Journal o f Immunological Methods, 24 (1978) 127--134 © Elsevier/North-Holland Biomedical Press



PETER H. SCHEUBER, MARIA MULLER, ELISABETH SIMON, HORST MOSSMANN and DIETRICH K. HAMMER Max-Planck-Institut fiir Immunobiologie, Freiburg i.Br. and Lehrstuhl fiir Hygiene und Technologie der ~Iilch, Universit~'t Miinchen, Munich, G.F.R. (Received 8 February 1978, accepted 18 May 1978)

A highly sensitive assay for determination of staphylococcal enterotoxin B (SEB) is based on inhibition of the immunospecific inactivation of SEB-bacteriophage conjugates by the free toxin. Threshold concentrations detectable ranged from 1 to 5 ng SEB/ml. The procedure is less time consuming and more than 100-fold more sensitive than immunodiffusion methods and has several advantages over the radioimmunoassay discussed. It has also been shown that treatment of SEB with a cross-linking reagent, glutaraldehyde, in the presence of rabbit serum albumin (RSA) results in loss of toxicity without appreciable diminution in immunogenicity. Cattle immunized with insoluble SEB derivatives produced considerable amounts of specific antibodies. SEB preferentially evokes both modified phage-inactivating and binding antibodies of the more negatively charged IgGs subclass, which are selectively concentrated within the dry mammary gland. Thus colostrum may provide a source for preparing anti-SEB antibodies on a large scale. INTRODUCTION T h e r e a c t i o n o f s t a p h y l o c o c c a l e n t e r o t o x i n B (SEB) with specific antib o d y serves as a sensitive m e t h o d f o r the d e t e c t i o n and q u a n t i t a t i o n o f the t o x i n . So far, specific a n t i b o d i e s have been p r e p a r e d primarily in the r a b b i t (Silverman, 1 9 6 3 ; Casman and B e n n e t t , 1964} a l t h o u g h o t h e r animals such as goats (Collins et al., 1 9 7 2 ) have also been used. One o f the m a j o r obstacles t o using native t o x i n s for i m m u n i z a t i o n is the need t o begin with very small doses in o r d e r t o avoid killing the animals. The length o f time r e q u i r e d t o p r e p a r e high titered antisera and the c o n s e q u e n t high costs i m p o s e great limitations o n the use o f these antisera for r o u t i n e diagnosis. A n u m b e r o f m e t h o d s f o r d e t e c t i o n and q u a n t i t a t i v e analysis o f SEB have been used w h i c h are m a i n l y based o n the r e a c t i o n o f the t o x i n with p r e c i p i t a t i n g antib o d y (Laurell, 1 9 6 6 ; Bergdoll, 1972). A l t h o u g h suitable f o r serial analysis, these have limitations b o t h in sensitivity at low SEB c o n c e n t r a t i o n s and in time c o n s u m e d in c a r r y i n g o u t the assay. O t h e r m e t h o d s such as r a d i o i m m u n o a s s a y (Collins et al., 1 9 7 2 ) and

128 hemagglutination (Silverman et al., 1968) have the advantage of greater sensitivity. Alternatively, reversed passive hemagglutination provides an apparently highly sensitive assay for detection of SEB (Silverman et al., 1968) but the reliability of the procedure is questionable (Sommerfeld and Terplan, 1975). Because of the high cost of commercial rabbit anti-SEB antisera it was the aim of the present study to prepare specific antibodies on a large scale and inexpensively by immunization of cattle. Cross-linking of SEB either in the absence or in the presence of a carrier protein was used to reduce the toxic activity of the immunogen without essentially altering its immunologic specificity. It is the main purpose of the present report to describe a highly sensitive assay for detection and quantitation of SEB based on inhibition of inactivation of SEB-bacteriophage conjugates by the bovine antiserum. MATERIALS AND METHODS

Antigens Staphylococcal enterotoxin B (SEB) was purified with minor modifications according to the procedure of Schantz et al. (1965) using chromatography on Bio-Gel P-60 (BioRad Laboratories, Richmond, CA) as the final purification step. This procedure permitted purification of SEB to homogeneity as assessed by both electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and ultracentrifugal analysis. Rabbit serum albumin (RSA) was prepared as described by Michael (1962).

