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SALMONELLA | Detection by Immunoassays
Detection by Immunoassays HP Dwivedi and G Devulder, bioMerieux, Inc., Hazelwood, MO, USA VK Juneja, Eastern Regional Research Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA Ó 2014 Elsevier Ltd. All rights reserved.
Antigenic Makeup of Salmonella
Enzyme-Linked Immunosorbent Assay
Salmonella serovars exhibit three different cell-surface antigens. The somatic (O) antigens, which are present on the outer membrane, are part of lipopolysaccharides (LPS) moieties. The heat-stable somatic antigens of Salmonella include both specific determinants and/or nondiscriminatory antigens. The Salmonella subspecies are divided into over 50 serogroups based on the presence of somatic (O) antigens, including A, B, C1, C2, D1, E1, E2, E3, and E4. Although specific somatic antigens (LPS) have considerable discriminatory power but cross-reaction can occur between the common antigens among the different serotypes of Salmonella. The anti-Salmonella antibodies could cross-react with the antigenically closely related species of certain genera. Gene clusters controlling the somatic antigen of Salmonella that has closer similarity with the somatic antigen of other genera are supposed to have originated from a common ancestor. The O-antigen modifications could possibly be attributed to the induction by prophage genes outside the gene cluster during the evolutionary process of species divergence. The flagellar (H) antigens, which are heat-labile proteins, are associated with peritrichous flagella. Variation of the flagellar antigens between two forms (biphasic antigenic variation), H1 and H2, in which the flagellar subunit consists of FliC or FliB protein, respectively, is noticed in serotypes such as S. Typhimurium. Phase variation is also common among Salmonella serotypes, and variable expression of flagellin proteins, resulting in the assembly of flagella with different structures, can be observed. In the case of biphasic organism, the phase inversion technique could be used to determine both phases. The capsular or virulence (Vi) antigen is also available to screen the Salmonella serotypes (group D). Salmonella serotypes such as Typhi, Paratyphi C, and Dublin exhibit the capsular antigens (Vi), which are referred to as K antigens in other enterobacteriaceae. Serotyping employing Salmonella antigens could be used for the differentiation among serotypes based on somatic, flagellar, and capsular antigens using slide agglutination or tube agglutination tests. The expression of Salmonella antigens could be affected by the components of the growth medium. For example, Salmonella enterica grown on solid medium containing iron, thiosulfate, hexoses, and amino acids is reported to undergo cell-surface differentiation such as increased flagellation and conversion from rough to smooth LPS. Similarly, peptone constituents of culture medium could induce morphological differences in S. Typhimurium, consequently affecting serological identification. Transfer of aflagellate Salmonella from nutritionally poor media deprived of optimum amounts of tyrosine into a rich nutrient broth could allow flagella synthesis, indicating that the aflagellate form is still able to produce flagella as reported in the study by Gray et al. (2006).
Enzyme-Linked Immunosorbent Assay (ELISA) tests are applied for the detection of Salmonella antigens or antibodies in a sample. However, most of the food Salmonella ELISAs are based on the detection of Salmonella antigens using antiSalmonella polyclonal or monoclonal antibodies as primary antibodies tethered to a solid support for the capture of antigens. Nonspecific immobilization of the Salmonella antigens in food could also be performed using passive adsorption on solid surfaces such as polystyrene microtiter plates. The primary antibodies bind specifically with the Salmonella antigens if present in food samples, resulting in specific antigen–antibody complexes. The detection of antigen– antibody complexes could be performed using secondary antibodies conjugated to an enzyme (such as alkaline phosphatase) in a sandwich format assay. The positive detection is facilitated by the development of a detectable color due to enzymatic reaction by conjugate, when an appropriate substrate (such as p-nitrophenyl phosphate) is applied to the conjugate. Many other combinations of conjugates and substrates could also be used such as Horse Radish Peroxidase conjugate and 3,30 ,5,50 -tetramethylbenzidine substrate. Washing steps are frequently performed to remove unbound antibodies and avoid any unspecified bindings to ensure specificity of the assay. Relative quantification of Salmonella antigens in unknown samples could be performed by comparing against the standard curve values to give a positive or negative call to a sample. ELISAs could be performed in various formats (such as direct and indirect) other than sandwich, which is described here. The direct application of some food samples on solid assay surfaces may result in nonspecific binding of matrix components, which in turn may interfere with antigen–antibody reactions or lead to nonspecific results. Several ELISAs have been developed to detect antigens of Salmonella spp. in foods. Besides primary antibodies used in the capture and immobilization of Salmonella antigens present in food samples, alternate ligands have been employed in enzyme immunoassay for the detection of Salmonella. For example, polymyxin-ELISA was reported for the detection of group D salmonellae (including S. Enteritidis), using polymyxin immobilized in the wells of microtiter plate as a high-affinity adsorbent for LPS antigens. Although the sensitivity of ELISAs depends on the food matrices being analyzed (among other factors), the typical limit of detection varies from as low as 104 to >105 CFU ml1. Sensitivity of the assay could further be affected by interference due to background flora in food matrices, expression of Salmonella antigens in the food, and growth rate of Salmonella strains. Specificity of ELISAs for Salmonella detection mostly relies on the specificity of the antibodies employed. Monoclonal antibodies could be more specific, but achieving inclusivity for more than 2400 Salmonella serotypes could be
