FEMS MicrobiologyImmunology 89 (1992)247-254 © 1992 Federation of European MicrobiologicalSocieties 0920-8534/92/$05.00 Published by Elsevier


FEMSIM 00212

Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization J o a q u l n G o y a c h e , Jos6 A. O r d e n , J o s e L. Blanco, Javier H e r n f i n d e z , A n a D o m 6 n e c h , G u i l l e r m o Su~rez and E s p e r a n z a G 6 m e z - L u c l a Departamento de Patolog{a Animal L Facultad de Veterinaria, Unit:ersidad Complutense, Madrid, Spain

Received 3 November 1991 Revision received6 January 1992 Accepted 27 January 1992 Key words: Staphylococcal enterotoxin B; Monoclonal antibody

1. S U M M A R Y

2. I N T R O D U C T I O N

A group of 14 monoclonal antibodies (mAbs) to staphylococcal enterotoxin B (SEB) were obtained by fusion of S p 2 / O myeloma cells with spleen ceils from female B A L B / c mice immunized with commercial SEB. The antibodies belonged to IgG~ and IgGzb subclasses. We evaluated the anti-SEB titres, competition assays and sensitivity of detection by indirect ELISA. Reactivity and cross-reactivity were also studied by indirect ELISA and confirmed by immunoblotting. All the mAbs reacted with SEB and with a second band which had a different electrophoretic mobility and probably represents an aggregate of SEB or SEB bound to membranes. Three mAbs reacted only with SEB and the rest showed cross-reactions with SEC~. No reactions were observed against any other serovar (SEA, SED and SEE) or other proteins.

Staphylococcal food poisoning is one of the main food-borne intoxications. This illness results from the ingestion of toxins pre-formed in food by toxicogenic strains of Staphylococcus aureus and is characterized by vomiting within 2 - 6 h after consuming contaminated foods. The staphylococcal enterotoxins (SEs) are a group of relatively small proteins (27-30 kDa) named A to E (SEA, SEB, SECI, SEC2, SEC3, SED and SEE). They are similar in biological activity and many physicochemical properties, but they differ serologically. The identified enterotoxins can be divided into two groups because of their amino acid composition: SEA, SED and SEE group and SEB-SECs group [1], so it is not surprising that cross-reacting antibodies have been detected in enterotoxin-specific polyclonal rabbit sera [2]. There are many reports on the development and use of monoclonal antibodies (mAbs) produced against SEs [2-18]. mAbs to all of the identified enterotoxins have been prepared [2], either specific or cross-reactive. Meyer et al. [14]

Correspondence to: G. Suarez, Departamento de Patologia

Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.

248 prepared a monoclonal antibody which reacts with all of the identified enterotoxins, but it also reacts non-specifically with other proteins [1,19]. Edwin et al. [7] produced mAbs which reacted specifically with SEA, SEB, SECt, SED and SEE. The present work describes an easy and realistic method for the production and characterization of mAbs against SEB and the desirable characteristics for their further use in detection systems.

3. M A T E R I A L S AND M E T H O D S 3.1. Staphylococcal enterotoxin B Lyophilized SEE (1.0 mg; Sigma) was rehydrated in 1 ml of 0.01 M phosphate buffer (pH 7.2) (PB). 3.2. Immunization Female B A L B / c mice (8- to 12-weeks-old) were injected intraperitoneally (0.5 ml) with 5, 10, 20 and 100 # g of purified SEB on days 0, 7, 14, 29 respectively. Freund's incomplete adjuvant was always used (1:1) except for the first time when SEB was emulsified in Freund's complete adjuvant. Finally, on day 42 (3 days before fusion), 100 # g of SEB in PS was injected intravenously. 3.3. Myeloma S p 2 / O - A g 14 ( S p 2 / O ) myeloma cells [20] were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% (v/v) of heatinactivated (56°C for 30 min) foetal calf serum (FCS), 4.5 g of glucose, 0.11 g of pyruvic acid sodium salt, 0.22 g of L-glutamine, 3.7 g of sodium bicarbonate, 200 mg of gentamicin (Gibco) and 60 mg of Tylocine "~u~(Gibco)/litre of medium, at 37°C in a 6% CO: water saturated atmosphere incubator. 3.4. Fusion S p 2 / O cells in the logarithmic phase of growth were pelleted by centrifugation at 1000 rpm for 10 min at room temperature, and washed three times with DMEM. The spleen was removed aseptically with sterile instruments and cells were released from the stroma by pressing gently with

