Production and caracterization of monoclonal antibodies for the detection of Salmonella enterica in chicken meat

Abstract

A panel of 13 monoclonal antibodies (MAbs) that react against outer membrane proteins of Salmonella Enteritidis was obtained. Two MAbs were classified as IgM, six were IgG2a, three were IgG3 and one was of the IgG2b isotype. The reactivity of the MAbs against different serovars of Salmonella enterica and other bacteria was investigated using an indirect ELISA. Five MAbs reacted only against Salmonella Enteritidis. Two MAbs presented crossed reactions with thermo-extracted antigens of Klebsiella pneumoniae, Citrobacter freundii and Enterobacter aerogenes. MAb 424H presented wide spectrum of reactivity, detecting antigens of Salmonella belonging to serogroups B, C, D, E and G. The detection limit of different serovars of Salmonella in a indirect ELISA with MAb 424H varied from 1.0 x 10 4 CFU/mL for Salmonella London to 1.4 x 10 6 CFU/mL for Salmonella Gallinarum and Salmonella Typhimurium. Evaluation of the performance of the ELISA with MAb 424H in the detection of Salmonella in samples of chicken meat artificially contaminated revealed that the ELISA was able to detect all serovars after sample enrichment using two levels of contamination. Samples of chicken meat not artificially contaminated analysed in parallel were negative for Salmonella in both the conventional and the ELISA methods.

Evaluation of an indirect ELISA for the detection of Salmonella in chicken meat

“…The post-enriched cultures were heated (100ºC/10 min) and used to sensitize four wells of polystyrene plates with 50 μL/well for 1h at 37ºC. The plates were washed three times with phosphate-buffered saline (0.01 M, pH 7.2) containing 0.05% Tween 20 (PBST), and two wells received MAb 424H, that react specifically with a putative outer membrane protein from salmonellae (27), and the other two were used without MAb for control of unspecific reactions. The wells received 50 μL of the MAb diluted 1:1000 in PBST and left for 1h at 37ºC.…”

“…The optical density (OD) was then read at 450 nm in a MR 700 microplate reader (Dynatech Laboratories, INC., Virginia). The ELISA cut off for positive samples corresponded to a mean OD above 0.095, or the 96 th percentile of 20 negative samples (27). As ELISA positive control, each plate contained four wells sensitized with a thermo-extracted antigen from Salmonella Enteritidis ATCC 13076.…”

Real Time and Conventional PCR for Characterization of Salmonella sp. from Imported Meat to Egypt

“…Implementation of immunological methods for detection of bacterial agents, spores, toxins and viruses expressed great success (Iqbal et al, 2000). Monoclonal antibodies have been used in the diagnosis of food borne pathogens as Salmonella enterica (Schneid et al, 2005), Salmonella typhi (Kumar et al, 2003). Most of immunological tests used for detection of foodborne pathogens are agglutination test (Matar et al, 1997) western blot test (Rasooly and Rasooly, 1998), enzyme immunoassay (EIA) (Borck et al, 2002), enzyme linked immunosorbent assay (ELISA) (Bennett, 2005).…”

Isolation and purification of O and H antigens from Salmonella Enteritidis as diagnostic tool

“…Many diagnostic tests have been developed for the detection of salmonella infections in poultry. Conventional methodology for identifying Salmonella is based on the isolation in pure cultures, and biochemical and serological tests (Fitzgerald et al 2003;Schneid et al 2005;Sonne-Hansen and Jenabian 2005). However, culture is expensive and time-consuming and also suffers because individual birds excrete Salmonella Enteritidis intermittently or may eliminate the infection altogether.…”

“…Salmonella Enteritidis is an invasive serotype, and immunoglobulin G (IgG) responses persist in birds that have been infected with Salmonella Enteritidis. Therefore, serology would be a superior method for screening birds that are intermittently culture-positive or that have eliminated Salmonella Enteritidis infection (McDonough et al 1998;Schneid et al 2005). The most used diagnostic tests are based on the detection of antibodies against the "O" chain of the S-LPS and/or flagellin (Barrow 1994;Ochoa-Reparaz et al 2005).…”