Polymerization of SEB SEB was cross-linked with glutaraldehyde either in the absence or the presence of RSA following the procedure of Avrameas and Ternynck (1969) using a molar ratio of RSA to SEB of 3 : 1.

Antisera Pregnant Simmentaler cattle 2--3 years of age were primed with either 20 mg SEB or 160 mg SEB-RSA polymer adsorbed on aluminium hydroxide gel (Fran~k and Simek, 1971) (AluGel S, Serva Heidelberg, G.F.R.) and emulsified in complete Freund's adjuvant (Difco, Detroit, MI, U.S.A.). A total of 10.0 ml emulsion was administered into multiple sites both intradermally and intramuscularly in 0.1 ml and 2.0 ml doses, respectively. Subsequent injections were given at 3--5-month intervals for 2 years following the immunization schedule outlined previously (Hammer et al., 1971). Antisera were obtained 2--3 weeks after each immunization step. The collection of the colostrum and the preparation of the colostral whey has been described previously (Hammer et al., 1968). A commercial rabbit anti-SEB antiserum was purchased from Makor, Ltd., Jerusalem, Israel.

Separation of IgG subclasses Bovine IgG and its subclasses were prepared from serum and colostral

129 whey by precipitation at 2 M (NH4)2SO4 followed by gel filtration (Sephadex G-200) and finally by chromatography on DEAE-Sephadex (Pharmacia AB, Uppsala, Sweden) as described in detail previously (KickhSfen et al., 1968).

Antibody determination Enzyme-linked irnrnunoassay (ELISA). SEB-enzyme conjugate was prepared by a two-step procedure according to Avrameas and Ternynck (1971). Horseradish peroxidase (HRP) grade, R z = 3.0 (Worthington, Freehold, NJ, U.S.A.) was first reacted with glutaraldehyde (2.5% in 0.1 M phosphate buffer pH 6.8) and purified SEB was then coupled at equimolar ratio in a second step. The solution was subjected to gel filtration on Sephadex G-75 superfine. Fractions were collected and the tubes containing the conjugate w e r e p o o l e d and stored at 4°C. Polystyrene tubes, 11 mm × 7 0 m m (Nunc, Roskilde, Denmark) were coated with either anti-SEB IgG or the IgG subclasses (10 gg protein/ml) thereof following the procedure of Engvall et al. (1971). A dilution of enzyme-conjugated SEB containing 10 t~g SEB and 14.3 pg HRP respectively in 1.0 ml was added and tubes were capped and rotated for 30 min at room temperature. Finally peroxidase activity was measured by adding the reagents in the following order: 0.6 ml of a 0.3% aqueous solution of H202, 58.8 ml of distilled water, 0.5 ml of o-dianisidine 1% in methanol with continuous stirring. The absorbance of each solution was then determined at 400 nm.

Inactivation of SEB-coated T4 bacteriophage Coupling of SEB to T4 bacteriophage (SEB-T4) was performed by reacting SEB with active phage (1 × 1013 PFU/ml) according to the procedure described by Haimovich et al. (1970) using glutaraldehyde as a cross-linking agent. The resulting modified bacteriophage preparation was tested for inactivation by either anti-SEB antiserum or the IgG fractions thereof following the agar double layer technique of Adams (1959).

Inhibition of inactivation of SEB-coated T4 bacteriophage In inhibition experiments different concentrations of soluble SEB in 0.6 ml phosphate buffer (0.05 M, pH 6.8) were allowed to react at 37°C for 1 h, with the amount of anti-SEB antiserum (0.2 ml) or the IgG fractions thereof that would have caused approximately 60% inactivation of the bacteriophage conjugate. SEB-T4 (0.2 ml containing 8.9 × 1012 PFU) was then added, and incubation continued for an additional 6 h at 37°C followed by dilution and determination of the surviving phage as described above. The percent inhibition was calculated from the extent of inactivation in the presence and in the absence of the inhibitor.


