Enzyme immunoassay for detection of Salmonellae in foods

Abstract

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique. The detection of salmonellae in foods and foodstuff involves a series of enrichment steps because these pathogens, when present in foods, are generally found in low numbers and are often sublethally injured. Therefore, detection methodologies for salmonellae must be sensitive and allow for the resuscitation and growth initiation of injured cells. Two methods of Salmonella detection are currently recommended by the Food and Drug Administration (3): (i) a pure culture technique (PCT) involving preenrichment, selective enrichment, and selective plating; and (ii) immunofluorescence (IF) after selective enrichment. The PCT is an expensive and time-consuming process that requires 4 or 5 days for analysis (3). In contrast, IF is more rapid; it is sensitive, but many false-positives are obtained because polyvalent OH antisera are used and anti-O antibodies cross-react with a wide spectrum of related enteric bacteria (16, 17). Enrichment serology (ES; 13), a third method used by some laboratories, is more specific than IF, but ES is more cumbersome and has not met with widespread acceptance. The list of additional techniques for the detection of salmonellae is lengthy; however, the acceptance of a new procedure requires that it demonstrate det Journal paper no.

Rapid and alternative methods for the detection of salmonellas in foods

“…typhimurium cells ml-' but the use of a salmonella polyvalent flagella antiserum caused false-positive results. The requirement for assay specificity has led to the purification of flagella antibodies, by removing IgM, and using phosphataselabelled secondary antibody to avoid the interference of bacterial peroxidases (Minnich et al 1982). The authors also found that fixation of cells from food broths to microtitre plates or microcuvettes was inconsistent and so centrifugation was used to remove unbound reagents.…”

Modern immunoassays in meat‐product analysis

“…Previous attempts to devise Salmonella-specific immunoassays using mixtures of polyclonal antibodies yielded many false positives because of the presence of cross-reacting components in the antisera [52]. Since antibodies generated against somatic antigens of Salmonella are primarily of the IgM type, use of purified IgG component of polyclonal antisera decreased the cross-reactivity [53] but did not completely eliminate it [54]. Also, batch-to-batch variations in the reactivity and titers of polyclonal antisera create problems in devising a standard test for routine quality control use.…”

Piezoelectric Biosensor for Detection of Salmonella typhimurium

“…Many attempts have been made to accelerate, simplify and improve the reliability of detection methods. Among these are the fluorescent antibody test (Rodrigues and Kroll, 1990;Tsai and Slavik, 1994), various enzyme-linked antibody tests (Minnich et al, 1982;Flowers et al, 1988), DNA hybridization methods (Fitts et al, 1983) and improved culturing methods (Poppe and Duncan, 1996). Most such tests have shown considerable degrees of success, but most require about 8-10 hr from initial sampling to final results.…”