the blunt end of a syringe plunger against a metallic screen. Debris and stroma connective tissues were removed by filtering the cell suspension through a screen. Red cells were lysed with buffer composed by 0.155 M, NH4CI, 0.1 mM N A 2 E D T A , 0.01 M KHCO~; 1.0 ml of the buffer to 40 ml of the cell suspension in DMEM. Splenocytes were pelleted by centrifugation and washed three times with DMEM. Spleen and S p 2 / O cells were counted and mixed at a ratio of 5:1 and pelleted by centrifugation. Polyethylene glycol 1450 (PEG) (Sigma) was melted and diluted 50% (w/v) in DMEM and filtered aseptically. The pellet was exposed to 1.0 ml of pre-warmed (37°C) PEG solution, which was added to the cells dropwise over a period of I rain with gentle stirring. The cell suspension was diluted by the slow addition of 50 ml of DMEM over a period of about 8 rain with gentle stirring. The preparation was kept at room temperature for 10 rain, then the cells were pelleted by centrifugation. The cells were distributed in 96-well tissue culture plates at 200 000 cells per well in DMEM containing hypoxanthine (1 × 10 4M), aminopterin (4 x I0 7M), thymidine (1.6 × 10 5) ( D M E M - H A T ) plus glycine (3 × 10 ~, M) [2122] and 20% of FCS in the presence of feeder layer cells (1000 mouse peritoneal macrophagcs per well) and kept at 37°C in a 6% CO~ watersaturated atmosphere incubator. 3.5. Screening The production of specific antibody-producing cells was assayed by EL1SA 10-15 days after fusion. Polystyrene 96-well microtitre ELISA plates were coated with 100/xl of 0.01 M PB (pH 7.2) containing 1 p.g of SEB ml-~ as the coating antigen for 18 h at 37°C. The wells were blocked during 1 h at 37°C with 3% bovine serum albumin (BSA) dissolved in PB. After three washes with PB-0.01% Tween 20 (PB-T), the plates were incubated with 100 p.1 of hybrid supernates for 2.5 h at 37°C. After three washes with PB-T, 50 /xl of rabbit anti-mouse IgG (H + L) antibodies conjugated with horseradish peroxidase (RAM-HRP) (Nordic) (1:1000 dilution in PB-T) were added and incubated at 37°C for 30 min. After eight

249 washes with PB-T, 100 p~l of substrate solution (40 mg of o-phenylenediamine dihydrochloride, 80 ml of citric acid buffer pH 4.5 and 40 #1 of hydrogen peroxide) were added. The A45onm was determined with a Micro-ELISA Autoreader MR-580 (Dynatech).

3.6. Cloning The positive hybrids were subcloned twice by the limiting dilution procedure in D M E M - H A T to ensure monoclonality and stability of the cells. Clones were expanded by sequential transfer into 24-well tissue culture, 25- and 75-ml cell culture flasks. In this procedure aminopterin was omitted from the medium (DMEM-HT). Clones were frozen and stored at -80°C. 3. 7. Ascites production Male a n d / o r female B A L B / c mice were primed by i.p. injection of Freund's incomplete adjuvant. After 4 - 7 days, 2-2.5 × 106 hybrid cells in 0.5 ml of D M E M - H T were inoculated into each mouse. Ascites was collected when mouse abdomens became distended. 3.8. Purification of m.Abs mAbs were partially purified from centrifuged ascites with 50% saturated ammonium sulphate. The precipitate was collected by centrifugation (5856 × g at 15°C for 20 rain) to remove denatured protein, and stored, frozen at - 8 0 ° C in 1-ml volumes. 3.9. Determination of mAbs titres Titres were determined by the indirect ELISA described above but with doubling dilutions of mAbs (from a previous decimal dilution) in PB. 3.10. Determination of antibody subclass The subclass of each antibody was deterrrrined by reaction in ELISA, as described above with R A M - H R P specific for each subclass: IgG~, IgG2~~, IgG2b, IgG 3 (1 : 3200, 1 : 800, 1 : 400 and 1 : 800 dilution in PB-T, respectively) (Serotec). 3.11. Cross-reactiHty of rnAbs by ELISA The indirect ELISA system was prepared as described above but with 1 p~g ml-z of crude SE

of each serological type (SEA, SEB, SECt, SED, SEE) kindly provided by M.S. Bergdoll (Food Research Institute, Madison, WI).