Encyclopedia of Food Microbiology, Volume 3
http://dx.doi.org/10.1016/B978-0-12-384730-0.00299-8
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SALMONELLA j Detection by Immunoassays
challenging. Similarly, polyclonal antibodies could be more inclusive, but there could be concern of cross-reactivity with an antigenically closer genus such as Citrobacter spp. The enrichment of food samples is required to achieve the limit of detection by enzyme-based immunoassays. For application in several immunoassays, heating of enriched samples is performed to release the antigens from bacteria attached to the food matrix. Many modified versions of ELISAs have been reported that require relatively smaller sample volumes and have improved detection limits with fewer reports of false-positive results. Further modifications have been reported to enhance the sensitivity and multiplexing ability of the assay and to make it more quantitative. In this effort, fluorogenic and electrochemiluminescent reporters have been employed in place of traditional chromogenic reporter substrates. Automated ELISA formats are amenable to performing large numbers of sample analyses with relative ease and rapidity. The choice of conjugate system also plays an important role in the application of ELISAbased detection in food matrices. For example, when using peroxidase conjugates, care should be taken as many food pathogens express intracellular peroxidases or catalases or both, thus resulting in nonspecific immune reaction. Alternatively, conjugates based on alkaline phosphate could be used. Salmonella concentration using immunoconcentration approaches could be performed upfront of enzyme immunoassay. These combined immunoconcentration-ELISA approaches could help reduce the time to detection and achieve enhanced detection sensitivity. The automated immunoconcentration platforms are commercially available, which can process several samples in a single run, thus improving the overall efficiency. In general, ELISAs are simple, easy to perform, scalable, and adaptable assays. Some of the commercial ELISAs provide simplicity of visual detection of color changes due to positive enzymatic reactions; however, many others are automated.
Table 1
Commercially available ELISA and immunoconcentration assays for Salmonella detection in foods are listed in Table 1.
Enzyme-Linked Fluorescent Assay Enzyme-Linked Fluorescent Assays (ELFAs) are based on enzymatic reactions similar to ELISA but instead use fluorogenic substrates such as 4-methylumbelliferyl phosphate (4MUP) as the reporter. 4MUP can detect much lower levels of alkaline phosphatase (109 M) by converting 4MUP into the fluorescent product 4-methylumbelliferone. However, colorimetric substrates such as p-nitrophenyl phosphate (PNP) can detect only 105 M of alkaline phosphatase as it converts PNP into the yellow pigmented p-nitrophenol. ELFA is reported to be more sensitive and faster than ELISA. The automation, rapidity, and ease of use of the automated ELFA formats have further increased ELFA’s popularity. Application of ELFA-based assays for the detection of Salmonella in various food matrices has been widely reported. Alternate ligands such as aptamers, recombinant phage proteins, and peptides could also be applied in conjunction with antibodies to enhance the sensitivities and specificities of ELFA. It must be noted that the sensitivity of ELFA still relies on the upfront enrichment of samples using broth media. Commercially available ELFAs for the detection of Salmonella in foods are listed in Table 1. It must be noted that all samples analyzed using enzyme immunoassays must be confirmed using culture-based procedures to conclude the test results. Until cultural confirmation procedures are completed, a positive immunoassay should be considered only a presumptive result. Immunoassays for detection are not to be confused with definitive serological methods, such as serotyping, which can be confirmatory.
Selected immunology based commercial products for detection and identification of Salmonella in food
Assay name and source
Technique
Target organism(s)
TECRA Salmonella VIA (3 M) TECRA Salmonella ULTIMA (3 M) VIP Goldä for Salmonella (BioControl Systems) Revealâ test systems Salmonella (Neogen Corp.) Reveal S. Enteritidis (Neogen) Oxoid Salmonella Latex Test (Oxoid, Thermo Fisher Scientific Inc.) Assurance EIA Salmonella (BioControl Systems) RapidCheck Salmonella spp. (SDIX) RapidCheck Salmonella Enteritidis (SDIX) Dynabeadsâ Anti-Salmonella antibody (Invitrogen, Life Technologies) BeadRetrieverä system (Invitrogen, Life Technologies) Pathatrix (Matrix MicroScience, Life Technologies) Bioline Salmonella Rapid Test Kit Methods (Bioline) Microgen Salmonella Latex Kit (KeyDiagnotics) Wellcolexâ Color Salmonella (Remel, Thermo Fisher Scientific Inc.) VIDASâ SLM (bioMerieux SA) VIDASâ ICS (bioMerieux SA) VIDASâ UP Salmonella (bioMerieux SA)
ELISA ELISA Lateral flow immunoassay Lateral flow immunoassay Lateral flow immunoassay Latex agglutination
Salmonella spp. Salmonella spp. Salmonella spp. Salmonella spp. Salmonella Enteritidis Salmonella spp.
