Evaluation of Immunogenicity of Purified Cell Wall-Associated 34 kDa Antigen of Mycobacterium avium subsp. paratuberculosis Infection Narges Kavid, 1 Rasool Madani, 1 Saman Hosseinkhani, 2 Nader Mosavari, 3 Fariba Golchinfar, 1 Tara Emami, 1 and Roohallah Keshavarz 3 The 34 kDa cell wall protein of Mycobacterium avium subsp. paratuberculosis ( MAP) has been suggested as a major species-specific immunodominant antigen in Johne’s disease. However to date, there has not been a purified 34 kDa protein isolated from bacterial lysates used in immunogenicity analysis. Therefore we attempted to assess the immunogenicity properties of the purified cell wall 34 kDa protein for the first time, and compare the results with previous studies. We used an ELISA test for evaluation of the immunogenicity of this 34 kDa antigen against MAP infection. All serum samples from cattle confirmed to be infected with MAP were positive and those from healthy cattle were negative with the present antigen in ELISA tests. The sensitivity and specificity of 34 kDa antigen were then evaluated in comparison with a standard commercial kit and whole cell wall extracts. The results indicated that the pure 34 kDa antigen specific to MAP with high specificity and sensitivity has a strong potential for use in serodiagnosis assays and screening of Johne’s disease. Introduction P aratuberculosis, or Johne’s disease ( JD), is an im- portant, chronic granulomatous entric disease of formed ruminants such as cattle, sheep, and goats, which is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recent reports suggest that MAP is associated with Crohn’s disease in humans, although its causative role in the human disease remains controversial. (1–3) Despite its heavy economic burden on the agriculture industry there are still no practical che- motherapeutic agents or efficacious vaccination programs against Johne’s disease. (4,5) The control of paratuberculosis relies on the identification of infected animals and culling them in the early stages of infection. Fecal culture is the most specific and standard method for diagnosis of para- tuberculosis. However this method requires up to 12 weeks or more before the results are obtained. (3) Due to the disadvan- tages of the current methods, rapid and robust methods based on MAP-specific antigens are required for identification and screening of infected animals. (6) Management and eradication programs to control Johne’s disease are severely hampered by the deficiency of a sensitive and specific test capable of detecting early host exposure to M. avium subsp. para- tuberculosis. (5) Current immunodiagnostics are based on crude antigen mixtures, and a more detailed dissection of the anti- genic make-up of M. avium subsp. paratuberculosis is necessary to enable development of improved tests. It has been more than 20 years since the hunt for an ideal serological test to monitor Johne’s diesease began. Many laboratories have consequently chosen to improve the effi- ciency of serological assays by replacing the current common ‘‘extract antigen’’ with ‘‘purified MAP-specific subunit anti- gens.’’ (7,8) Different approaches have been applied in the search for such antigens. (9–11) The antigens used in conven- tional ELISAs generally consist of complex, ill-defined mix- tures of proteins, lipids, and carbohydrates, including a purified protein derivative, (12) cell wall–derived lipoar- abinomannan, (13) or protein antigens (14) obtained by extensive physical and chemical disruption of MAP cells. (4) Bannantine and colleagues evaluated multiple M. avium subsp. para- tuberculosis recombinant proteins in an ELISA test for sheep. According to their results even an antigen mixture prepared for ELISA that contained proteins encoded by Map2737, Map0862, Map0865, and Map0852 was not sufficient to detect all infected animals in large herds. (4) Hence it is largely ac- cepted that major improvements are required in the currently available serodiagnostic tests to allow for an efficient control program to be set up. (15,16) In 1992 the 34 kDa cell wall protein was identified as a MAP species-specific immunodominant antigen, which belongs to the major antigen complex of M. paratuberculosis named A36 complex. (17,18) The 34 kDa protein of the A36 complex was found to contain B cell epi- topes specific to paratuberculous cattle and has a major role in the pathogenesis of the organism. (17,19,20) Other studies using 1 Department of Biotechnology, 3 Department of PPD Production, Razi Vaccine and Serum Research Institute, Karadj, Iran. 2 Department of Biochemistry, Tarbiat Modares University, Tehran, Iran. HYBRIDOMA Volume 31, Number 3, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/hyb.2011.0108 163