3.12. Cross-reactiL,ity of rnAbs by immunoblotting The procedure recently described [23] for polyclonal antibodies was modified for mAbs. 3.12.1. Electrophoresis. S D S - P A G E electrophoresis of crude SEs (SEA, SEB, SECj, SED and SEE) was done with 8-25 gradient following the instructions of PhastSystem (Pharmacia) on PhastGels with sample applicators (8/1). 3.12.2. lmmunoblot. Electrophoresis was followed by electroelution of proteins with a transfer apparatus (LKB, model 2005 Transphor) onto nitrocellulose papers (Schleicher & Schull) [24]. Papers were blocked with 0.01 M phosphate buffered saline, 0.15 M NaC1, 0.5% Tween-20 (pH 7.3) (PBS-T) for 30 min at room temperature and exposed to mAb for 2 h at 37°C. After three washes with PBS-T, papers were incubated for 30 rain at 37°C with R A M - H R P (1:500 dilution in PBS-T). Papers were washed 8 times with PBS-T and exposed to the substrate solution (50 ml of PBS, 25 mg of di-amino-benzidine and 50 #I of 33% H202). 3.13. Inhibition test Inhibition study of mAbs was done by the indirect ELISA, after incubating double concentration of mAbs with different amounts of homologous enterotoxin (0, 1, 10, 100 and 1000 ng SEB ml-~ in PB-T) (1:1); thus 200 ~1 of mAb at a dilution corresponding to 50% of its titration curve was preincubated with 200/_d of SEB dilutions for 1.5 h at 37°C with shaking. These mixtures were added in triplicate to wells of 96-polystyrene plates coated with 100 pA of SEB (1 /~g ml I). The ELISA was developed as described above. 3.14. Sensiticity of detection This parameter was determined by the indirect ELISA technique. The microtitre plates were coated with 100 #1 of various levels of SEB (0, 1, 5, 10, 50, 100, 500 and 1000 ng SEB ml -~) and a constant concentration of mAb. All the levels were tested in duplicate.

250 4. R E S U L T S A N D D I S C U S S I O N F o u r t e e n h y b r i d o m a s p r o d u c i n g m A b s antiSEB w e r e s e l e c t e d on the basis of c o n s t a n t l y p r o d u c i n g positive results by E L I S A . T h e s e hyb r i d o m a s w e r e c l o n e d twice a n d e x p a n d e d . C h a r a c t e r i z a t i o n was d o n e f r o m p a r t i a l l y p u r i f i e d m a terial from ascites. E i g h t of t h e s e a n t i b o d i e s bel o n g e d to IgG~ subclass, while the o t h e r s w e r e IgG2b ( T a b l e 1). A b s o r b a n c e s in each case (IgG~ or IgG2b) w e r e m a r k e d l y h i g h e r t h a n in o t h e r subclasses ( d a t a n o t shown). T h e p e r c e n t a g e of m A b s b e l o n g i n g to IgGzb subclass (42.85%) is much h i g h e r t h a n t h o s e d e s c r i b e d by o t h e r aut h o r s for SEs [2,9,17] a n d most a u t h o r s d e s c r i b e a n t i b o d i e s b e l o n g i n g only to l g G 1 subclass [6,10,14]. T h e t i t r a t i o n curves of e a c h m A b with h o m o l o gous S E w e r e p l o t t e d after c o a t i n g the p l a t e s with 0.1 # g S E B / w e I 1 . T h e titre was d e f i n e d as the r e c i p r o c a l of the highest dilution r e n d e r i n g an a b s o r b a n c y > 0.1 at 450 nm as s u g g e s t e d by B o h a c h et al. [3]. T i t r e s r a n g e d b e t w e e n 40 (B12) a n d 1280 (BS) ( T a b l e 1); titres w e r e m u c h lower than those described by other authors [3,5,9,10,13]. T o study the influence of a low c o a t i n g c o n c e n t r a t i o n in t h e s e results, p l a t e s w e r e