Enzyme immunoassay Lateral flow immunoassay Lateral flow immunoassay Immuno-magnetic beads
Salmonella spp. Salmonella spp. Salmonella Enteritidis Salmonella
Immuno-magnetic separation (IMS) system IMS system ELISA Latex slide agglutination test Latex agglutination test (detection and serogrouping) ELFA Immunoconcentration ELFAa
Salmonella and other foodborne pathogens Salmonella and other foodborne pathogens Salmonella spp. Salmonella spp. Salmonella spp.
a
Combine specific phage protein technology.
Salmonella spp. Salmonella spp. Salmonella spp.
SALMONELLA j Detection by Immunoassays
Lateral Flow Immunoassay (LIA) The Salmonella antigens present in food samples can be visualized using specific antibody coated test strips. The test strip for lateral flow assay has Salmonella-specific capture antibodies impregnated in a solid support such as a nitrocellulose membrane at a defined distance from the sample application slot. The detection antibodies coupled to colloidal latex or gold particles are placed near the sample application slot. When an enriched food sample is applied, the Salmonella antigens bind with the detection antibody, and the complex moves laterally toward the impregnated capture antibody due to capillary action. The antigenic complex and detection antibody in the moving fluid segregates into two different capture zones, one specific for the Salmonella antigen–antibody complex and the other specific for the unbound detection antibody. A positive test result is visually evident (usually by two colored lines) when a Salmonella-specific reaction occurs. This differs from the visible signal caused by the detection of an antibody-only control (such as a single line). Detection using these strips is rapid and takes around 5-10 min. Lateral flow immunoassays have been reported for the detection of S. enterica from a variety of food matrices. Several lateral flow immunoassays for the detection of foodborne Salmonella are commercially available (Table 1). Lateral flow assays are usually reported to have a high limit of detection; thus sample enrichment prior to test is required. There are reports of a relatively higher number of false-positive results using LIA as compared to more traditional microtiter plate ELISA methods. Serotype-specific LIA for the detection of S. Enteritidis in poultry products such as eggs are also commercially available. When applying such assays, it is important to differentiate S. Enteritidis from closely related serotypes of Salmonella such as non-Enteritidis group D1 Salmonella (such as S. Berta or S. Dublin). Further, many virulent phage types of S. Enteritidis have been reported; thus the inclusivity of these in the S. Enteritidis-specific assay becomes important. Advancements such as automated readers could further improve the usefulness of this assay format. Overall, the ease and rapidity of using LIA makes it convenient to perform preliminary screening of pathogen contamination in foods and environmental samples, despite its comparatively lower specificity and sensitivity.
Latex Agglutination Assay Latex agglutination (LA) is an immunoassay that is performed mostly for the primary culture screening of Salmonella by mixing isolated colony from plate with sensitized latex beads linked to antibodies specific for Salmonella antigens. Visible clumping indicative of a positive reaction can be compared with positive and negative control samples. Attention must be paid to observe the isolates that autoagglutinate; otherwise they can interfere with interpretation of the result. LA assays are rapid and easy to perform, providing results within a few minutes. These assays are mainly used for the presumptive confirmation of the isolated colonies and could help reduce samples for further confirmation. However, LA can also be performed for detection of Salmonella directly from enriched food samples. Specificity of LA assays relies on the specificity of antibodies incorporated in the
341
assay. Many latex agglutination kits, including Salmonella serogroups specific tests, are commercially available for the detection of foodborne pathogens (Table 1).
See also: Salmonella: Introduction; Salmonella: Detection by Classical Cultural Techniques.
