Detection of Staphylococcal Enterotoxins by Enzyme-Linked

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  • APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 885-8900099-2240/86/050885-06$02.00/0Copyright C 1986, American Society for Microbiology

    Detection of Staphylococcal Enterotoxins by Enzyme-LinkedImmunosorbent Assays and Radioimmunoassays: Comparison of


    MARY L. EVENSON, AND MERLIN S. BERGDOLLFood Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706

    Received 18 October 1985/Accepted 5 February 1986

    Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxinwere examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin perml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifyingan effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate andremoved the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated tohorseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the samesystem by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected inone assay system. Two effective systems were described for each of types B and D. Each of these systems, whencompared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to comparefavorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins Aand C2 were examined for a variety of food extracts; no abnormal interference could be detected from theseextracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specificpolyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Singlemonoclonal antibodies generaUly did not perform as well in the RIA as did the homologous toxin-specificpolyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity andprecision of the polyclonal system for the detection of each toxin by the RIA.

    Detection of staphylococcal enterotoxins in food samplesis dependent upon rapid, reliable, and sensitive immunolog-ical assay systems. Three basic types of immunologicalassays have been applied to the detection of staphylococcalenterotoxins in food: (i) immunodiffusion assays, (ii)radioimmunoassays (RIAs), and (iii) enzyme-linked immu-nosorbent assays (ELISAs).The most commonly used assay is the microslide assay

    (5), a double-gel immunodiffusion assay performed on amicroscope slide. Because the sensitivity of this assay is low(about 0.1 Rg of toxin per ml of food extract), the extractfrom a 100-g food sample must be concentrated about 1,500times to reliably detect 1 ng of toxin per g of food sample.This procedure can require up to 3 days to complete (18).

    Several investigators have successfully applied the RIA tothe analysis of food samples for the presence of staphylo-coccal enterotoxins (2). The RIA affords sensitivity (1 ng oftoxin per ml) without concentration of the food extract.Several variations of the RIA exist, but the competitive RIAis the most commonly used method for quantitative mea-surement of a specific compound in a sample. The compet-itive RIA requires the availability of purified, radioisotopi-cally labeled antigen which competes with the unlabeledantigen in the sample for reactivity with the specific anti-body. A special license is required for this work, anddisposal of radioactive waste is a major problem.The ELISA combines the sensitivity of the RIA with the

    universality of the microslide assay. Many ELISA systems

    * Corresponding author.t Present address: Department of Oncology, McArdle Laboratory

    for Cancer Research, University of Wisconsin-Madison, Madison,WI 53706.

    have been reported for the detection of staphylococcalenterotoxins in food samples (9, 10). Fey et al. compared thebasic versions of the ELISA used to detect staphylococcalenterotoxin and concluded that the double-antibody sand-wich system is superior to the other versions of the ELISA(9). In this assay, antigen-specific immunoglobulin G (IgG)coated onto a solid-phase support removes the antigen fromthe test material. A separate preparation of antigen-specificIgG, which is conjugated to an appropriate enzyme, is usedto probe for the captured antigen. Subsequent reaction of theenzyme with the substrate results in a quantitative, colori-metric measurement of the antigen present.To date, all of the immunological assays described for the

    detection of staphylococcal enterotoxins in food sampleshave used enterotoxin-specific polyclonal antibodies. Sev-eral reports (7, 14, 25) have described the preparation ofmonoclonal antibodies (MAbs) reactive with the variousserological types of enterotoxin. However, none of thesereports has described the adaptation of these antibodies topractical assay systems for the detection of staphylococcalenterotoxins in food samples.The antibody sandwich ELISA (10) and the RIA (15) have

    been used in our laboratory for the detection of enterotoxinin food samples for several years. This paper describes theadaptation of MAbs to these two assay systems for thedetection of staphylococcal enterotoxins A to E (designatedSEA, SEB, SEC1 SEC2, SEC3, SED, and SEE). The resultsobtained with each assay system were compared with thoseobtained with the homologous polyclonal rabbit system.These MAb-based systems might prove to be useful substi-tutes for the polyclonal rabbit antibody-based systems cur-rently in use.


