Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

  • Varshney, Brajesh C. (Intas Biopharmaceuticals Ltd.) ;
  • Ponnanna, N.M. (R&D, Biotechnology, National Dairy Development Board) ;
  • Sarkar, Pranati A. (R&D, Biotechnology, National Dairy Development Board) ;
  • Rehman, Pragna (R&D, Biotechnology, National Dairy Development Board) ;
  • Shah, Jigar H. (R&D, Biotechnology, National Dairy Development Board)
  • Published : 20070300
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Abstract

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

Keywords

Acknowledgement

We are extremely grateful to Dr. Chitrita DebRoy, Senior Research Associate & Deirector, Gastroenteric Disease Center, Wiley Lab, Penn State University, USA and to Dr. Bhushan Jayarao, Associate Professor, Cooperative Extension, Department of Veterinary Science, Penn State University (USA) for their guidance and help provided in preparing the manuscript. The Department of Microbiology and Biotechnology, M.S. University, Vadodara, India, facilitated the electron microscopic studies. We are thankful to the National Dairy Development Board, Anand, Gujarat for providing the facilities used in the present work.

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