c o a t e d with 1 /xg S E B / w e l l as d o n e by Edwin et al. [6] for S E A . All titres w e r e i n c r e a s e d , t h o u g h the levels w e r e always d i s c r e t e ( r a n g i n g b e t w e e n 640 for B7 and B12 and 40960 for B1, B3 a n d B l l ; T a b l e 1). To explain these low results, n o n - p r e c i p i t a t e d ascites from 8 clones w e r e tested. T i t r e s of ascites a n a l y z e d by the first system (0.1 # g S E B / w e l l ) r a n g e d b e t w e e n 103 for B8 a n d 107 for B4, B7 and B14. T h u s we believe that the low a m o u n t of p r o t e i n was d u e to deficient ascites p r e c i p i t a t i o n . In this w o r k c r o s s - r e a c t i o n s b e t w e e n all m A b s were s t u d i e d by indirect E L I S A , c o a t i n g with 0.1 /xg S E / w e l l of each of the s e r o v a r ( S E A , SEB, S E C l, S E D a n d SEE). R e s u l t s are shown in T a b l e 1. S o m e m A b s r e a c t e d exclusively with the h o m o l o g o u s toxin ( S E B ) (B5, B12 and B14), but most showed c r o s s - r e a c t i o n s with S E G I . This fact d e s c r i b e d by o t h e r s [2,10,17] is not surprising w h e n the h o m o l o g i e s in a m i n o acid s e q u e n c e of these two m o l e c u l e s are c o n s i d e r e d [2]. N o reactions were seen against any o t h e r serovar. T h e m A b s did not react with B S A when used as c o a t i n g a n t i g e n (3% in PB). However, the reactivity n o t e d in E L I S A could not always be interp r e t e d as being specific to S E [7,8]. T h u s m A b s w e r e also a n a l y z e d by i m m u n o b l o t t i n g to d e m o n -

Table l Characterization of mAbs mAb B1 B2 B3 B4 B5 B6 B7 B8 B9 BI0 BII B12 BI3 B14

IgG subclass 2b 2b 1 1 2b 1 2b 1 1 1 1 2b 1 2b

Titre " 0.1


Ascites b

160 320 640 160 160 160 160 1280 64(1 160 320 40 120 320

40 960 20 480 40 960 20 480 10240 1280 640 10 240 2 560 2560 40960 640 1280 1280

ND 106 ND 107 104 ND 107 103 ND ND 104 ND ND 10 7

Reactivity ELISA


Detection (ng SEB/well)

Inhibition Total SEB (ng)

B, C t B,C t B,C l B, C I B B, C E B, C t B, C t B, C 1 B, C I B, C~ B B. C 1 B

B, C B,C B,C B, C B B B, C B B, C B B B B, C B

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.1 0.1 0.1 0.1 0.5

5 5 50 5 0.5 500 50 5[) 5 5 5 > 500 0.5 (/.5

I 1 1 I

I 1


" Titre was determined by two systems: plates were coated with 0.1 tag or 1 tag S E B / w e l I . b Ascites titre was determined coating the microtitre plates with 0A tag SEB/welI. c Abbreviations: IMB: Immunoblotting, ND: Not Done.





Fig. 1. Reactivity and cross-reactivity by immunoblotting. (A): reactivity of mAb B13 with crude SEB (Lane I) and SEC t (Lane 2); (B) reactivity of mAb B5 with SEB.

strate the specific binding of the mAbs to the SEs. Fig. 1 shows reactivities of mAbs B5 and B13 by immunoblotting. In some cases (B6, B8, B10 and B l l ) the results obtained by ELISA and immunoblot were different (Table 1). With SEB, two bands could always be seen, one corresponding to SEB ( = 30 kDa) and another of a slightly higher molecular weight, which probably corresponds to the precursor described by Tweten and Iandolo [25,26] (Fig. 1). This fact was previously described by Edwin et al. [7] for a mAb reacting with all the five serovars of SE. mAb B13 reacts more strongly with the toxin than with the precursor, while B5 reacts similary with both bands (Fig. 1). No mAb in our study reacted with the minor bands (=-15.5 kDa and = 13 kDa) reported previously by Spero et al. [27] and others [13,17]. These minor bands are believed to result from nicking of the toxin by contaminating proteases at the cysteine loop [13,17]. These two bands clearly appear in SEB used for immunizing (Fig. 2, line 5). mAbs produced by Lin et al. [13] reacted with the 15.5 kDa fragment.

