Effects of Adjuvant and Immunization Route on Antibody Responses against Naja Naja oxiana Venom.

Naja naja oxiana (NNO) is one of the important venomous species in Iran. The current snakebite treatment is antivenom therapy that deals with hyper immunization of horses with crude or fractionated snake venom plus traditional adjuvants, like Freund's adjuvant. For improvement of antivenom production, it has been suggested to use different adjuvant systems or immunization procedures. In this study, humoral immune responses against immunogenic fractions of NNO venom (NNO3 and NNO4) and crude venom have been compared by usage of different adjuvant and immunization routes. Additionally, a new indirect enzyme-linked immunosorbent assay (ELISA) was set up for the detection of specific antivenom antibodies. This study was conducted on six different groups of female Dutch rabbits that were hyperimmunized using crude and fractionated NNO venom, along with Freund's and MF59 adjuvants through subcutaneous or intramuscular route. The immunization was performed four times with 10-day intervals and the levels of specific antibodiees were detected by indirect ELISA. The statistical analysis reveals a negligible variation in the antivenom titers among the venom-inoculated groups, regardless of the adjuvant type or the immunization route. Finally, it was concluded that the fractions are efficient for antivenom production, and it is possible to use MF59 adjuvant via subcutaneous routes as an alternative to Freund's adjuvants considering its fair immunopotentiation capacity and safety in animals.

Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance.