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

Monoclonal and Polyclonal Antibody SystemsNANCY E. THOMPSON,t* MEENAKSHI RAZDAN, GERHARDT KUNTSMANN, JEAN M. ASCHENBACH,

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 per

ml. 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 same

system 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 a

microscope 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 various

serological 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.

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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.

MATERIALS AND METHODS

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, con-tained in hybridoma culture supernatant fluid, was tested forreactivity with heterologous toxin types by titrations againstthe 125I-labeled heterologous toxin types, using the reactionwith the homologous toxin type as a control. Antibodies thatshowed a significant reaction with the 125I-labeled heterolo-gous toxin type were then used in a competition RIA (15)with that heterologous toxin type to determine the degree ofcross-reactivity with the antibody (25). Most antibodies werealso tested by the ELISA method described by Meyer et al.(14).

Antibody affinity. An estimation of the affinity constant foreach antibody was calculated by the competitive RIAmethod described by Muller (16).Antibody sandwich ELISA. Purified antibodies were con-

jugated to horseradish peroxidase (Sigma Chemical Co.) bythe method of Nakane and Kawaoi (17), except that theIgG-enzyme conjugate was not reduced with NaBH4, 5 mgof horseradish peroxidase was used per 10 mg of IgG, andthe enzyme-aldehyde was reacted with IgG for 3 h at roomtemperature and then overnight at 4°C.ELISAs were performed in 96-well Linbro plates (Flow

Laboratories, Inc., McLean, Va.) by the method of Freed etal. (10), except that plates were coated with 50 p.1 of a

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5-,ug/ml solution of the coating antibody, the substrate wasreacted for 30 min at room temperature, and the enzyme-substrate reaction was not quenched but read immediatelyon a Dynatech Microplate Reader (model MR 600). Standardenterotoxin solutions were prepared from crude toxin prep-arations.

Competition RIA. Competition RIAs were performed es-sentially as described by Miller et al. (15). Briefly, eachantibody preparation was titrated to determine the concen-tration that would yield 50% of the maximum binding of12,000 cpm of the 125I-labeled homologous enterotoxin. Theamount of antibody was reacted overnight in 1.0 ml of RIAbuffer at 4°C with unlabeled, purified enterotoxins at con-centrations ranging from 0 to 20 ng of enterotoxin per ml.The reaction was then challenged with 12,000 cpm of 1251_labeled enterotoxin and reacted for 1.5 h at room tempera-ture before the protein A-containing cells were added.Food extracts. Ham, cheese, sausage, and egg noodles

were purchased from a local supermarket, and extracts wereprepared by the method of Miller et al. (15). Skim milk wasalso purchased and used without treatment.

RESULTS AND DISCUSSION

Characterization of MAbs. The MAbs used in this studyare described in Table 1. Some of these antibodies have beenused in a previous study (25).The screening procedure for isolating these MAbs used

staphylococcal protein A as the immunosorbent. Antibodiesof the mouse IgGl subclass are reputed not to bind to proteinA (11). However, many investigators have reported a reac-tion between protein A and this IgG subclass (13), andclearly, a large number of IgGl antibodies can be isolated bythis method (Table 1). With the exception of antibody 1C3,these IgGl antibodies show lower binding to the 125I-labeledhomologous toxin than do antibodies of the IgG2a and IgG2bsubclasses when protein A is used as an immunosorbent(Table 1). Generally, the recovery of 1251-labeled toxin by

12 3 4 65 6^8

FIG. 1. Autoradiogram of a nitrocellulose blot containing SEAafter electrophoresis on SDS-PAGE. Lanes 1, 2, and 3 contained 25,10, and 1 ,ug of SEA, respectively; this portion of the blot was

reacted with antibody IA, probed with 1251-labeled SEA, and an

autoradiogram was prepared. Lanes 4, 5, and 6 contained 25, 10 and1 jig of SEA, respectively; this portion of the blot was stained with0.1% amido black.

TABLE 2. Effective combinations of MAbs in assay systems forthe detection of staphylococcal enterotoxins

Toxin - ELISA0 Effective poolstype Coating Probing of MAbsb in

antibody antibody RIAs

SEA 2A 1A or 4A 1A, 2A, 4ASEB 2B (6B) 6B (3B) 2B, 3B, 6BSEC 1C3 4C2 or 2C2 2C2, 4C2, 1C3SED 3D (4D) 1D (3D) 1D, 3D, 4DSEE 2A 5E or4E 2E, 4E, 5Ea Antibodies in parentheses denote the alternative system described in the

text.b Supernatant fluids combined in equal proportions.

