Preparation and Purification of Polyclonal Antibodies against Mycobacterium Avium Paratuberculosis Antigens in Rabbit

Background & Objective: Johne’s disease is the chronic granulomatous enteritis of ruminants, and a major health hazard worldwide. In recent years, researchers have focused on mycobacterium avium subsp. paratuberculosis )MAP( antigens in diagnostic tests. Identification of antibodies against MAP antigens is, therefore, effective for the diagnosis or preparation of vaccine. The aim of this study was to prepare and purify polyclonal antibodies against MAP antigens. Materials & Methods: A New Zealand white rabbit was immunized at a certain time period with MAP antigens and Fre-und’s adjuvant. After the immunization of the animal, the rabbit was bled to obtain enriched serum. Immunoglobulins were obtained via sedimentation with ammonium sulfate 35% and then IgG was purified by ion exchange (DEAE-cellulose) chromatography. Serologic test was used to evaluate the interaction of antigens and antibodies. Results: Ion exchange chromatography of IgG showed one peak, and SDS_PAGE of IgG showed a single band. Serologic test was applied and clear precipitation lines were appeared up to 1:16 dilution, which indicated the high quality of the product. Conclusion: In this study, the humoral immune response was induced well by immunization with MAP antigens in a New Zealand white rabbit and polyclonal antibodies were produced in high titers. Polyclonal antibodies are relatively inexpen-sive and easy to produce in large quantities and can connect to the more connective sites, resulting in better sensitivity. Identification of polyclonal antibodies via immunological tests can play a significant role in studying MAP disorders.

[1]  M. Collins,et al.  Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants. , 2009, Veterinary microbiology.

[2]  S. Gumber,et al.  Evaluation of the immunogenicity of recombinant stress-associated proteins during Mycobacterium avium subsp. paratuberculosis infection: Implications for pathogenesis and diagnosis. , 2009, Veterinary microbiology.

[3]  N. Ahmed,et al.  Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease. , 2008, Veterinary immunology and immunopathology.

[4]  É. Álvarez,et al.  Bovine IgG1 antibodies against Mycobacterium avium subsp. paratuberculosis protein p34-cx improve association of bacteria and macrophages. , 2008, Veterinary research.

[5]  F. V. van Zijderveld,et al.  Secreted antigens of Mycobacterium avium subspecies paratuberculosis as prominent immune targets. , 2006, Veterinary microbiology.

[6]  G. Jungersen,et al.  Longitudinal study of interferon-gamma, serum antibody and milk antibody responses in cattle infected with Mycobacterium avium subsp. paratuberculosis. , 2004, Veterinary microbiology.

[7]  C. Buergelt,et al.  Identification of Mycobacterium avium subspecies paratuberculosis by Polymerase Chain Reaction in Blood and Semen of a Bull with Clinical Paratuberculosis , 2004 .

[8]  Gathumbi,et al.  Production of ultrasensitive antibodies against aflatoxin B1 , 2001, Letters in applied microbiology.

[9]  J. Skvor,et al.  Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose. , 1998, Journal of chromatography. B, Biomedical sciences and applications.

[10]  M. Resmini,et al.  Polyclonal catalytic antibodies. , 2002, Biochemical Society transactions.

[11]  D. Watson,et al.  Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep , 1991, Journal of clinical microbiology.

[12]  E. Spangler,et al.  Characterization of Mycobacterium paratuberculosis antigenic proteins. , 1985, American journal of veterinary research.

[13]  H. V. Van Kruiningen,et al.  Ruminant paratuberculosis (Johne's disease): the current status and future prospects. , 1984, The Cornell veterinarian.

[14]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[15]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.