Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese

Abstract

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorptionglutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h. This sandwich ELISA could be a rapid and sensitive screening method for the detection of staphylococcal enterotoxins in food for the agri-food industries.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

“…TSST-1, SEA, SEB and SEC 1 were detected by a capture ELISA method used previously which was adapted from that described by Morisette et al [7,9]. The puri¢ed toxins (Toxin Technology, USA) were tested in the ELISA to determine the lower limit of detection and to assess cross-reactivity of the anti-sera for heterologous toxins.…”

Detection of Renibacterium salmoninarum in salmonid kidney samples: a comparison of results using double‐sandwich ELISA and isolation on selective medium

“…In tissue samples as well as food samples, lipids can interfere with immunoreagents and give high background values. Detergents are widely used to diminish this effect (Yolken 1982;Morissette, Goulet & Lamoureux 1991). In the ELISA reported here, a solvent is used to extract lipids from the samples, leaving a clear aqueous phase for testing and a solid layer of fat that can be discarded.…”

Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System

“…A number of reports have been published on the detection of staphylococcal enterotoxins in foods by immunodiffusion [8], latex agglutination [20], radioimmunoassay [9], competitive ELISA [29] as well as double antibody sandwich ELISA [11,12,30]. Among these, the sandwich ELISA was shown to be the most satisfactory method for SEB detection [10,12,31]. The double antibody sandwich ELISA allowed the detection of toxin at around 1 ng/g of sample in several types of foods [10,11,30,32,33].…”

“…Among these, the sandwich ELISA was shown to be the most satisfactory method for SEB detection [10,12,31]. The double antibody sandwich ELISA allowed the detection of toxin at around 1 ng/g of sample in several types of foods [10,11,30,32,33]. However, in many of the cases, antibodies used in the immunodetection system of SEs were monoclonal obtained from hybridoma clones [30,32].…”

“…Different immunological methods have been proposed for detection of staphylococcal enterotoxins in food samples including immunodiffusion assays [8], radioimmunoassay [9] and enzyme linked immunosorbent assays [10][11][12]. But the important bottleneck is the availability of high titre and specific antibodies for the development of sensitive and specific detection system against staphylococcal enterotoxins.…”