Inhibition of Adhesion ofEscherichia coliK88ac Fimbria to Its Receptor, Intestinal Mucin-Type Glycoproteins, by a Monoclonal Antibody Directed against a Variable Domain of the Fimbria

Abstract

Strains of enterotoxigenic Escherichia coli that express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants of K88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants and may be referred to as conserved epitopes, whereas others are unique to a specific variant and may be referred to as variable epitopes. In this study, monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by enzyme-linked immunosorbent and immunoblot assays. Fab fragments were prepared from these MAbs and were tested for their ability to block the binding of K88-positive bacteria and purified fimbriae to porcine enterocyte brush border vesicles and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (

Characterization of the Binding Specificity of K88ac and K88ad Fimbriae of Enterotoxigenic Escherichia coli by Constructing K88ac/K88ad Chimeric FaeG Major Subunits

“…One study indicated that a minimum peptide including amino acids 64 to 107 was needed to produce a MAb that blocked K88ac binding specifically and suggested that the peptide including amino acids 64 to 107 was a variant-specific antigen (39). In contrast, Bakker et al reported that neither the K88ac nor K88ab fimbria changed its binding activity to the erythrocyte of several different animals after the first 80 or 128 amino acids at the N terminus were switched (3).…”

“…Twenty microliters of prepared total proteins was used for detection of the FaeG subunit in a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot assay (36,42,44). Transferred membrane blottings were blocked with 2% bovine serum albumin (BSA) overnight at 4°C and then incubated with a mixture of hybridoma supernatants of several anti-K88ac and anti-K88ad monoclonal antibodies (MAbs) ( (30,39). Multiple anti-K88ac and anti-K88ad MAbs were used to ensure that all chimeric FaeG proteins would be recognized by the antibody.…”

“…A 200-mesh copper grid (EMS, Hatfield, PA) was incubated with each bacterial suspension by floating a grid on the top of a drop of diluted bacterial suspension (10 7 CFU/ml) for 30 to 60 min. Each bacterium-coated grid was separately rinsed in PBS (with 2% BSA and 0.05% Tween 20) three times and then individually incubated with a mixture of hybridoma supernatants of MAbs, namely, 36/41 (K88ac), 30/17 (K88ac), 17/44 (K88ac and K88ad), 99/150 (K88ad), and 221/38 (K88ac and K88ad) (30,39), for 30 min. After three washes with PBS (with 2% BSA and 0.05% Tween 20) and another three washes with PBS, each grid was separately incubated with gold-conjugated (20 nm) goat-anti-mouse IgG for 30 min.…”

Heat-Labile Enterotoxin PromotesEscherichia coliAdherence to Intestinal Epithelial Cells

“…The supernatant was concentrated by dialysis against solid polyethylene glycol and precipitated by adding 2.5% citric acid to give a final pH of 4.0. The precipitate was resuspended in PBS and analyzed by immunoblotting using an anti-FaeG monoclonal antibody (43).…”

Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína

“…Experimentos realizados por Sun et al (2000) demonstraram que a subunidade FaeG é a responsável pela aderência da bactéria a receptores localizados nos enterócitos, e que anticorpos anti-FaeG são capazes de inibir a adesão. No entanto, estes autores não descartaram a possibilidade da proteína FaeC também ter um papel importante pois esta está localizada na extremidade da fímbria.…”