Submitted to: UJNR Food & Agricultural Panel Proceedings
Publication Type: Proceedings
Publication Acceptance Date: November 10, 2004
Publication Date: November 22, 2004
Citation: Tabatabai, L.B., Zehr, E.S. 2004. Avidin-binding proteins of haemophilus parasuis: identification of biotin carboxyl-carrier protein. UJNR Food & Agricultural Panel Proceedings, November 22, 2004, Abstract #5. Technical Abstract: Enzyme immunoassay-based detection methods using avidin-HRP conjugates are convenient methods for detecting binding of biotin-labeled probes to proteins of interest. H. parasuis, however, expresses high background levels of avidin-binding protein or proteins which could not be eliminated entirely by employing standard protocols for reducing high background staining. After endoprotease LysC digestion and N-terminal sequencing of HPLC-purified fragments, we established that the major background protein band is a homolog of the H. influenzae biotin carboxyl binding protein (BCCP). Depending on the Western detection method applied, interference due to BCCP was substantially reduced with chicken egg white/d-biotin blocking followed by detection with the avidin-HRP system. However, when a chemiluminescent method was used to amplify the signal, the BCCP protein band was present as a very intense band at an apparent molecular weight of 22,000. Utilizing the avidin-HRP detection system resulted in faint binding of the transferrin probe to the transferrin binding protein. By substituting the Super Signal® chemiluminescent system, however, binding of the probe to the transferrin'binding protein was observed. Interestingly, the avirulent strain expressed the transferrin-binding protein constitutively, whereas the virulent field strains expressed the binding protein only in the absence of 2,2'-dipyridyl.