IgGl antibodies can be increased by the addition of anti-mouse IgG prepared in rabbits before the addition of theprotein A (25).The estimated affinity constants for this group of antibod-

ies ranged from 2.3 x 108 to 8.3 x 1010 liters/mol. Thus, allantibodies seemed of relatively high affinity, suitable forimmunodiagnostic procedures. The high affinity of theseMAbs was not unexpected, because approximately 0.3 ng of1251I-labeled enterotoxin per ml was used in each assay duringthe screening of the hybridomas. Therefore, the parametersof the screening method were adjusted so that only antibod-ies capable of detecting that level of enterotoxin would bedetected.

Specificity of antibodies. Each antibody was determined tobe specific for its homologous enterotoxin by the Westernblot technique. The autoradiogram in Fig. 1 shows thereaction of antibody 1A with several concentrations ofpurified SEA.

All antibodies were tested for reactivity with heterologoustoxin types by the RIA method described in Materials and

2AI1A PCR

2A/4A

0-.

0

.O

0

ci I 2 3 4 5

SEA CONCENTRATION (ng/mI)FIG. 2. Standard curves generated from absorbance reading of

ELISA systems for the detection of 0 to 5 ng of SEA per ml. Thefirst MAb system (A) used antibody 2A for coating and antibody 1Afor probing. The second MAb system (A) used antibody 2A forcoating and antibody 4A for probing. These systems were run

simultaneously with the SEA-specific polyclonal rabbit IgG (-) forboth coating and probing. The absorbance reading for 5 ng of SEA,using the 2A/1A system, was consistently off scale.

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TABLE 3. Analysis of enterotoxins added to food extracts by ELISA, using polyclonal and monoclonal antibody systemsAbsorbance'

ToxinMenstrum concn SEA SEC2

(ng/ml) Polyclonal Monoclonalb Polyclonal Monoclonal'

Buffer 0 0.127 0.041 0.070 0.0480.6 0.219 0.169 0.114 0.1321.25 0.351 0.314 0.148 0.1402.5 0.466 0.503 0.254 0.2815.0 0.855 0.842 0.401 0.636

Ham extract 0 0.149 0.027 0.093 0.1640.6 0.285 0.189 0.144 0.1641.25 0.378 0.337 0.248 0.2022.5 0.601 0.549 0.490 0.3585.0 0.895 0.934 0.741 0.747

Cheese extract 0 0.123 0.052 0.082 0.0950.6 0.218 0.223 0.140 0.1861.25 0.367 0.166 0.372 0.2492.5 0.548 0.275 0.416 0.4015.0 0.742 0.628 0.662 0.592

Sausage extract 0 0.130 0.045 0.054 0.0790.6 0.085 0.125 0.132 0.1001.25 0.223 0.212 0.195 0.1352.5 0.392 0.389 0.457 0.2825.0 0.567 0.623 0.696 0.554

Egg noodle extract 0 0.245 0.090 0.081 0.0690.6 0.185 0.281 0.143 0.1441.25 0.407 0.399 0.195 0.1562.5 0.686 0.641 0.383 0.2905.0 0.971 1.023 0.644 0.578

Milk 0 0.183 0.062 0.043 0.0330.6 0.263 0.160 0.065 0.0081.25 0.346 0.234 0.130 0.0112.5 0.508 0.347 0.260 0.0835.0 0.743 0.753 0.561 0.306

a Average of duplicate wells for each concentration of enterotoxin.b Antibody 2A was used for coating, and conjugated 1A was used for probing.c Antibody 1C3 was used for coating, and conjugated 4C2 was used for probing.

Methods. A summary of the cross-reactions observed by theRIA method appears in Table 1.

