Letters in Applied Microbiology 1991, 13, 175-178

ADONIS 0266825491OO114M

Protein A insensitive ELISA detection of staphylococcal enterotoxin I3 V . R O G E M O N D *J, . M . D A C O S T AC A S T R O *T. , D E L A U N A YC* ., C A Z A U X& * R . M . F . G U I N E T t $ Centre d'lmmunochimie Microbienne, *Nouueaux Deueloppements, Behring Diagnostic., tlnstitirt Pasteur de Lyon, Le Poirier, Lentillp, 69210 L'Arbresle, France

AITl0.5: received and accepted 17 June 1991

R O G E M O N DV , . , D A COSTAC A S T R OJ.M., , DELAUNAY T., , C A Z A U XC. , & G U I N E TR, . M . F 1901 Protein A ~nsensitiveELISA detection of staphylococcal enterotoxin B. Letters in Applied Microbiology 13, 175-178. A sandwich enzyme-linked imrnunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ndml of enterotoxin B was detected and interference by protein A was prevented by the use of rat rnonoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than loo0 ng/rnl.

Staphylococcal enterotoxins are a group of proteins that are responsible for staphylococcal food poisoning. Several immunoassays have been described for the detection of the enterotoxins in food extracts and culture supernatant fluids : the most sensitive methods (sensitivity of 1 to 0.1 ng/ml) are radioimmunoassay (RIA: Johnson et al. 1973; Bergdoll & Reiser 1980), enzyme-linked immunosorbent assay (ELISA: Saunders & Bartlett 1977 ; Stiffler-Rosenberg & Fey 1978; Fey et al. 1984), reverse passive latex agglutination (RPLA,: Igarashi et al. 1985; Fujikawa & Igarashi 1988). These methods are frequently hampered by false positive results due to protein A, produced concurrently with enterotoxins, by most strains of Staphylococcus aureus (Forsgren 1970). Thus, the official method (AOAC 1984) remains the microslide immunodiffusion assay (Cassman et al. 1969) which is less sensitive (100 ng/ml) and less rapid than the other immunoassays. In the present cornmunication we report the development of an ELISA test for the detection of enterotoxin B with no interference due to

1Corresponding author

protein A. This test uses rat monoclonal antibodies of the IgG2a isotype which showed no reactivity with protein A even at concentrations greater than loo0 ng/ml.

Materials and Methods Purified enterotoxin B (SEB) was purchased from Sigma Chimie (L'Isle d'Abeau, France); purified enterotoxin A (SEA), C (SEC), D (SED) and E (SEE), were purchased from Serva Fine Chemicals (St Germain en Laye, France). Monoclonal antibodies were obtained by fusing spleen cells from a female LOU rat, hyperimmunized with purified SEB, with rat myeloma cells (IR 983 F). Hybridoma supernatant fluids were screened by ELISA on 96-well polystyrene plates coated overnight, at 37"C, with 50 pl SEB (2 ,u'g/ml in citrate buffer). Antigen-specific IgG2a antibodies were selectively revealed with mouse anti-rat y2a monoclonal antibodies conjugated to peroxidase. Hybridomas producing suitable antibodies were expanded, subcloned, produced and purified as described by Bazin (1990).



Rogemond et al.

Table 1. Characterization of anti-SEB monoclonal antibodies Monoclonal antibodies


BB4 BE4 BF 1 CBlO CD5 CF12 DD8 DE4 DE8

IgG2a, K IgGZa, K IgGZa, K IgG2a, K IgG2a, I IgG2a, K IgGZa, K IgG2a, K IgGZa, K

Detection limit (ng/mU

80 15 2 2 >20 000 1-1.5 2000 1-13 1

(diluted 1 : 200 in PBS-T) for 30 rnin at 37°C. After three washes, the wells were filled with 100 pl of streptavidin peroxidase (France Biochem, Meudon, France) (diluted 1/10 OOO in PBS-T) for 5 rnin at room temperature. The subsequent steps were as described above. INTERFERENCE BY PROTEIN A

To assess the influence of protein A in the sandwich ELISA, protein A (Sigma Chimie, L'Isle d'Abeau, France) was added to the wells instead of the SEB at concentrations increasing from 0.1 to 2500 ng/ml.



