Establishment Of A Sandwich ELISA For The Detection Of Bovine And Ovine MBM

Bovine Spongiform Encephalopathy(BSE)is designated as list B disease in the OIE International Animal Health Code and list A disease in China, considering its potential impact to animal production and public health. BSE has been causing huge damage to the world beef and dairy industry, especially in some developed country, and has been a serious public health issue in some countries like UK, where BSE has killed more 150 people since the end of 1990's.Epidemiological studies in UK ascribed the occurrence and the spreading of BSE to feeding cattle with meat and bone meals (MBM) derived from ruminants. To reduce the risk of BSE transmission, most country in the world has banned the use of ruminant ingredients to ruminants. For practice, it is necessary to have valid techniques to check the ruminant feed for any contamination of ruminant MBM. In this study, we developed a sandwich ELISA for ruminant MBM, basing on monoclonal antibodies to skeletal troponin I.Skeletal troponin I (sTnI) is even distributed in skeletal muscle(5mg/g). It is a thermostable and salting-in protein that stands the very high temperature process in MBM production, and appears to be an ideal biomarker for MBM tracing. sTnI have two sub-types fast-sTnI and slow-sTnI.. No sequences of bovine and ovine sTnI available in GenBank, we compared the similar sequences of other mammalians and found that both fast-sTnI and slow-sTnI of these mammalians showed 90% homology in their sequences, enabling us to clone the bovine fast-sTnI and slow-sTnI genes respectively by using the most conservative sequences as primers. DNA sequencing reviewed that the cloned sequences had 90% homology with their counterpart genes of other mammalians. The cloned sequences were then inserted into plasmid pET-28a(+) to generate recombinant plasmid pET-28a-fast-sTnI and pET-28a-slow-sTnI and expressed in E.coli BL21(DE3). The expressed proteins were purified and used as immunogen to...