Protein d e t e r m i n a t i o n F o r d e t e r m i n a t i o n of the protein c o n t e n t of chromatographic fractions the following ex t i nc t i on coefficients ~ t~'~% c m , 2S0nm) were used: SEB = 14.4 (Bergdoll, 1972); IgG2 = 13.5; IgG1 = 13.6; IgG s = 13.7 ( K i c k h f f e n et al., 1968). As an additional parameter a modification of the biuret m e t h o d was e m p l o y e d as described by K i c k h f f e n et al. (1968). RESULTS

Immunization Antisera were obtained from pregnant cows receiving a total o f 5 injections (160 mg cross-linked SEB-RSA). Two to 6 h after the first injection all animals showed clinical s y m p t o m s characterized by a rise in t e m p e r a t u r e of 1--2°C and diarrhea. T he illness was mild and lasted only 12 h. With subseq u e n t injections of SEB-RSA there were no clinical s y m p t o m s indicating t h at the cattle became i m m une to SEB. Antisera from 4 cattle collected after each injection were pooled and inactivation o f phage conjugates was determined as a function of serum dilution. As shown in Fig. 1, the antiserum pool obtained 2--3 weeks after priming caused 50% inactivation of SEB-T4 at a dilution of 1.7 × 10 -s. There was a marked increase in ant i body activity after the fifth injection, the antisera being 6--7 times m or e effective than after the primary dose. In contrast, the inactivation titer of a commercial rabbit anti-SEB antiserum was only 4 × 10-4 and thus appr oxi m at el y 25 times less active than antisera p r o d u c e d in cattle. In addition the bovine antisera were f ou nd to precipitate with SEB over a broad range o f antigen concentrations.

Injection r



second third



forth fifth



2 4 6 8 10 (X" 10s ) Reciprocol serum di{ution causing 50% inactivation o1 SEB-T 4

Fig. 1. I n a c t i v a t i o n o f S E B - c o n j u g a t e d T4 phage b y a n t i s e r a (pools) collected f r o m 4 c a t t l e after each o f 5 i n j e c t i o n s w i t h c o p o l y m e r i z e d SEB-RSA. A n t i s e r a were o b t a i n e d 2 w e e k s a f t e r each i m m u n i z a t i o n . R e c i p r o c a l s e r u m d i l u t i o n s causing 50% i n a c t i v a t i o n are given ( o r d i n a t e ) .


Distribution of anti-SEB antibody among IgG subclasses Modified phage technique. Previous experiments had indicated that bovine antibodies to both basic and negatively charged antigens are associated predominantly with the more negatively charged IgG s subclass (Mossmann et al., 1973). Based on this observation the following experiment (summarized in Fig. 2) was performed, to determine whether anti-SEB antibodies have a similar distribution among the IgG subclasses. Antisera were obtained from 2 cows injected 4 times with SEB-RSA and bled 14 days after the last injection. In addition, colostrum was collected immediately after delivery. The 7S IgG fraction from antiserum and colostral whey was separated on DEAESephadex into the IgG2, IgG1 and IgG s subclass (Kickh6fen et al., 1968), and the a m o u n t of anti-SEB antibody was determined by inactivation of SEB-T4 phage conjugates. It is apparent from Fig. 2 that more than 70% of SEB-T4 inactivating antibodies were present in the IgGs subclass. On the other hand, only 11--14% could be identified as IgG2 and IgG1 class antibodies, respectively. In addition, the data illustrated in Fig. 2 indicate that the magnitude of the neutralizing activity of the IgGs fraction from colostrum was comparable with that of the corresponding fraction of the dam's serum.

Enzyme-linked immunoassay In antisera collected after 5 injections the binding capacity of the unfractionated 7S IgG was found to range from 168 txg to 360 pg/20 mg IgG. Coincident with the results obtained by the modified phage inactivation

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1 10-I

i 10-I

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Fig. 2. D i s t r i b u t i o n o f a n t i - S E B a n t i b o d i e s a m o n g IgG subclasses o f 2 cows i n j e c t e d 4 t i m e s w i t h S E B - R S A . R e c i p r o c a l d i l u t i o n s o f IgG f r a c t i o n s (20 m g / m l ) i n a c t i v a t i n g 50% SEB-T4 are s h o w n . Fig. 3. I n h i b i t i o n o f i n a c t i v a t i o n o f SEB-T4 phage c o n j u g a t e b y a n t i - S E B a n t i s e r u m w i t h various c o n c e n t r a t i o n s o f soluble SEB. I n h i b i t o r was r e a c t e d w i t h a n t i s e r u m (1.3 × 10 -s d i l u t i o n ) for 1 h at 37°C, f o l l o w e d b y t h e a d d i t i o n o f SEB-T4, i n c u b a t i o n for 6 h at 37°C a n d s u b s e q u e n t plating.

