Appendix 39
Monoclonal antibody based detection of 3A containing non-structural proteins in Al (OH)3 adjuvanted Foot-and-Mouth disease vaccines Fuat Özyörük, Ünal Parlak, Gülhan Aynagöz, Hidayet Bozoğlu Foot-and-Mouth Disease Institute, Ankara-Turkey
Abstract To detect 3A containing non-structural proteins [3A, 3AB and 3ABC (3A-NSPs)] in foot and mouth disease (FMD) vaccines, and to find out how they are eliminated are required. This study proposes a standard method based on enhanced detection of 3A-NSPs in Al(OH)3 adjuvanted FMD vaccines by an amplified Western blot assay (AWB). Limit of detection of AWB was found 11.5 ng/lane using anti-3A monoclonal antibody (MAb) 2C2 and recombinant 3ABC. Cellular debris (CD) of five baby hamster kidney cells (BHK-21) infected or non-infected with different type of FMDV, and elution fluids acquired from fifteen batches of FMD vaccines were analyzed for their reactivity with MAb 2C2 by AWB. All infected CD were reactive with Mab 2C2 at the molecular weight (MW) of 28 kDa identical to hypothetical MW of NSP-3AB, whereas none of the FMD vaccines tested had bands reactive with Mab 2C2. The results indicated that NSP-3AB was sedimented together with BHK-21CD, and FMD vaccines tested did not contain 3A-NSPs more than 12.3 ng/lane in the context of detection limit. Introduction Purity of the FMD vaccine is mainly associated with effective separation of virus in supernatant from CD (1, 4). It is important that vaccine be free from cellular proteins as well as non-structural viral proteins (NSP). Presence of NSPs in FMD vaccines would result in ambiguity whether or not vaccinated animals have FMD history, as NSP antibody response is the most reliable way to differentiate infected/convalescing from vaccinated animals in the field to facilitate prevention of new outbreaks. Many publication have revealed that anti-NSP 3ABC, 3AB, 3A, 2C responses in animals are specific indication of FMD history other than vaccination (2, 3, 8-11, 14). However these findings cannot justify that estabilshment of NSP detection procedures in FMD vaccine plants is unnecessary; because at what stage of the production, how, and how much of these NSPs are eliminated are not known except for 2C. In one study, absence of anti-2C antibodies from the sera of vaccinated animals, due to association of 2C with cellular debris that is separated from the virus harvest during vaccine production, have been reported (8). Present study proposed to establish a standard method based on enhanced detection of 3A-NSPs in FMD vaccines and CD of baby hamster kidney BHK-21 by an anti-3A monoclonal antibody. The results indicated that NSP-3AB was sedimented together with BHK-21 CD, and FMD vaccines examined did not contain 3ANSPs more than 12.3 ng/ml.
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