title: Immunization of horses with Crotalus durissus terrificus (South American rattlesnake) venom. A comparison of four different procedures.
abstract: 1. A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use of Freund's adjuvant, A1(OH)3 and liposomes as adjuvants. The antibody titer was assessed by enzyme linked immunosorbent assay (ELISA) and the neutralizing potency by the neutralizing median effective dose (ED50). 2. The inoculation schedule used in horses to obtain antivenom serum consisted of sc injections of a 7.5 mg venom starting dose in 5.0 ml sterile saline emulsified with an equal volume of Freund's complete adjuvant. One week later, 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant was injected. This was followed by three doses of 2.5 mg venom in 12.0 ml sterile saline at 2-day intervals. This immunization procedure, based on low doses of antigen (37.5 mg/horse) emulsified with Freund's adjuvant, produced a more protective and sustained immune response when compared with other procedures using A1(OH)3 or liposome emulsions with either low (50.0 mg/horse in A1(OH)3 and 5.0 mg/horse in liposome) or high (870.0 mg/horse in A1(OH)3 and 20.0 mg/horse in liposome) antigen doses. The ED50 values evaluated at the end of the procedure were 15.4 microliters serum/20 g mouse when antigen was emulsified with Freund's adjuvant; 21.7 microliters serum/20 g mouse when 870.0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 microliters serum/20 g mouse when 50.0 mg antigen/horse was emulsified with A1(OH)3. When antigen was emulsified with liposome, the immune serum was ineffective against the lethal effects of C. d. terrificus venom. 3. The inoculation schedule used in horses to obtain hyperimmune serum consisted of reimmunization with sc booster injections of 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant. One week later, 2.5 mg venom in 12.0 ml sterile saline was inoculated at 2-day intervals. This reimmunization schedule, based on low doses of antigen (15.0 mg/horse) emulsified with Freund's incomplete adjuvant or with saline, produced a protective and sustained immune response, regardless of the initial immunization procedure. The ED50 evaluated for each of the animals five days after the reimmunization period was never more than 20 microliters serum/20 g mouse. 4. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/l (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil.
authors.forename: T V
authors.initials: TV
authors.lastname: Freitas
authors.forename: C L
authors.initials: CL
authors.lastname: Fortes-Dias
authors.forename: C R
authors.initials: CR
authors.lastname: Diniz
authors.forename: D T
authors.initials: DT
authors.lastname: Velarde
authors.forename: C F
authors.initials: CF
authors.lastname: Freitas
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000997:Antivenins / Q000276:immunology*; D003435:Crotalid Venoms / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006736:Horses / Q000276:immunology*; D007115:Immunization Schedule; D007117:Immunization, Secondary; D008081:Liposomes / Q000276:immunology; D051379:Mice; D009500:Neutralization Tests
chemical_list: D000997:Antivenins; D003435:Crotalid Venoms; D008081:Liposomes