Further Reading Ali, A., Ali, R., 1983. Enzyme-linked immunosorbent assay for anti-DNA antibodies using fluorogenic and colorigenic substrates. Journal of Immunological Methods 56, 341–346. Banada, P.P., Bhunia, A.K., 2008. Antibodies and immunoassays for detection of bacterial pathogens. In: Zourob, M., Elwary, S., Turner, A. (Eds.), Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York. Blais, B.W., Martinez-Perez, A., 2008 Feb. Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay. Journal of Food Protection 71 (2), 392–396. Bohaychuk, V.M., Gensler, G.E., King, R.K., Wu, J.T., McMullen, L.M., 2005. Evaluation of detection methods for screening meat and poultry products for the presence of food borne pathogens. Journal of Food Protection 68 (12), 2637–2647. Briggs, J., Dailianis, A., Hughes, D., Garthwaite, I., 2004. Validation study to demonstrate the equivalence of a minor modification (TECRA ULTIMA protocol) to AOAC method 998.09 (TECRA Salmonella visual immunoassay) with the cultural reference method. Journal of AOAC International 87 (2), 374–379. Chapman, P.A., Ashton, R., 2003. An evaluation of rapid methods for detecting Escherichia coli O157 on beef carcasses. International Journal of Food Microbiology 87 (3), 279–285. Cheesbrough, S., Donnely, C., 1996. The use of a rapid Salmonella latex serogrouping test (spectate) to assist in the confirmation of ELISA-based rapid Salmonella screening tests. Letters in Applied Microbiology 22 (5), 378–380. Cox, N.A., 1988. Salmonella methodology update. Poultry Science 67, 921–927. Cox, N.A., Fung, D.Y.C., Bailey, J.S., Hartman, P.A., Vasavada, P.C., 1987. Miniaturized kits, immunoassays and DNA hybridization for recognition and identification of food borne bacteria. Dairy and Food Sanitation 7, 628–631. Crowley, E., Bird, P., Fisher, K., Goetz, K., Benzinger Jr., M.J., Agin, J., Goins, D., Johnson, R.L., 2011. Evaluation of VIDAS Salmonella (SLM) easy Salmonella method for the detection of Salmonella in a variety of foods: collaborative study. Journal of AOAC International 94 (6), 1821–1834. de Paula, A.M.R., Gelli, D.S., Landgraf, M., Destro, M.T., Franco, B., 2002. Detection of Salmonella in foods using Tecra Salmonella VIA and Tecra Salmonella UNIQUE rapid immunoassays and a cultural procedure. Journal of Food Protection 65, 552–555. Dwivedi, H.P., Jaykus, L.A., 2011. Detection of pathogens in foods: the current state-of-the-art and future directions. Critical Reviews In Microbiology 37 (1), 40–63. Gracias, K.S., McKillip, J.L., 2004. A review of conventional detection and enumeration methods for pathogenic bacteria in food. Canadian Journal of Microbiology 50, 883–890. Gray, V.L., O’Reilly, M., Müller, C.T., Watkins, I.D., Lloyd, D., 2006. Low tyrosine content of growth media yields aflagellate Salmonella enterica serovar Typhimurium. Microbiology 152 (Pt 1), 23–28. Guard-Petter, J., 1997. Induction of flagellation and a novel agar-penetrating flagellar structure in Salmonella enterica grown on solid media: possible consequences for serological identification. FEMS Microbiology Letters 149 (2), 173–180. Hoerner, R., Feldpausch, J., Gray, R.L., Curry, S., Islam, Z., Goldy, T., Klein, F., Tadese, T., Rice, J., Mozola, M., 2011. Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance tested method 960801. Journal of AOAC International 94 (5), 1467–1480. Ishikawa, E., Kato, K., 1978. Ultrasensitive enzyme immunoassay. Scandinavian Journal of Immunology 8, 43–55. Leng, S., McElhaney, J., Walston, J., Xie, D., Fedarko, N., Kuchel, G., 2008. ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63 (8), 879–884.
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Leung, W., Chan, C.P., Rainer, T.H., Ip, M., Cautherley, G.W.H., Renneberg, R., 2008. InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections. Journal of Immunological Methods 336 (1), 30–36. Marjan, W., Woude, V., Bäumler, A.J., 2004. Phase and antigenic variation in bacteria. Clinical Microbiology Reviews 17 (3), 581. Milley, D.G., Sekla, L.H., 1993. An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli. Applied and Environmental Microbiology 59 (12), 4223–4229. Perepelov, A.V., Liu, B., Guo, D., Senchenkova, S.N., Shahskov, A.S., Feng, L., Wang, L., Knirel, Y.A., 2011 Jul. Structure elucidation of the O-antigen of Salmonella enterica O51 and its structural and genetic relation to the O-antigen of Escherichia coli O23. Biochemistry (Moscow) 76 (7), 774–779. Shekarchi, I.C., Sever, J.L., Nerurkar, L., Fuccillo, D., 1985. Comparison of enzymelinked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus. Journal of Clinical Microbiology 21 (1), 92–96.
Valdivieso-Garcia, A., Riche, E., Abubakar, O., Waddell, T.E., Brooks, B.W., 2001. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. Journal of Food Protection 64, 1166–1171. Voogt, N., Wannet, W.J., Nagelkerke, N.J., Henken, A.M., 2002. Differences between national reference laboratories of the European community in their ability to serotype Salmonella species. European Journal of Clinical Microbiology & Infectious Diseases 21, 204–208. Weeratna, R.D., Doyle, M.P., 1991. Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food. Applied and Environmental Microbiology 57 (10), 2951–2955. Yolken, R.H., Stopa, P.J., 1979. Enzyme-linked fluorescence assay: ultrasensitive solid-phase assay for detection of human rotavirus. Journal of Clinical Microbiology 10, 317–321.