The isotype was determined by ELISA using mouse anti-rat isotype monoclonal antibodies. Detection limits of MAbs were determined by ELISA. Fifty pl of purified MAb at concentrations from 0.5 to 20 OOO ng/ml were incubated for 30 min on polystyrene plates which had been coated overnight with 100 ng SEB. After three washes with 0.1% Tween 20 in phosphatebuffered saline (PBS-T), rabbit anti-rat IgG coupled to peroxidase (Biosys, Compiegne, France) was added for 30 min. The plates were washed with PBS-T and the peroxidase activity was detected by incubation with 100 pl of tetramethylbenzidine (TMB, Behring Diagnostic, Rueil, France) (peroxidase substrate) for 30 rnin in the dark. The reaction was stopped by addition of 100 pl 0.5 mol/l H,SO, and the optical density (O.D.) was determined at 450 nm. Cross reactivities with SEA, SEC, SED and SEE were studied by ELISA as described above. BIOTINYLATION OF


MAbs were biotinylated with biotinaminocaproyl-N-hydroxysuccinimide ester (biotine-X-NHS, France Biochem, Meudon, France) according to the method previously described by Da Costa Castro et al. (1987). DOUBLE ANTIBODY SANDWICH ELISA

ELISA was performed in polystyrene plates by incubating 100 p1 of a solution of a MAb (2.5 pg/ml in carbonate-bicarbonate buffe, at 37°C overnight. After washing with PBS-T, 100 pl of SEB (0.1 to 100 ndml in PBS-T) were added for 30 min at 37°C. The plates were rinsed and then incubated with 100 pl of a biotinylated MAb

Results and Discussion M O N O C L O N A L ANTIBODIES

Nine MAbs were purified and characterized. Eight MAbs were found to be y2a, K isotype and one MAb was 72a, 1 isotype (Table 1). A detection limit was determined for each MAb (Table I). Cross-reactivities with the other staphylococcal enterotoxins SEA, SEC, SEB, SEE were tested in the same manner by ELISA. None of the MAbs recognized enterotoxins other than SEB. INDIRECT S A N D W I C H ELISA

Two MAbs, CF12 and DE4, were selected for use in a sandwich ELISA. These MAbs had detection limits for SEB of about 1 ng/ml. They were coupled to biotin via amino groups and used as conjugates for the detection of SEB. Each MAb was used as coating antibody to coat the plate and, after biotinylation, as probing antibody to detect the captured toxin. The assay did not work in a homologous sandwich with the same antibody for both capture and conjugate. Thus the epitopes recognized by two different MAbs were not the same, i.e. the test acted well in a double site immunosassy using antibodies CF12 and DE4. The best system was CF12 as coating antibody and DE4 as probing antibody, detecting less than 1 ng/ml of SEB (Table 2). The opposite system was a little less sensitive, with 1.2 ng/ml of SEB detected. The amounts of protein A interfering in these sandwich immunoassays (Table 2) showed that these two systems are virtually insensitive to protein A. Indeed, about 2 pg/ml was necessary to give a false positive

Protein A insensitive S E B ELISA Table 2. Detection limits* of SEB and protein A in the SEB ELISA system Detection limit Capture antibody (2.5 ppiml)

Biotinylated conjugate (1 : 2CO)

CF12 DE4 CF12 DE4


SEB (ngiml)

Protein A (ngiml)

0.85 1.20 > 100 > 100

1900 1800-2000 ND ND

* Detection limit: SEB concentration (ngiml) for 0 . D : 0.1 + O.D.,, - S.D.,, I O.D. 5 0 . 1 + O.D.,,

+ S.D.,,

O.D.,,, mean value obtained for assay without SEB (4 wells); S.D.,, , standard deviation. ND, Not determined.

resu!t. By comparison, 3 ng/ml of protein A was suffcient to interfere in a previous ELISA system using polyclonal rabbit IgGs both as capture antibodies and as biotinylated conjugate (unpublished result). This result was not in agreement with the findings of Hahn et al. (1986) who found that the use of a biotin-rabbit IgG conjugate prevented interference due to protein A in an ELISA system for the detection of enterotoxins A and B. The binding to the Fc region of the IgGs of many species by protein A can lead to false positive results in i:mmunodetection (Koper et al. 1980). That is the reason why some authors used sample treatments such as chromatography (Berdal et al. 1981; Lapeyre et al. 1987) or incubation with insoluble porcine IgG (Fey & Buckhard 1981). We chose not to use timeconsuming treatments and therefore developed rat monoclonal antibodies of the IgG2a isotype, described as insensitive to protein A. In a similar way, Notermans et al. (1982) used sheep IgGs in a sandwich ELISA which was not affected by protein A interference, but this result was disputed (Hahn et al. 1986). The ELISA test we have developed is rapid, sensitive and little subject to protein A It could be useful for the detection of SEB in food extracts where protein A interference appears to be less important than in culture supernatant fluids (Freed et al. 1982). We are now trying to obtain rat MAbs against the other staphylococcal enterotoxins to complete our test. References ASSOCIATIONOF

O F F I r I A I . ANALYTICAL CHEMISTS 1984 Oficial Methods of ,4nalysis 14th edn. Arlington, VA: Association of Analytical Chemists.