It was originally thought that some lower affinity reactionscould be detected by the use of an ELISA procedure. TheMAbs, with the exception of the SED antibodies, weretherefore tested in the ELISA system described by Meyer etal. (14). No additional cross-reactions could be detected bythis ELISA method. Enterotoxin D could not be testeddirectly by the Meyer ELISA system. No reaction wasdetected with any of the SED-specific MAbs in this assay.Clearly, these antibodies react with SED, as evidenced byreaction in the Western blot and in the antibody sandwichELISA and RIA (described below). It is possible that ourpreparation of SED did not coat the microtiter plate effec-tively under these conditions.Antibody sandwich ELISA. The primary limitation of

adapting a MAb to the antibody sandwich ELISA was thattwo different MAbs had to be used. One antibody (thecoating antibody) was used to coat the plate and removed theenterotoxin from the test solution. The other antibody (theprobing antibody) was conjugated to horseradish peroxidaseand used as a probe to detect the captured toxin. Thus, itwas necessary to identify an effective pair of antibodies for

each toxin type. These effective pairs are listed in Table 2.Our experience with this system has indicated that thereciprocal combination (i.e., reversing the coating and prob-ing antibodies) generally results in an assay system withlowered sensitivity. The reasons for this are not immediatelyclear. However, all of the coating antibodies listed in Table2 will generate usable standard curves when the probe usedis the homologous enzyme-conjugated toxin-specific poly-clonal rabbit IgG; most of the antibodies designated asprobing antibodies (Table 2) do not work well as coatingantibodies in conjunction with the conjugated polyclonal IgG(data not presented). This seems to indicate that someantibodies are more effective than others at coating thepolystyrene plate under the conditions used for this proce-dure. However, various coating conditions were not exam-ined in this study.

Typical standard curves over the range of 0 to 5 ng of SEAper ml are shown in Fig. 2. This assay used the SEA-specificpolyclonal system and the two most effective monoclonalsystems (coating with antibody 2A and probing with conju-gated 1A [designated 2A/1A] and coating with antibody 2Aand probing with conjugated 4A [designated 2A/4A]). Bothof these systems compare favorably with the polyclonal

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system, although the 2A/1A system seems to be somewhatmore sensitive.

Similar curves were generated by the other pairs listed inTable 2. In all cases, the ELISA generated by the MAb pairsin Table 2 were comparable to those generated by theenterotoxin-specific polyclonal IgG.The most effective MAb pair of the detection of SEB was

coating with 2B and probing with conjugated 6B. However,an alternative system that seemed to be only slightly lesssensitive was coating with 6B and probing with conjugated3B.Although we isolated many MAbs reactive with at least

one of the subtypes of SEC, for the purposes of devising anassay system for SEC, we selected antibodies that reactedwell with all of the subtypes of SEC. All subtypes of SECcould be detected by coating with antibody 1C3 and probingwith either conjugated 2C2 or conjugated 4C2. However,probing with conjugated 4C2 seemed to be somewhat moresensitive.

Enterotoxin D could be detected by coating with antibody3D and probing with conjugated 1D or by coating withantibody 4D and probing with conjugated 3D (Table 2).

Enterotoxin E could be detected by coating with antibody2A and probing with either antibody SE or antibody 4E(Table 2). The 2A/4E system could also be used to detectSEA.Although each system has yet to be tested in a large

number of practical assay situations, the ELISA systems forenterotoxins A and C were tested in food extracts preparedfrom ham, cheese, sausage, and egg noodles; skim milk wasalso tested. The data in Table 3 compared the results of the2A/1A and the 1C3/4C2 systems with results obtained fromtheir respective polyclonal antibody systems for the detec-tion of SEA and SEC2 in these food extracts. The anomalousstandard curve generated by the SEC-specific MAb systemin milk is probably a result of the fact that some milk samplescontain low antibody titers toward SEC (24).Thus, we have described effective MAb pairs that can be

used in the antibody sandwich ELISA for the detection ofstaphylococcal enterotoxins. To date we have not detectedany reactivity of these antibodies with other macromoleculesthat would preclude their use in this assay system. In fact,the use of two antibodies that must react with the antibody atdifferent steps in the assay procedure should increase thespecificity of the assay system.We have also shown that it is possible to assay for both

SEA and SEE in the same ELISA system by the use ofcross-reacting MAbs. Realistically, it should be possible togenerate two ELISA systems (an SEA/SED/SEE systemand an SEB/SEC system) capable of detecting all of theenterotoxins. Antibody 1E reacted well with SEE and SEAbut only weakly with SED (Table 1). Likewise, antibody 6Breacted with all three subtypes of SEC, but these reactionswere approximately threefold lower than its reaction withSEB (Table 1). Thus, neither antibody 1E nor antibody 6Bseemed a good candidate for the development of a cross-reacting system. Another approach would be to coat with apool of the coating antibodies (Table 2) and probe with a poolof the probing antibodies (Table 2). This approach has notbeen tried.