132 technique, anti-SEB antibodies were associated predominantly with the more negatively charged IgGs and the ratio of binding capacity in the IgGs and IgG2 subclass was approximately 2 : 1 . Comparisons of binding activity of the IgG~ fractions from the dam's serum and colostrum showed only minor differences.

Inhibition of SEB-T4 phage inactivation for quantitation of SEB The specificity of the anti-SEB antibody/SEB phage assay was demonstrated by inhibition of anti-SEB antiserum by soluble SEB (Fig. 3). In addition, inhibition of phage inactivation served as a test system for the sensitive detection and quantitation of SEB. The inhibitory capacity of free SEB was evaluated with a concentration of anti-SEB antisera which alone would have caused 60% inactivation of the bacteriophage conjugate. For quantitation of SEB its inhibitory efficiency was determined at the level of 25% inhibition of phage inactivation. A threshold concentration of as little as 3 ng SEB/ml was capable of inhibiting the inactivation of phage conjugates by the corresponding antibody. These findings show that inhibition of inactivation of modified phage by bovine antisera may serve as a highly sensitive assay for detection and quantitation of SEB in food extracts. DISCUSSION Previous reports have shown that the use of the toxin rather than the toxoid resulted in better antibody formation (Bergdoll, 1972). The present results show that considerable antibody responses may be induced in cattle with 20 mg doses of SEB polymer. Since in pilot experiments SEB in the a m o u n t given behaved as a toxic agent when administered by parenteral routes, reduction of the toxic activity was necessary. This was accomplished by cross-linking SEB with glutaraldehyde either in the presence or the absence of the carrier protein RSA. Glutaraldehyde reacts with free amino groups to give insoluble derivatives of cross-linked molecules (Avrameas and Ternynck, 1969). On the other hand, there is good evidence that chemical modification of SEB with either acetic anhydride or succinic anhydride has profound effects on the immunological activity of the molecule which are closely related to loss of toxicity and directly proportional to the amino groups modified (Chu et al., 1969). Insolubilization of SEB with glutaraldehyde resulted in loss of toxicity without appreciably affecting the immunological activity suggesting that only a small number of free amino groups on the molecule are reactive with glutaraldehyde. Following the first injection of SEB-RSA a considerable antibody response was achieved, SEB-phage inactivation titers ranging from 1 × 10 -s to 2 × 10 -5. On further injection the antibody response increased by a factor of 6--7 and the antisera obtained were approximately 25 times more active


than rabbit anti-SEB antisera commercially available. Analysis of the distribution of anti-SEB antibody among the IgG subclasses showed that the bulk of binding antibody (70%) was associated with the IgG~ subclass whereas 11--14% were identified as IgG2 and IgG1 respectively. A similar approach using inactivation of SEB-phage conjugates for detection of antibody activity provided further evidence that a basic antigen like SEB (pI 8.6) preferentially stimulates negatively charged IgG, antibodies. These results extend and confirm previous findings (Sela and Mozes, 1966; Maurer et al., 1970; Mossmann et al., 1973). In addition, our findings together with those reported previously (Lascelles and McDowell, 1974; Kemmler et al., 1975) indicate a marked selective concentration of IgG~ antibodies within the m a m m a r y gland. Thus colostrum might be a convenient large scale source of anti-SEB antibodies providing relative inexpensive reagents for routine detection and quantitation of enterotoxins in foods. Assuming 700--800 g IgG present in the m a m m a r y gland at parturition (Dixon et al., 1961), the a m o u n t of antibody therein would be sufficient to bind between 10 and 15 g SEB. The present studies were also designed to determine whether inhibition of inactivation of SEB-bacteriophage conjugates by antibody provides an alternative sensitive m e t h o d for detection and quantitation of SEB. Our results show that the threshold concentration of SEB detectable by such inhibition assay is in the range 1--5 ng. This m e t h o d of determining SEB appears to have the advantage of being less time consuming and more sensitive than precipitin or immunodiffusion tests. Separation of free and bound SEB is not necessary and iodination of the toxin, which may have some effect on immunological specificity, is avoided. The major drawback of radioimmunoassay methods is that a highly purified preparation of SEB is required as test antigen (Bergdoll and Bennett, 1975). In the modified phage assay the same conjugate may be used over prolonged periods and this obviates the need for frequent calibration. Inhibition of modified phage inactivation thus provides a highly sensitive assay suitable for direct application to detection of SEB in food extracts, requiring little if any concentration of the latter. ACKNOWLEDGEMENTS This investigation was supported by a Grant InSan 0875-V-043 of the Frauenhofer Gesellschaft, Mfinchen. REFERENCES Adams, M.H., 1959, in: Bacteriophages, ed. M.H. Adams (Interscience, New York) p. 27. Avrameas, S. and T. Ternynck, 1969, Immunochemistry 6, 53. Avrameas, S. and T. Ternynck, 1971, Immunochemistry 8, 1175. Bergdoll, M.S., 1972, in: The Staphylococci, ed. J.O. Cohen (John Wiley, New York) p. 301.