BAZIN,H. (ed.) 1990 Rat Hybridomas and Rat Monoclonal Antibodies p. 515. Boca Raton, FL: CRC Press. 0.& OMLAND, T. 1981 A sandBERDAL, B.P., OLSVIK, wich ELISA method for the detection of Staphylococcus aureus enterotoxins. Acta Pathologica Microbiologica Scandinavica, Sect. B, 89,411-415. BERGDOLL, M.S. & REISER,R. 1980 Application of radioimmunoassay for detection of Staphylococcus aureus enterotoxins in foods. Journal of Food Protection, 43, 68-72. CASSMAN,E.P., BENNETT.R.W., DORSEY,A.E. & STONE,S.E. 1969 The microslide gel double diffusion test for the detection of staphylococcal enterotoxins. Health Laboratory Science 6, 185-198. DA COSTACASTRO, J.M., DESCHAMPS, F., BENBACHIR, M., HENRICHSEN, J., VOLLE,P.J. & GUINET, R.M.F. 1987 Highly sensitive biotin-avidin sandwich ELISA for the rapid detection of pneumococcal capsular polysaccharide antigens. Journal of Immunological Methods 104, 265-270. G. 1981 Measurement of FEY, H. & BUCKHARD, staphylococcal protein A and detection of protein A carrying Staphylococcus strains by a competitive ELISA method. Journal of Immunological Methods 47,99-102. FEY,H., PF’ISTER, H. & RUEGG,0. 1984 Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C and D. Journal of Clinical Microbiology 19, 34-38. FORSGREN, A.S. 1970 Significance of protein A production by Staphylococci. Infection and Immunity 2, 972-973. FREED,R.C., EVENSON, M.L., REISER,R.F. & BERGDOLL, M.S. 1982 Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in food. Applied and Environmental Microbiology 44, 1349-1355.

FUJIKAWA, H. & IGARASHI, H. 1988 Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high density latex particles. Applied and Environmental Microbiology 54, 23452348. P., LENZ,W. & BRANDIS, H. HAHN,I.F., PICKENHAHN, 1986 An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. Journal oflmmunological Methods 92, 25-29. IGARASHI, H., SHINGAKI, M.. FUJIKAWA, H., USHIODA, T. 1985 Detection of staphylococH. & TERAYAMA, cal enterotoxin in food poisoning outbreaks by reversed passive latex agglutination. Zentralblatt f u r Bakteriologie, Mikrobiologie und Hygiene, Abt. 1, SUPPI,14, 255-257. JOHNSON, H.M., BUKOVIC,S.A. & KAUFFMANN, P.E. 1973 Staphylococcal enterotoxins A and B: solidphase radioimmunoassay in food. Applied Microbiology 26, 309-313. KOPER,S.W., HAGENAARS, A.M. & NOTERMANS, S. 1980 Prevention of cross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus nureus enterotoxin B in culture filtrates and foods. Journal of Food Safety 2, 35-45.


V . Rogemond et al.

LAPEYRE, C., KAVW,S.V. & STRASBERG, A.D. 1987 A novel approach to prevent the interference of protein A in immunoassay of enterotoxins. Letters in Applied Microbiology 5, 55-59. NOTERMANS, S., TIMMERMANS, P. & NAGEL,J. 1982 Interaction of staphylococcal protein A in enzymelinked immunosorbent assay for detecting staphylococcal antigens. Journal of Immunological Methods 55, 35-41.

SAUNDERS, G.C. & BARTLETT, M.L. 1977 Double antibody solid-phase immunoassay for the detection of staphylococcal enterotoxin A. Applied and Environmental Microbiology 34,518-522. STIFFLFX-ROSENBERG, G. & FEY,H. 1978 Simple assay for staphylococcal enterotoxins A, B and C: modification of enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 8,473479.

Protein A insensitive ELISA detection of staphylococcal enterotoxin B.

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodie...
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