A Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Salmonella Using Biotinylated Monoclonal Antibodies

Abstract

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 10(4) CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.

Lipid fingerprinting of Bacillus spp. using online MALDI-TOF mass spectrometry

“…A variety of traditional methods, such as nucleic acid hybridization, 3 polymerase chain reaction (PCR), 4 enzyme-linked immunosorbent assays (ELISA), 5,6 immunofluorescence assays (IFAs), 7 dielectrophoretic impedance analysis, 8 and several other methods, 9 have been used for collecting, isolating and identifying pathogens from different specimens and samples. Such traditional methods are able to reliably differentiate bacteria at the species and sub-species levels, but their usefulness is hindered by the prolonged time (hours to days) and their expensive cost.…”

Detection of 7S and 11S soy protein fractions by monoclonal antibody-based immunoassays in food

“…Soy protein has one of the best essential amino acid standards, amongst proteins of vegetal origin, being sufficiently similar to the essential amino acid standard of high quality proteins of animal origin. Soy protein fractions may be detected by several methods, including polyacrylamide gel electrophoresis (Burks et al 1988), capillary electrophoresis (Wong et al 1994, Ló pez-Tapia et al 1999, high performance liquid chromatography (Garcia et al 1998), immunoblotting, ELISA (Burks et al 1988) and immunoassays (Lipp et al 2000, Valdivieso-Garcia et al 2001. The advantages of using monoclonal antibodies-based immunoassays are the low cost, rapidity of the analysis, good In this work, the extraction and purification of the 7S and 11S soy protein fractions were successfully carried out as indicated by SDS-PAGE showing the molecular weights of the subunits a (63.17 kDa), a?…”

Cultural, Serological, and Genetic Methods for Identification of Bacteria

“…The use of monoclonal instead of polyclonal antibodies may increase specificity. 49,52,58 However, because the same epitope can be present in more than one species, a monoclonal antibody against one species may cross-react with other bacteria. 50 For this reason serological methods are not always successful for detection of bacteria in environmental samples and nucleic acid-based methods are now commonly used.…”

“…For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA. 49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria. 58,59 Immunofluorescence assays (IFAs) may be sensitive enough to identify even a single bacterial cell in a contaminated sample.…”

“…49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria. 58,59 Immunofluorescence assays (IFAs) may be sensitive enough to identify even a single bacterial cell in a contaminated sample. 51,60 There are two types of IFAs, direct and indirect.…”