Antigenic quantitation of type 1 fimbriae on the surface of Escherichia coli cells by an enzyme-linked immunosorbent inhibition assay

Abstract

Type 1 fimbriae from two strains of Escherichia coli, K-12-derived CSH50 and a clinical isolate VL-2, were purified by a simplified procedure, which should be applicable to a variety of bacterial strains. After mechanical removal from the cells, the fimbriae were sedimented in the ultracentrifuge and resuspended in 5 M urea to disaggregate cell membranes and flagella, leaving the urea-resistant fimbriae intact. After several hours at 37 degrees C, this crude fimbrial suspension was diluted to 1 M urea, and the intact fimbriae were sedimented through a 1 M urea-1 M sucrose cushion. The pellet was found to be pure fimbriae by sodium docecyl sulfate-polyacrylamide gel electrophoresis, with apparent subunit molecular weights of 17,000 for the fimbriae from K-12 strain CSH50 and 19,000 for those from the clinical isolate VL-2. High-titer rabbit antiserum raised against CSH50 fimbriae was specific for fimbriae by indirect ferritin labeling and immunoprecipitation and was used to develop an enzyme-linked immunosorbent assay. Competitive inhibition of antifimbrial antiserum in the enzyme-linked immunosorbent assay by a known amount of either purified fimbriae or fimbriae-bearing bacteria permitted precise quantitation of fimbrial antigen in cultures of strain CSH50, thereby providing a simple means of determining the effects of environmental conditions on the synthesis of type 1 fimbriae.

Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae of Escherichia coli.

“…Purification of Type 1 Fimbriae. CSH50 fimbriae were purified according to the method of Dodd and Eisenstein (10) . Briefly, bacteria were washed with 0 .5% NaCl and resuspended in 5 mM Tris buffer, pH 7 .8 .…”

Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon

“…Enzyme-linked immunosortient assay inhibition determinations were done as previously described (Dodd and Eisenstein. 1982;Eisenstein etat.. 1983).…”

New Enzyme Immunoassays for Specific Assay and General Detection of Trypanosoma cruzi, Epimastigotes

“…The serological identification of an infectious bacterial species by the classical agglutination had been studied extensively (8). The detection of an immunological equilibrium of antigen and antibody by immunoassay methods has shown rapid progress (4,13,18), and many efforts have been made to develop an immunoassay applicable for the identification of the species of an unknown microorganism (5,7,14). Although the enzyme-linked immunosorbent assays (ELISA) have been applied for the identification of several viral species (6,9), immunoassay has seldom been the method of choice to identify the species of a bacterium, mold, or parasite.…”