Introduction: Salmonella infection (salmonellosis) is a common bacterial disease that affects the intestinal tract. Several methods like Multiplex or real-time PCR, ELISA, and Agglutination are used to identify these bacteria. However, normally rapid, cost effective and easy diagnostic methods such as agglutination test is recommended. In Iran, positive control antiserums used in diagnostic kits work based on polyvalent agglutination and are against O and H antigens. The purpose of this research was to produce specific anti-sera against O and H antigens for using in agglutination and ELISA kits.
Methods: New Zealand white rabbits were immunized by intravenous injections of inactivated bacterial O and H antigens adjusted to a cell density equivalent to a turbidity of a McFarland number 3 standard. Serum collection was performed 7 days after the last injection. Collected Antisera were tested with positive human specimens as well as cross-reaction antibodies. Absorption method was used to obtain specific anti-sera against O and H antigens. Produced Anti-O and Anti-H antibodies were mixed with bacterial H and O antigens respectively and incubated for 1 hours in 37˚c. The Mixture was centrifuged and the supernatant was collected. Furthermore, in order to use these antisera in specific kits such as ELISA, Immunofluorescence etc., purification methods like Ammonium sulphate precipitation, tangential Flow Filtration and Chromatography were performed. This study was approved by the ethics committee of Pasteur Institute of Iran (Code: IR.PII.REC.1399.006).
Results: Results of agglutination test before and after adsorption showed cross-reaction before adsorption and no cross-reaction with H and O antigens with monospecific antisera against O and H after adsorption, respectively. Moreover, high quality and quantity of mono-specific antibody was obtained after purification.
Conclusions: Serum-based assays are recommended for the timely diagnosis of the disease since these assays are specific, sensitive, inexpensive, and rapid. Therefore, the produced antiserum in the present research can be used in primary screening of salmonella infections based on agglutination tests which are cost effective and simple. In addition, purified anti-sera can be used in the development of ELISA and Immunofluorescence assays.