134 Bergdoll, M.S. and R.W. Bennett, 1975, Appl. Microbiol. 2 3 , 4 4 1 . Casman, E.P. and R.W. Bennett, 1964, Appl. Microbiol. 12,363. Chu, F.S., E. Crary and M.S. Bergdoll, 1969, Biochemistry 8, 2890. Collins, W.S., J.F. Metzger and A.D. Johnson, 1972, J. Immunol. 108,852. Dixon, F.J., W.O. Weigle and J.J. Vazquez, 1961, Lab. Invest. 10,216. Engvall, E., K. Jonsson and P. Perlmann, 1971, Biochim. Biophys. Acta 251,427. Fran~k, F. and L. Simek, 1971, Eur. J. Immunol. 1 , 3 0 0 . Haimovich, J., E. Hurwitz, N. Novik and M. Sela, 1970, Biochim. Biophys. Acta 207, 115. Hammer, D.K., B. Kickh6fen and G. Henning, 1968, Eur. J. Biochem. 6 , 4 4 3 . Hammer, D.K., B. KickhSfen and T. Schmid, 1971, Eur. J. Immunol. 1,249. Kemmler, R., H. Mossmann, U. Strohmaier, B. KickhSfen and D.K. Hammer, 1975, Eur. J. Immunol. 5 , 6 0 3 . KickhSfen, B., D.K. Hammer and D. Scheel, 1968, Hoppe-Seyler Z. Physiol. Chem. 349, 1755. Lascelles, A.K. and G.H. McDowell, 1974, Transplant. Rev. 19,170. Laurell, C.-B., 1966, Anal. Biochem. 15, 45. Maurer, P.H., C.F. Merryman and W.A. Stylos, 1970, Int. Arch. Allergy Appl. Immunol. 139,435. Michael, S.E., 1962, J. Biochem. 8 2 , 2 1 2 . Mossmann, H., K. Bartsch, E. Riide, B. KickhSfen and D.K. Hammer, 1973, Eur. J. Immunol. 3 , 2 9 3 . Schantz, E.J., W.G. Roessler, J. Wagman, L. Spero, D.A. Dunnery and M.S. Bergdoll, 1965, Biochemistry 4 , 1 0 1 1 . Sela, M. and E. Mozes, 1966, Proc. Natl. Acad. Sci. U.S.A. 55,445. Silverman, S.J., 1963, J. Bacteriol. 8 5 , 9 5 5 . Silverman, S.J., A.R. Knott and M. Howard, 1968, Appl. Microbiol. 16, 1019. Sommerfeld, P. and G. Terplan, 1975, Arch. Lebensmittelhyg. 26, 121.

Inhibition of immunospecific inactivation of staphylococcal enterotoxin B-bacteriophage conjugate by bovine antibodies: a sensitive assay for staphylococcal enterotoxin B.

Journal o f Immunological Methods, 24 (1978) 127--134 © Elsevier/North-Holland Biomedical Press 127 INHIBITION OF IMMUNOSPECIFIC INACTIVATION OF STA...
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