Statistical Evaluation == A two-sided Student’st-test or one-way analysis of variance accompanied by Tukeys or Dunnet multiple assessment tests were performed using GraphPad Prism (version 8.0) for Home windows (GraphPad Software, NORTH PARK, CA). Finally, it had been figured the fractions are effective for antivenom creation, which is possible to utilize MF59 adjuvant via subcutaneous routes instead of Freunds adjuvants taking into consideration its reasonable immunopotentiation capability and protection in pets. Keywords:Adjuvant, Antivenom, Immunization, Nanja naja oxiana == Intro == Snake bite is known as to be one of the most essential noninfectious deadly real estate agents all around the globe. The World Wellness Organization (WHO) offers approximated that about 2.7 million venomous snake bites happen each season worldwide, leading to 80,000 to 138,000 fatalities. Nearly all these bites happen in Africa, South-East Asia, and South Asia (1). Appropriately, three quarters from the lands in Iran are possibly suitable for existence of diverse varieties of venomous snakes (2). Probably the most medically important venomous snake family in Iran is definitely Elapidae, especially Asian cobra (Naja naja oxiana), and Viperidae (2,3). A unique and reliable treatment against snake venom activity is Bimatoprost (Lumigan) definitely antivenom administration which is prepared in a second immunized sponsor (4,5). It is important to increase the antivenom effectiveness to enhance the survival rate of victims. Some styles have been taken to create potent and safe antivenom in horses along with other hosts, including use of appropriate adjuvant, use of specific fractions of venom, and toxin changes to increase immunogenic properties and diminish the harmful effects of venom (5,6). Another significant issue is related to the care and health of the sponsor animal. Although horse is the most desired sponsor animal for commercial antivenom production, but they may suffer from severe complications during venom/adjuvant combination inoculation, such as cells damages at injection site, fibrosis, fistula, swelling, and granuloma. These could affect the quality of antivenom and reduce the production yield (7,8). Concerning animal welfare and to save time and production costs, use of proper adjuvant with the least side effects and the most immune reinforcement has a great deal of importance for antivenom production. Traditionally, Freunds total and incomplete adjuvants (FCA/FIA) have been used for antivenom production, which often cause local reactions (9). However, MF59 as an oil in water emulsion adjuvant, has been confirmed and licensed for some common vaccines, such as influenza and hepatitis (10). Most previous studies MAPKAP1 possess recommended the use of crude venom in antivenom preparation. However, some earlier studies possess emphasized the use of harmful fractions and their effectiveness in the hyper-immunization process (6,11,12). This study targeted to investigate some guidelines for antivenom production, including the effect of adjuvant type, antigen use, and route of injection. For this purpose, an alternative process was developed for hyperimmunization of rabbits with Naja naja Bimatoprost (Lumigan) oxiana (NNO) venom by a MF59 adjuvant. Harmful fractions were used instead of whole venom; moreover, MF59 adjuvant was tried for antivenom production inside a rabbit model, to obtain more immune stimulation with Bimatoprost (Lumigan) less side effects. Moreover, these plans were tried in two routes, namely intramuscular (IM) and subcutaneous (SC) routes. == 2. Materials and Methods == == 2.1. Venom Preparation == The NNO venom was from Venomous Animal Division of Razi Vaccine and Serum Study Institute (RVSRI), Iran where it was prepared by milking some northeast Iranian NNO snakes. After collection, the venom was lyophilized and stored in -20 C. Some of the crude venom was used for fractionation by chromatography with gel filtration method. == 2.2. Venom Fractionation == Sephadex G50 was inflamed in 0.1 M ammonium acetate and packed in two series column.