    Vol. 51, No. 5


    TABLE 1. Characterization of MAbs reactive with staphylococcal enterotoxins

    Homologous % Binding of Antibody Estimated affinity Cross-reactivity byAntlbody-toxin toxin subclass (liter/mol)a RIAb

    1A SEA 29 IgGl 9.3 x 108 None2A SEA 28 IgGl 7.6 x 109 SEE3A SEA 77 IgG2a 9.3 x 108 None4A SEA 21 IgGl 2.3 x 108 None2B SEB 30 IgGl 1.5 x 109 None3B SEB 22 IgGl 3.2 x 108 None6B SEB 23 IgGl 5.1 x 109 (SEC,, SEC2, SEC3)2C2 SEC2 56 IgG2b 2.3 x 109 SEC,, SEC34C2 SEC2 63 IgG2a 2.3 x 109 SEC,, SEC31C3 SEC3 81 IgGl 1.7 x 109 SEC,, SEC21D SED 24 IgGl 2.4 x 109 None3D SED 27 IgGI 4.9 x 109 None4D SED 22 IgGl 1.8 x 109 None1E SEE 66 IgG2a 4.9 x 108 SEA, (SED)2E SEE 39 IgGl 8.3 x 1010 None4E SEE 36 IgGl 2.7 x 109 SEASE SEE 50 IgGl 2.3 x 1010 None

    a Detennined on the homologous toxin type by the method of Muller (16).b Toxin types in parentheses show weak reactivity with the antibody.


    Enterotoxins. The staphylococcal enterotoxins were puri-fied at the Food Research Institute as previously described (1,3, 4, 6, 19, 22, 23). The homogeneity of each toxin wasdetermined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) in the native form and afterreduction with 2-mercaptoethanol. Each toxin was iodinatedby the method of Miller et al. (15), using Na'251 (New EnglandNuclear Corp., Boston, Mass.). Crude toxins were preparedby chromatography of each culture supernatant fluid onAmberlite CG-50 (Sigma Chemical Co., St. Louis, Mo.).

    Antibodies. Specific polyclonal rabbit serum was preparedto each of the enterotoxins by the injection of the purifiedprotein into New Zealand White rabbits by the procedure ofRobbins and Bergdoll (21). The IgG-containing fractionswere isolated by precipitation with 35% (NH4)2SO4 withsubsequent chromatography on a column (2.5 by 100 cm) ofSephacryl S-300 (Pharmacia Fine Chemicals, Piscataway,N.J.) in phosphate-buffered (0.01 M) saline (0.15 M NaCl).

    Cell lines producing MAbs reactive with each enterotoxinwere prepared as described previously (25). Cultures werescreened for the production of enterotoxin-specific antibod-ies by the method of Miller et al. (15) for the titration ofantisera. Briefly, culture medium (100 ,ul) from each well wasdiluted into 1.0 ml of RIA buffer. Approximately 12,000 cpmof 1251I-labeled enterotoxin was added to each tube; thisresulted in an enterotoxin concentration of approximately0.3 ng/ml. After incubation for 1.5 h at room temperature,200 ,ul of a 10% solution of protein A-containing cells wasadded and incubated for 20 min at room temperature. Thecells were removed by centrifugation, the supernatant fluidswere aspirated, and the pellets were counted for 1 min in agamma counter. The nonspecific binding of 125I-labeledenterotoxin to the cells was usually around 400 cpm; there-fore, samples binding 1,000 cpm or more were considered tobe presumptive positives. Cells in each presumptive positivewell were cloned twice by limiting dilution to ensuremonoclonality and stability of the cell line.Each MAb was purified by precipitation of the IgG from

    ascites fluid by the addition of 35 to 45% (NH4)2SO4 and

    subsequent chromatography on a column (2.5 by 100 cm) ofSephacryl S-300 in phosphate-buffered saline. The subclassof each antibody was determined by reaction in double-gelimmunodiffusion assays with subclass-specific antisera(Miles Laboratories, Elkhart, Ind.).SDS-PAGE and Western blotting. Discontinuous SDS-

    PAGE was performed by the method of Laemmli (12), usinga 12.5% running gel (140 by 125 by 1.5 mm).

    Enterotoxin from gels was blotted onto nitrocellulose(Bio-Rad no. 162-0114) under basic conditions by the methodof Towbin et al. (26). Electroblotting was performed at 100mA overnight. Antigen-antibody reactions were detected bythe immunoradiographic technique of Renart et al. (20),except that 1% gelatin was used in the blocking buffer, 50 p.lof ascites fluid was used as the source of antibody, and thereaction was probed with 50 ml of 1251I-labeled homologousenterotoxin (0.012 ,uCi/ml).

    Determination of cross-reactivity. Each antibody, c