Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

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.

Fig. 1

Electron micrograph of E. coli strain SAR-14 showing fimbriae. 1% phosphotungstic acid stain. × 40,000.

Fig. 2

SDS-PAGE of crude and purified fimbrial preparations. Lanes: 1, heat-treated bacterial pellet; 2, proteins in heat-treated bacterial supernatant; 3, ammonium sulfate precipitate; 4 & 5, proteins eluted from Sepharose CL-4B column fractions 1 & 2, respectively; 6 & 7, protein profile following DOC treatment of fraction 1 supernatant & pellet, respectively; 8 & 9, protein profile following DOC treatment of fraction 2 supernatant & pellet, respectively; M, Marker (10 kDa protein ladder).

Fig. 3

Elution profile of fimbrial extracts of E. coli on a Sepharose CL-4B column (1.1 × 60 cm) equilibrated with phosphate-urea buffer. (Inset): Dot blot assay of fimbrial extracts to reference K99 monoclonal antibodies. lanes: 1, heat extract; 2, ammonium sulfate precipitate; 3, ammonium sulfate supernatant; 4 & 5, gel filtration column elutes 1 & 2, respectively.

Fig. 4

Fast protein liquid chromatography: elution profile of the E. coli K99 antigen from a cation exchange column. Column, MonoS HR 5/5; equilibration buffer, 10 mM PBS, pH 7.2; elution buffer, 10 mM PBS with 250 mM NaCl.; fraction size, 1.0 ml; flow rate, 1 ml/min.

Fig. 5

Western blot of E. coli fimbrial proteins probed with reference K99 MAbs. Lanes: M, molecular weight marker (10 kDa protein ladder); 1, heat extract; 2, ammonium sulfate precipitate; 3 & 4, gel filtration column fractions 1 & 2, respectively; 5, FPLC purified K99 antigen.

Fig. 6

Western blot of E. coli fimbrial proteins probed with different MAbs to the K99 antigen. Panel A: Reference MAb (CVL), Panel B: MAb derived from clone 5.6.12, Panel C: MAb derived from clone 3.8.1, Lanes: M, molecular weight marker (10 kDa benchmark protein ladder); 1 & 2, gel filtration column fractions 1 & 2, respectively; 3, FPLC purified K99 antigen.

Fig. 7

Western blot of whole cell lysates of Gram negative bacteria probed with tissue culture-derived K99 MAbs (NEK99-5.6.12). lanes: M, molecular weight marker (10 kDa benchmark protein ladder); 1, E. coli (SAR-14 strain); 2, Pasteurella; 3, Enterobacter; 4, Pseudomonas; 5, Klebsiella.