Fig. 2. SDS-PAGE electrophoresis. Lane 1, crude SEE; lane 2, crude SED; lane 3, crude SECt,; lane 4, crude SEB; lane 5, purified SEB; lane 6, crude SEA; lane 7, purified SEA; lane 8 low molecular weight marker. Proteins from bottom to top are: a-lactalbumin (14400), trypsin inhibitor (20100), carbonic anhydrase (30000), ovalbumin (43000), bovine serum albumin (67000), phosphorylase (94 000).

As regards dilution curves, data shown in Figs. 3 and 4 are typical of high affinity antibodies, i.e. a plateau of maximal absorbancy followed by a steep decrease at higher dilutions [28, 29]. However, the dissociation constant (K d) determined

Absorbance (450 nm)


0.5 0.4









"-~-" B6 B7 0.










t 320






Dilution (t,n)

Fig. 3. Titration curves of mAbs B1 to B7. ELISA was performed coating the plates with 0.1 # g SEB/well.


1450 nrn)


Absorbance (450 nrn)

0.3 0.6 ~


", \'

05 ~ 0.4 0.3



~/ +





,. \...



",', t,





~'- -++ ~ _ ~







_ _

Blo Bll B12 B13

0.15 0.1-




0.05 0




Fig. 4. T i t r a t i o n

80 160 320 Dilution (1;n)

curves o f m A b s


Mab+PB M a b + l Mab+10 Mab+100 Mab+1000 Monoclonal Antibody + $EB (ng/ml) (1:1)

1280 2560

B8 to B]4.


Fig. 6. I n h i b i t i o n of m A b s B8 to B14 with SEB. Double c o n c e n t r a t i o n of each m A b was i n c u b a t e d with different a m o u n t s of SEB (1:1) as d e s c r i b e d in MAIH~b*,I.S .XXb M~rHODS (section 3.13). The a b s o r b a n c e s for m A b s without toxin (control) were s h o w e d in the m A b + PB lane.


p e r f o r m e d coating the p l a t e s with 0.1 # g S E B / w e l l .

by the method described by Friguet et al. [30] for partially purified mAbs was not exceedingly high (10 -7 to 10 - s M). These mAbs could always detect low quantities of SE by indirect E L I S A (mainly 0.1 ng SEB/weI1) (Table 1), as levels were higher than those without SE or coated with BSA at 3% in PB-T (data not shown). This suggests that, at least theoretically, they could be used succesfully for the detection of SEB. Most mAbs were inhibited in their ability to bind SEB with very low quantities of SEB, especially in the case of B5, BI3 and B14 (0.5 ng SEB) (Table 11. We considered that the binding ability

of each mAb was inhibited when thc absorbancies were about half of the reference absorbance (mAb + PB) (Figs. 5 and 6). Because of the relatively high affinity of the mAbs for the SEB, it was enough to employ low amounts of toxin to show competition. mAbs described in this study could be used in detection systems in food extracts as they do not react with proteins other than SE or the putative precursor to SEB, so they are potentially useful for the development of specific detection methods.

Absorbance (450 nm)




0.25 B2


I [SB Ba




~ e e





This study was supported by C A I C Y T grant BT-85-0036. The authors are indebted to the D e p a r t m e n t of Biochemistry and Molecular Biology IV (U.D. Chemistry) of the Veterinary Sciences School (U.C.M.) for the loan of the semiautomated electrophoresis system and transfer apparatus.

0 Mab÷l Mab÷10 Mab*100 Mab+1000 Monoclonal Antibody + SEB (ng/rnl) (1:1)


Fig. 5. I n h i b i t i o n of m A b s BI to B7 with SEB. D o u b l e c o n c e n t r a t i o n of each m A b was i n c u b a t e d with different a m o u n t s of SEB ( 1 : 1 ) as d e s c r i b e d in MATEmALS ,XYb MIrH ODS (section 3.131. The a b s o r b a n c e s for m A b s w i t h o u t toxin (control) were s h o w e d in the m A b + PB lane.

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Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization.

A group of 14 monoclonal antibodies (mAbs) to staphylococcal enterotoxin B (SEB) were obtained by fusion of Sp2/O myeloma cells with spleen cells from...
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