Antigenic Makeup of Salmonella
Enzyme-Linked Immunosorbent Assay
Salmonella serovars exhibit three different cell-surface antigens. The somatic (O) antigens, which are present on the outer membrane, are part of lipopolysaccharides (LPS) moieties. The heat-stable somatic antigens of Salmonella include both specific determinants and/or nondiscriminatory antigens. The Salmonella subspecies are divided into over 50 serogroups based on the presence of somatic (O) antigens, including A, B, C1, C2, D1, E1, E2, E3, and E4. Although specific somatic antigens (LPS) have considerable discriminatory power but cross-reaction can occur between the common antigens among the different serotypes of Salmonella. The anti-Salmonella antibodies could cross-react with the antigenically closely related species of certain genera. Gene clusters controlling the somatic antigen of Salmonella that has closer similarity with the somatic antigen of other genera are supposed to have originated from a common ancestor. The O-antigen modifications could possibly be attributed to the induction by prophage genes outside the gene cluster during the evolutionary process of species divergence. The flagellar (H) antigens, which are heat-labile proteins, are associated with peritrichous flagella. Variation of the flagellar antigens between two forms (biphasic antigenic variation), H1 and H2, in which the flagellar subunit consists of FliC or FliB protein, respectively, is noticed in serotypes such as S. Typhimurium. Phase variation is also common among Salmonella serotypes, and variable expression of flagellin proteins, resulting in the assembly of flagella with different structures, can be observed. In the case of biphasic organism, the phase inversion technique could be used to determine both phases. The capsular or virulence (Vi) antigen is also available to screen the Salmonella serotypes (group D). Salmonella serotypes such as Typhi, Paratyphi C, and Dublin exhibit the capsular antigens (Vi), which are referred to as K antigens in other enterobacteriaceae. Serotyping employing Salmonella antigens could be used for the differentiation among serotypes based on somatic, flagellar, and capsular antigens using slide agglutination or tube agglutination tests. The expression of Salmonella antigens could be affected by the components of the growth medium. For example, Salmonella enterica grown on solid medium containing iron, thiosulfate, hexoses, and amino acids is reported to undergo cell-surface differentiation such as increased flagellation and conversion from rough to smooth LPS. Similarly, peptone constituents of culture medium could induce morphological differences in S. Typhimurium, consequently affecting serological identification. Transfer of aflagellate Salmonella from nutritionally poor media deprived of optimum amounts of tyrosine into a rich nutrient broth could allow flagella synthesis, indicating that the aflagellate form is still able to produce flagella as reported in the study by Gray et al. (2006).
Enzyme-Linked Immunosorbent Assay (ELISA) tests are applied for the detection of Salmonella antigens or antibodies in a sample. However, most of the food Salmonella ELISAs are based on the detection of Salmonella antigens using antiSalmonella polyclonal or monoclonal antibodies as primary antibodies tethered to a solid support for the capture of antigens. Nonspecific immobilization of the Salmonella antigens in food could also be performed using passive adsorption on solid surfaces such as polystyrene microtiter plates. The primary antibodies bind specifically with the Salmonella antigens if present in food samples, resulting in specific antigen–antibody complexes. The detection of antigen– antibody complexes could be performed using secondary antibodies conjugated to an enzyme (such as alkaline phosphatase) in a sandwich format assay. The positive detection is facilitated by the development of a detectable color due to enzymatic reaction by conjugate, when an appropriate substrate (such as p-nitrophenyl phosphate) is applied to the conjugate. Many other combinations of conjugates and substrates could also be used such as Horse Radish Peroxidase conjugate and 3,30 ,5,50 -tetramethylbenzidine substrate. Washing steps are frequently performed to remove unbound antibodies and avoid any unspecified bindings to ensure specificity of the assay. Relative quantification of Salmonella antigens in unknown samples could be performed by comparing against the standard curve values to give a positive or negative call to a sample. ELISAs could be performed in various formats (such as direct and indirect) other than sandwich, which is described here. The direct application of some food samples on solid assay surfaces may result in nonspecific binding of matrix components, which in turn may interfere with antigen–antibody reactions or lead to nonspecific results. Several ELISAs have been developed to detect antigens of Salmonella spp. in foods. Besides primary antibodies used in the capture and immobilization of Salmonella antigens present in food samples, alternate ligands have been employed in enzyme immunoassay for the detection of Salmonella. For example, polymyxin-ELISA was reported for the detection of group D salmonellae (including S. Enteritidis), using polymyxin immobilized in the wells of microtiter plate as a high-affinity adsorbent for LPS antigens. Although the sensitivity of ELISAs depends on the food matrices being analyzed (among other factors), the typical limit of detection varies from as low as 104 to >105 CFU ml1. Sensitivity of the assay could further be affected by interference due to background flora in food matrices, expression of Salmonella antigens in the food, and growth rate of Salmonella strains. Specificity of ELISAs for Salmonella detection mostly relies on the specificity of the antibodies employed. Monoclonal antibodies could be more specific, but achieving inclusivity for more than 2400 Salmonella serotypes could be
