM, Zangirolami TC, Tanizaki MM, Cabrera-Crespo J. Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up. J Ind Microbiol Biotechnol 2008; 35(11): 1217-22.
  14. Peltola H. Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: Global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 2000; 13(2): 302-17.
  15. Chu C, Schneerson R, Robbins JB, Rastogi SC. Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates. Infect Immun 1983; 40(1): 245-56.
  16. Madore DV, Anderson P, Baxter BD, Carlone GM, Edwards KM, Hamilton RG, et al. Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 1996; 3(1): 84-8.
Journal of Isfahan Medical School
Volume 34, Issue 375 - Serial Number 375
January and February 2016
Pages 238-244

  • Receive Date 11 July 2015
  • Accept Date 06 April 2022