|EDA, SHIGETOSHI - University Of Tennessee|
|SCOTT, M - University Of Tennessee|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/9/2009
Publication Date: 8/9/2009
Citation: Eda, S., Scott, M.C., Bannantine, J.P. 2009. Analysis of Mycobacterium avium subsp. paratuberculosis Antigens Used in an In-house Enzyme-linked Immunosorbent Assay for Johne's Disease [abstract].
Technical Abstract: We developed a novel enzyme-linked immunosorbent assay (ELISA), called EVELISA, for the detection of MAP infection in cattle which showed a higher sensitivity than current ELISA test. We previously reported that the use of heat-killed M. flavascens for pre-absorption of cross-reactive antibodies improved specificity of the EVELISA. Further, we showed that EVELISA antigens can also be used for detection antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in a microfluidic system (the 9th International Colloquium on Paratuberculosis). The EVELISA uses surface antigens removed from the bacteria with 80% ethanol followed by gentle vortex agitation. To test the hypothesis that the high sensitivity of EVELISA was due to MAP-specific cell surface antigens, we used thin layer chromatography (TLC) to compare the extract of MAP against that of a series of bacteria that are either closely related to MAP or known to cause false positive antibody reaction in commercially available ELISAs. Surface antigen extracts were fractionated using the Folch wash method followed by cold-acetone precipitation and loaded onto aluminum-backed silica-gel-60 plates, then developed with a series of solvents, including ceric sulphate/ammonium molybdate, ninhydrin and alpha-naphthol solution. MAP-specific molecules were detected in the chloroform fraction of the Folch wash and were not precipitated by the cold acetone treatment. Fractions obtained after the Folch wash and acetone precipitation were tested for anti-MAP antibody detection in an ELISA format. We found that none of the fractions could achieve the level of EVELISA sensitivity, indicating that the cocktail of antigens in EVELISA was the key for EVELISA’s high sensitivity. The protein components contained in the aqueous fraction are being analyzed by using polyclonal antibodies produced against the MAP extract.