Encyclopedia of Food Microbiology, Volume 3
http://dx.doi.org/10.1016/B978-0-12-384730-0.00299-8
339
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SALMONELLA j Detection by Immunoassays
challenging. Similarly, polyclonal antibodies could be more inclusive, but there could be concern of cross-reactivity with an antigenically closer genus such as Citrobacter spp. The enrichment of food samples is required to achieve the limit of detection by enzyme-based immunoassays. For application in several immunoassays, heating of enriched samples is performed to release the antigens from bacteria attached to the food matrix. Many modified versions of ELISAs have been reported that require relatively smaller sample volumes and have improved detection limits with fewer reports of false-positive results. Further modifications have been reported to enhance the sensitivity and multiplexing ability of the assay and to make it more quantitative. In this effort, fluorogenic and electrochemiluminescent reporters have been employed in place of traditional chromogenic reporter substrates. Automated ELISA formats are amenable to performing large numbers of sample analyses with relative ease and rapidity. The choice of conjugate system also plays an important role in the application of ELISAbased detection in food matrices. For example, when using peroxidase conjugates, care should be taken as many food pathogens express intracellular peroxidases or catalases or both, thus resulting in nonspecific immune reaction. Alternatively, conjugates based on alkaline phosphate could be used. Salmonella concentration using immunoconcentration approaches could be performed upfront of enzyme immunoassay. These combined immunoconcentration-ELISA approaches could help reduce the time to detection and achieve enhanced detection sensitivity. The automated immunoconcentration platforms are commercially available, which can process several samples in a single run, thus improving the overall efficiency. In general, ELISAs are simple, easy to perform, scalable, and adaptable assays. Some of the commercial ELISAs provide simplicity of visual detection of color changes due to positive enzymatic reactions; however, many others are automated.
Table 1
Commercially available ELISA and immunoconcentration assays for Salmonella detection in foods are listed in Table 1.
Enzyme-Linked Fluorescent Assay Enzyme-Linked Fluorescent Assays (ELFAs) are based on enzymatic reactions similar to ELISA but instead use fluorogenic substrates such as 4-methylumbelliferyl phosphate (4MUP) as the reporter. 4MUP can detect much lower levels of alkaline phosphatase (109 M) by converting 4MUP into the fluorescent product 4-methylumbelliferone. However, colorimetric substrates such as p-nitrophenyl phosphate (PNP) can detect only 105 M of alkaline phosphatase as it converts PNP into the yellow pigmented p-nitrophenol. ELFA is reported to be more sensitive and faster than ELISA. The automation, rapidity, and ease of use of the automated ELFA formats have further increased ELFA’s popularity. Application of ELFA-based assays for the detection of Salmonella in various food matrices has been widely reported. Alternate ligands such as aptamers, recombinant phage proteins, and peptides could also be applied in conjunction with antibodies to enhance the sensitivities and specificities of ELFA. It must be noted that the sensitivity of ELFA still relies on the upfront enrichment of samples using broth media. Commercially available ELFAs for the detection of Salmonella in foods are listed in Table 1. It must be noted that all samples analyzed using enzyme immunoassays must be confirmed using culture-based procedures to conclude the test results. Until cultural confirmation procedures are completed, a positive immunoassay should be considered only a presumptive result. Immunoassays for detection are not to be confused with definitive serological methods, such as serotyping, which can be confirmatory.
Selected immunology based commercial products for detection and identification of Salmonella in food
Assay name and source
Technique
Target organism(s)
TECRA Salmonella VIA (3 M) TECRA Salmonella ULTIMA (3 M) VIP Goldä for Salmonella (BioControl Systems) Revealâ test systems Salmonella (Neogen Corp.) Reveal S. Enteritidis (Neogen) Oxoid Salmonella Latex Test (Oxoid, Thermo Fisher Scientific Inc.) Assurance EIA Salmonella (BioControl Systems) RapidCheck Salmonella spp. (SDIX) RapidCheck Salmonella Enteritidis (SDIX) Dynabeadsâ Anti-Salmonella antibody (Invitrogen, Life Technologies) BeadRetrieverä system (Invitrogen, Life Technologies) Pathatrix (Matrix MicroScience, Life Technologies) Bioline Salmonella Rapid Test Kit Methods (Bioline) Microgen Salmonella Latex Kit (KeyDiagnotics) Wellcolexâ Color Salmonella (Remel, Thermo Fisher Scientific Inc.) VIDASâ SLM (bioMerieux SA) VIDASâ ICS (bioMerieux SA) VIDASâ UP Salmonella (bioMerieux SA)
ELISA ELISA Lateral flow immunoassay Lateral flow immunoassay Lateral flow immunoassay Latex agglutination
Salmonella spp. Salmonella spp. Salmonella spp. Salmonella spp. Salmonella Enteritidis Salmonella spp.
