Maximum 1 contained many contaminating proteins along with the K99 antigen. was developed to detect the K99 pilus antigen [7]. DNA gene probes specific for genes encoding toxins and adhesins of ETEC [27] and multiplex polymerase chain reaction (PCR) for the quick testing of ETEC toxins [24,26] have also been used with a fair amount of success. However, these tests require RIPA-56 proper facilities and some amount of scientific experience to conduct and interpret the test results. Therefore, we developed a simple but specific test to detect K99+ recovered from feces of diarrheic calves. The K99 fimbrial antigen was isolated and purified, monoclonal antibodies (MAbs) were produced against K99, and a co-agglutination test was developed to detect K99+ were isolated from fecal samples collected from diarrheic calves. The isolates were cultivated in Minca-Isovitalex medium as explained by Guinee et al. [6]; the medium was supplemented with 1 g of candida draw out (Oxoid) per liter of medium. K99+ isolates were initially recognized by agglutination checks using K99 antiserum from the National Institute of General public Health and Environmental Safety (Netherlands), and consequently confirmed by electron microscopy. K99 antigen was isolated and purified from a field isolate, designated SAR-14, which exhibited strong agglutination with K99 antiserum. The research K99 (F5) MAb was procured from your Central Veterinary Laboratory (CVL), UK. Electron microscopy Electron microscopy was carried out as explained by Korhonen et al. [11]. SAR-14, a crazy strain of was produced in 3.0 l of Minca-Isovitalex broth for 17 h at 37 (O.D.660 = 1.6). The bacteria were then harvested by centrifugation at 6,000 g and resuspended in phosphate urea buffer (50 mM phosphate buffer, pH RIPA-56 7.2 with 2M urea) at O.D.660 = 100. The suspension was heated at 60 for 20 min and centrifuged at 30,000 g for 15 min. The sediment was discarded, while the K99 antigen in the supernatant was precipitated with ammonium sulfate, separated and dialyzed as per Morris et al. [17]. Gel filtration chromatography A glass column (Pharmacia, Sweden) measuring 60 cm in length by 1 cm in diameter was packed with Sepharose CL-4B (Pharmacia, Sweden) to a bed volume of 35 ml having a peristaltic pump. The packed column was washed with sodium phosphate buffer (50 mM, pH 7.2) and equilibrated with several column quantities of phosphate buffer containing 2M urea (PUB). The salt-precipitated bacterial proteins (in PUB) were gently loaded RIPA-56 within the column and 60 fractions of 1-ml were collected. Spectrophotometric readings of each fraction were taken Rabbit polyclonal to ALX3 at 280 nm. Fractions constituting individual peaks were pooled and analyzed for K99 antigen. Concentrated, pooled fractions were dialyzed for 72 h against phosphate-deoxycholate (DOC) buffer (phosphate buffer, pH 7.5 comprising 0.5% sodium deoxycholate) after addition of DOC to the fraction [0.5% DOC (w/v)]. The purity of the fractions was checked by SDS-PAGE. Fast protein liquid chromatography (FPLC) The FPLC system (Amersham Pharmacia Biotech, USA) equipped with cation exchange column MonoS HR 5/5 was utilized for purification. The column was equilibrated in RIPA-56 buffer A (10 mM phosphate buffer, pH 7.2), and bound proteins were eluted in buffer B (10 mM phosphate buffer containing 250 mM NaCl) having a phosphate buffer-NaCl gradient of 0-100%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE gels were prepared by the Laemmli method [13] with the changes of Lugtenberg et al. [14]. Electrophoresis was carried out after loading 2 g of sample per lane, along with a lane of standard molecular excess weight markers (10 kDa ladder; Gibco BRL, USA). Gels were stained with Coomassie Amazing Blue. Immunoblotting Crude and purified protein fractions were subjected to Western blotting as explained by Sambrook et al. [20]. Proteins were separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membrane using LKB 2117 Electrophoresis unit, NOVABLOT (Pharmacia, Sweden). Membranes were incubated with obstructing answer (1% skimmed milk powder in distilled water) for 2 h to avoid non-specific binding. The research anti-K99 MAbs (CVL, UK) were diluted 1 : 500 and.