Burton W. Blais

Use of polymyxin-coated polyester cloth in the enzyme immunoassay of Salmonella lipopolysaccharide antigens

Polyester cloth coated with polymyxin B was used to capture Salmonella typhimurium lipopolysaccharide antigens which were then quantitatively or qualitatively assayed using a specific antibody-peroxidase conjugate. This simple, rapid method can be used to assay a large number of samples by employing a large sheet of the polymyxin-coated cloth onto which multiple samples can be blotted. The method is reproducible and economical, since polymyxin B is relatively inexpensive, stable and available in pure form.

Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods

A rapid and economical semi-quantitative test for Salmonella cells in foods is proposed. Food samples containing different levels of Salmonella cells were homogenized and serially diluted in enrichment broths and then incubated for about 20 h at 37 degrees C. The presence of Salmonella cells in each dilution was assayed by capturing deoxycholate-extracted Salmonella lipopolysaccharides on a sheet of polymyxin-coated polyester cloth, followed by colorimetric detection with an anti-Salmonella antibody-enzyme conjugate. The minimum dilutions which resulted in no detectable growth were correlated with the extent of Salmonella contamination in the food samples.

Use of a hydrophobic cloth for enzyme immunoassay

As an adsorbent for enzyme immunoassay, a macroporous polyester cloth was superior to the conventional non-porous microtiter plate in that it provides a much larger surface area for immunoreactions (thus, faster reactions), can accommodate a larger volume of sample, and permits easier washing. These attributes should enable polyester cloth to provide rapid and simple field assays.

Use of detergents in the preparation of Salmonella samples for enzyme immunoassay on polymyxin-coated polyester cloth

Heating Salmonella cells in the presence of detergents such as deoxycholate or Triton x-100 greatly increased the sensitivity of an enzyme immunoassay for lipopolysaccharide antigens using polymyxin-coated polyester cloth as the solid phase for antigen capture. The presence of a 100-fold excess of E. coli lipopolysaccharide in the test sample did not affect the detection of the Salmonella lipopolysaccharide. The sensitivity of the assay using deoxycholate-heat treatment of the antigens followed by detection on polymyxin-coated cloth was equivalent to that of an assay using antibody-coated polyester cloth for antigen capture.

Extraction of Salmonella antigens in non-sedimentable forms for enzyme immunoassay

Heating Salmonella typhimurium in ethylenediaminetetraacetate was found to dissociate its antigens into forms that are non-sedimentable at 10,000 x g. The treatment caused a marked increase in the rate of immunoreaction and the sensitivity in an enzyme immunoassay for the detection of Salmonella antigens. The method permits the extraction of Salmonella antigens from solid-rich samples and the preparation of solid-free samples by means of centrifugation. When the level of the antigens in the supernatant was too low for the immunoassay, the antigens were readily concentrated by passing a large volume of the supernatant through a macroporous hydrophobic cloth coated with anti-Salmonella antibody. Using this method, the detection of as few as 10 Salmonella cells per gram of chicken meat was possible within a total of 18 h, which included 16 h of enrichment in either tetrathionate, selenite cystine, or nutrient broths.

Use of hydrophobic cloths as antibody adsorbents for enzyme immunoassay: detection of Brucella antigens

A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.

Rapid Enzyme Immunoassay of Chicken EGG Yolk IgG

A simple and rapid enzyme immunoassay for specific antibodies in chicken egg yolk is described. As a model system, the levels of anti-Salmonella IgG in the yolk of eggs obtained from various produce retailers were compared. Polyester cloth coated with Salmonella typhimurium lipopolysaccharide was used to capture specific egg yolk antibodies, which were then detected using an anti-chicken IgG-peroxidase conjugate. This assay, requiring less than 30 min to complete, revealed considerable differences in the relative levels of anti-Salmonella IgG in the egg yolks. Anti-Salmonella IgG levels were generally lower in eggs obtained from large produce retailers than in eggs obtained from a small local farm. This assay may provide a rapid and economical means of monitoring the levels of Salmonella contamination in chicken rearing facilities.

Activation of polyester cloth for the immobilization of a polysaccharide antigen

A polysaccharide antigen was immobilized on polyester cloth using cyanuric chloride as a coupling agent, yielding an antigen-coated solid phase suitable for enzyme immunoassay. The method should be generally useful for coating polyester cloth with amino or hydroxyl containing antigens or haptens which otherwise adsorb poorly to the cloth.

Affinity purification and biotinylation of antibodies on antigen-coated polyester cloth

Anti-Salmonella antibodies in an antiserum were immunoadsorbed onto lipopolysaccharide-coated polyester cloth, biotinylated and then eluted. The biotinylated affinity purified antibody required less than 2 hours to prepare, and when used in combination with a streptavidin-alkaline phosphatase conjugate permitted the detection of 106Salmonella cells/ml in an enzyme immunoassay.