title: Development and Optimization of a Homemade ELISA Kit for Detection of Antibodies Against Haemophilus influenzae Type b.
abstract: BACKGROUND
  Haemophilus influenzae type b (Hib) infection has high morbidity and mortality rate, especially in children under 5 years of age. Enzyme-linked immunosorbent assay (ELISA) technique is the most used method to detect antibodies against H. influenzae. Available commercial ELISA kits are expensive and not always readily available, particularly for epidemiological studies.
  
  
  OBJECTIVE
  This study was performed to develop and optimize a homemade ELISA kit for the detection of Hib anti-polyribosylribitol phosphate (PRP) antibodies in children.
  
  
  METHODS
  To develop and optimize an indirect ELISA method, pure PRP was prepared. The PRP was coupled to bovine serum albumin, using sodium periodate. Then optimal conditions for ELISA, including coating antigen concentration and peroxidase labeled conjugate concentrations, incubation temperature and incubation time, were determined. To confirm the efficacy of optimized kit, 83 serum samples from non-vaccinated children, aged less than 6 years were collected and analyzed, using homemade and commercial ELISA.
  
  
  RESULTS
  The optimal conditions were considered to perform ELISA. Comparison between results obtained from optimized ELISA kit and commercial ELISA kit showed a good agreement.
  
  
  CONCLUSIONS
  Taking into account these data, we elaborated a homemade ELISA kit that is an efficacious and cost-effective substitute for commercial kit, in disease control and diagnosis.
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Seyed Fazlolah
authors.initials: SF
authors.lastname: Mousavi
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Sara
authors.initials: S
authors.lastname: Fatemi
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.affiliation: Center for Communicable Disease Control, Tehran, IR Iran.
authors.forename: Seyed Mohsen
authors.initials: SM
authors.lastname: Zahraei
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Elnaz
authors.initials: E
authors.lastname: Nikanpour
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Mohamad Ali
authors.initials: MA
authors.lastname: Malekan
authors.affiliation: Diagnosis Biotechnology Unit, Production and Research Complex, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Khabiri
authors.affiliation: Department of Bacteriology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Janani
keywords: Child; Enzyme-Linked Immunosorbent Assay; Haemophilus influenza; Polyribosylribitol Phosphate