Enzyme immunoassay Lateral flow immunoassay Lateral flow immunoassay Immuno-magnetic beads
Salmonella spp. Salmonella spp. Salmonella Enteritidis Salmonella
Immuno-magnetic separation (IMS) system IMS system ELISA Latex slide agglutination test Latex agglutination test (detection and serogrouping) ELFA Immunoconcentration ELFAa
Salmonella and other foodborne pathogens Salmonella and other foodborne pathogens Salmonella spp. Salmonella spp. Salmonella spp.
a
Combine specific phage protein technology.
Salmonella spp. Salmonella spp. Salmonella spp.
SALMONELLA j Detection by Immunoassays
Lateral Flow Immunoassay (LIA) The Salmonella antigens present in food samples can be visualized using specific antibody coated test strips. The test strip for lateral flow assay has Salmonella-specific capture antibodies impregnated in a solid support such as a nitrocellulose membrane at a defined distance from the sample application slot. The detection antibodies coupled to colloidal latex or gold particles are placed near the sample application slot. When an enriched food sample is applied, the Salmonella antigens bind with the detection antibody, and the complex moves laterally toward the impregnated capture antibody due to capillary action. The antigenic complex and detection antibody in the moving fluid segregates into two different capture zones, one specific for the Salmonella antigen–antibody complex and the other specific for the unbound detection antibody. A positive test result is visually evident (usually by two colored lines) when a Salmonella-specific reaction occurs. This differs from the visible signal caused by the detection of an antibody-only control (such as a single line). Detection using these strips is rapid and takes around 5-10 min. Lateral flow immunoassays have been reported for the detection of S. enterica from a variety of food matrices. Several lateral flow immunoassays for the detection of foodborne Salmonella are commercially available (Table 1). Lateral flow assays are usually reported to have a high limit of detection; thus sample enrichment prior to test is required. There are reports of a relatively higher number of false-positive results using LIA as compared to more traditional microtiter plate ELISA methods. Serotype-specific LIA for the detection of S. Enteritidis in poultry products such as eggs are also commercially available. When applying such assays, it is important to differentiate S. Enteritidis from closely related serotypes of Salmonella such as non-Enteritidis group D1 Salmonella (such as S. Berta or S. Dublin). Further, many virulent phage types of S. Enteritidis have been reported; thus the inclusivity of these in the S. Enteritidis-specific assay becomes important. Advancements such as automated readers could further improve the usefulness of this assay format. Overall, the ease and rapidity of using LIA makes it convenient to perform preliminary screening of pathogen contamination in foods and environmental samples, despite its comparatively lower specificity and sensitivity.
Latex Agglutination Assay Latex agglutination (LA) is an immunoassay that is performed mostly for the primary culture screening of Salmonella by mixing isolated colony from plate with sensitized latex beads linked to antibodies specific for Salmonella antigens. Visible clumping indicative of a positive reaction can be compared with positive and negative control samples. Attention must be paid to observe the isolates that autoagglutinate; otherwise they can interfere with interpretation of the result. LA assays are rapid and easy to perform, providing results within a few minutes. These assays are mainly used for the presumptive confirmation of the isolated colonies and could help reduce samples for further confirmation. However, LA can also be performed for detection of Salmonella directly from enriched food samples. Specificity of LA assays relies on the specificity of antibodies incorporated in the
341
assay. Many latex agglutination kits, including Salmonella serogroups specific tests, are commercially available for the detection of foodborne pathogens (Table 1).
See also: Salmonella: Introduction; Salmonella: Detection by Classical Cultural Techniques.