Competitive RIA. The RIA method of Miller et al. (15) waschosen to investigate the behavior of these MAbs in the RIA.This method seems to be the most sensitive, most rapid,least expensive, and easiest to perform of the many RIAmodifications available.The low binding of the homologous 125I-labeled toxin by

0

m 50t30

10 _

l I 1111111 1 1 1 111110O1 0.5 10 50 10

SEA CONCENTRATION (ng/mI)

FIG. 3. Logit-log plot of a competitive RIA for the detection ofSEA by a pool of monoclonal antibodies 1A, 2A, and 4A and bySEA-specific polyclonal rabbit serum. Percent B/Bo is the "25I-labeled SEA bound when in competition with the specific amount ofunlabeled SEA/'251-labeled SEA bound when no unlabeled SEA waspresent (x 100).

most of the antibodies listed in Table 1 limits their usefulnessin this RIA. In this assay it was necessary to dilute theantibody to 50% of the maximum binding. For low-bindingantibodies, this decreases the effective range of the standardcurve and also decreases the slope of the standard curve,resulting in a loss of precision of the assay. While it ispossible to increase the recovery of 125I-labeled toxin byadding a secondary antibody to the system after the initialMAb toxin reaction has been completed (25), experiencewith this approach in our laboratory has indicated that thisdoes not improve the slope of the standard curve. Thus, themajority of the MAbs listed in Table 1 do not comparefavorably with the homologous toxin-specific polyclonalrabbit serum in the RIA.However, as Erhlich et al. have noted (8), the affinity of

MAbs can be enhanced by creating pools of the antigen-specific MAb. We examined the MAbs listed in Table 1 andgrouped them into pools that generated standard curvescomparable to the homologous toxin-specific polyclonalrabbit serum. These effective pools are listed in Table 2, anda representative standard curve, using the SEA MAb pool, isshown in Fig. 3.As in the ELISA system, the use of pools would be

expected to increase the specificity of the assay. However, apool must consist of three or more MAbs; a pool of only twoMAbs can yield a bimodal standard curve (data not pre-sented).

Finally, we also examined some of the cross-reactingMAbs in the RIA. A pool of antibodies 2A, 1E, and 4E canbe used to detect either SEA or SEE. However, the antibodyconcentration must be adjusted for each toxin type toachieve the 50% binding value. Therefore, the practicality ofthis system is somewhat questionable.

In conclusion, we have described both ELISA and RIAsystems, using MAbs that are capable of detecting 1 ng ofenterotoxin per ml of food sample. Both of these systemscompare favorably with the polyclonal rabbit antibody sys-tems currently in use.There are two advantages of using the MAb systems over

the polyclonal systems: (i) the MAb system provides an

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unlimited supply of highly uniform reagents; and (ii) cross-reacting MAbs can be adopted to these assay systems. Theuse of MAbs reduces the number of assays needed to detectall of the serological types of enterotoxins.

ACKNOWLEDGMENTSWe thank Raoul Reiser and. Kelli Stahlnecker for purifying the

enterotoxin and Ruth Robbins for preparing the enterotoxin-specificpolyclonal rabbit sera.

This research was supported by the College of Agriculture andLife Sciences, University of Wisconsin, and by contributions fromvarious companies and associates of the food industries.

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3. Borja, C. R., and M. S. Bergdoll. 1967. Purification and partialcharacterization of enterotoxin C produced by Staphylococcusaureus strain 137. Biochemistry 6:1467-1473.

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5. Casman, E. P., and R. W. Bennett. 1965. Detection of staphy-lococcal enterotoxin jn food. Appl. Microbiol. 13:181-189.

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15. Miller, B. A., R. F. Reiser, and M. S. Bergdoll. 1978. Detectionof staphylococcal enterotoxins A, B, C, D, and E in foods byradioimmunoassay, using staphylococcal cells containing pro-tein A as immunoadsorbent. Appl. Environ. Microbiol.36:421-426.

16. Muller, R. 1983. Determination of affinity and specificity ofanti-hapten antibodies by competitive radioimmunoassay.Methods Enzymol. 92:589-601.

17. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labeled anti-body. A new method of conjugation. J. Histochem. Cytochem.22:1084-1091.

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20. Renart, J., J. Reiser, and G. R. Stark. 1979. Transfer of proteinsfrom gels to diazabenzyloxymethyl-paper and detection withantisera: a method for studying antibody specificity and antigenstructure. Proc. Natl. Acad. Sci. USA 76:3116-3120.

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