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.Objectives:This study was performed to develop and optimize a homemade ELISA kit for the detection of Hib anti-polyribosylribitol phosphate (PRP) antibodies in children.Materials and 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.