Further Reading Ali, A., Ali, R., 1983. Enzyme-linked immunosorbent assay for anti-DNA antibodies using fluorogenic and colorigenic substrates. Journal of Immunological Methods 56, 341–346. Banada, P.P., Bhunia, A.K., 2008. Antibodies and immunoassays for detection of bacterial pathogens. In: Zourob, M., Elwary, S., Turner, A. (Eds.), Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York. Blais, B.W., Martinez-Perez, A., 2008 Feb. Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay. Journal of Food Protection 71 (2), 392–396. Bohaychuk, V.M., Gensler, G.E., King, R.K., Wu, J.T., McMullen, L.M., 2005. Evaluation of detection methods for screening meat and poultry products for the presence of food borne pathogens. Journal of Food Protection 68 (12), 2637–2647. Briggs, J., Dailianis, A., Hughes, D., Garthwaite, I., 2004. Validation study to demonstrate the equivalence of a minor modification (TECRA ULTIMA protocol) to AOAC method 998.09 (TECRA Salmonella visual immunoassay) with the cultural reference method. Journal of AOAC International 87 (2), 374–379. Chapman, P.A., Ashton, R., 2003. An evaluation of rapid methods for detecting Escherichia coli O157 on beef carcasses. International Journal of Food Microbiology 87 (3), 279–285. Cheesbrough, S., Donnely, C., 1996. The use of a rapid Salmonella latex serogrouping test (spectate) to assist in the confirmation of ELISA-based rapid Salmonella screening tests. Letters in Applied Microbiology 22 (5), 378–380. Cox, N.A., 1988. Salmonella methodology update. Poultry Science 67, 921–927. Cox, N.A., Fung, D.Y.C., Bailey, J.S., Hartman, P.A., Vasavada, P.C., 1987. Miniaturized kits, immunoassays and DNA hybridization for recognition and identification of food borne bacteria. Dairy and Food Sanitation 7, 628–631. Crowley, E., Bird, P., Fisher, K., Goetz, K., Benzinger Jr., M.J., Agin, J., Goins, D., Johnson, R.L., 2011. Evaluation of VIDAS Salmonella (SLM) easy Salmonella method for the detection of Salmonella in a variety of foods: collaborative study. Journal of AOAC International 94 (6), 1821–1834. de Paula, A.M.R., Gelli, D.S., Landgraf, M., Destro, M.T., Franco, B., 2002. Detection of Salmonella in foods using Tecra Salmonella VIA and Tecra Salmonella UNIQUE rapid immunoassays and a cultural procedure. Journal of Food Protection 65, 552–555. Dwivedi, H.P., Jaykus, L.A., 2011. Detection of pathogens in foods: the current state-of-the-art and future directions. Critical Reviews In Microbiology 37 (1), 40–63. Gracias, K.S., McKillip, J.L., 2004. A review of conventional detection and enumeration methods for pathogenic bacteria in food. Canadian Journal of Microbiology 50, 883–890. Gray, V.L., O’Reilly, M., Müller, C.T., Watkins, I.D., Lloyd, D., 2006. Low tyrosine content of growth media yields aflagellate Salmonella enterica serovar Typhimurium. Microbiology 152 (Pt 1), 23–28. Guard-Petter, J., 1997. Induction of flagellation and a novel agar-penetrating flagellar structure in Salmonella enterica grown on solid media: possible consequences for serological identification. FEMS Microbiology Letters 149 (2), 173–180. Hoerner, R., Feldpausch, J., Gray, R.L., Curry, S., Islam, Z., Goldy, T., Klein, F., Tadese, T., Rice, J., Mozola, M., 2011. Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance tested method 960801. Journal of AOAC International 94 (5), 1467–1480. Ishikawa, E., Kato, K., 1978. Ultrasensitive enzyme immunoassay. Scandinavian Journal of Immunology 8, 43–55. Leng, S., McElhaney, J., Walston, J., Xie, D., Fedarko, N., Kuchel, G., 2008. ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63 (8), 879–884.
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Leung, W., Chan, C.P., Rainer, T.H., Ip, M., Cautherley, G.W.H., Renneberg, R., 2008. InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections. Journal of Immunological Methods 336 (1), 30–36. Marjan, W., Woude, V., Bäumler, A.J., 2004. Phase and antigenic variation in bacteria. Clinical Microbiology Reviews 17 (3), 581. Milley, D.G., Sekla, L.H., 1993. An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli. Applied and Environmental Microbiology 59 (12), 4223–4229. Perepelov, A.V., Liu, B., Guo, D., Senchenkova, S.N., Shahskov, A.S., Feng, L., Wang, L., Knirel, Y.A., 2011 Jul. Structure elucidation of the O-antigen of Salmonella enterica O51 and its structural and genetic relation to the O-antigen of Escherichia coli O23. Biochemistry (Moscow) 76 (7), 774–779. Shekarchi, I.C., Sever, J.L., Nerurkar, L., Fuccillo, D., 1985. Comparison of enzymelinked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus. Journal of Clinical Microbiology 21 (1), 92–96.
Valdivieso-Garcia, A., Riche, E., Abubakar, O., Waddell, T.E., Brooks, B.W., 2001. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. Journal of Food Protection 64, 1166–1171. Voogt, N., Wannet, W.J., Nagelkerke, N.J., Henken, A.M., 2002. Differences between national reference laboratories of the European community in their ability to serotype Salmonella species. European Journal of Clinical Microbiology & Infectious Diseases 21, 204–208. Weeratna, R.D., Doyle, M.P., 1991. Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food. Applied and Environmental Microbiology 57 (10), 2951–2955. Yolken, R.H., Stopa, P.J., 1979. Enzyme-linked fluorescence assay: ultrasensitive solid-phase assay for detection of human rotavirus. Journal of Clinical Microbiology 10, 317–321.