FEMS MicrobiologyLetters 80 (1991) 35-40 © 1991 Federation of European MicrobiologicalSocieties0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100222Q

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FEMSLE 04417

Murine monoclonal antibodies reactive with staphylococcal enterotoxins A, B, C2, D, and E K u n i h i r o S h i n a g a w a 1, T h o r u K a n a z a w a , N a o n o r i M a t s u s a k a ~, Shunji Sugii 2 and Kumiko Nagata 3 1 Department of Veterinary Medicine, Faculty of Agriculture, lwate University, Morioka, lwate, 2 Department of Serology and Immunology, School of Medical Technology, Kitasato University, Kanagawa and 3 Department of Bacteriology, Hyogo College of Medicine, Mukogawa, Hyogo, Japan

Received 11 December1990 Accepted 9 January 1990 Key words: Enterotoxin; Monoclonal antibody; Staphylococcus aureus

1. SUMMARY By fusion of mouse spleen cells immunized with five different staphylococcal enterotoxins (SEA, SEB, SEC2, SED, and SEE) with myeloma cells, we obtained 15 hybridomas producing monoclonal antibodies (mAbs). Four mAbs were reactive with both SEA and SEE, whereas 8 mAbs were reactive with SEB and SEC 2. One mAb reacted with SEA, SED, and SEE. The other two mAbs were found to be reactive with all five serotypes of SEs. The mAbs specific for five serotypes of SEs were found to be most reactive with SED, reactive with SEA, and slightly less reactive with SEB, SEC2, and SEE. Those mAbs with specificities for all serotypes of SEs may be valuable to prepare immunoadsorbent(s) for isolation of SEs and to detect SEs in foods and clinical

Correspondence to: K. Shinagawa, Iwate University, Faculty of Agriculture, Department of VeterinaryMedicine, 3-18-8 Ueda, Morioka, Iwate 020, Japan.

specimens involved in outbreaks of staphylococcal food poisoning.

2. I N T R O D U C T I O N Staphylococcal enterotoxins (SEs) are known as bacterial exotoxins with a molecular mass of 27-30 kDa which cause emesis and diarrhea in humans and primates [1,2]. SEs are antigenically classified into A (SEA), B (SEB), Ch (SEC 1, SEC 2, and SEC3) , D (SED), and E (SEE) [1,2]. Serological cross-reactions have been found between SEA and SEE [3-5] and between SEB and SEC 1 [6-9]. Monoclonal antibodies (mAbs) are useful to study such immunological cross-reaction between SEB and SEC 1 [9-12]. Moreover, such mAbs with dual specificity will be valuable to isolate two different serotypes of SEs by immunoaffinity chromatography. This has been recently confirmed by the finding that a mAb reactive with both SEA and SEE can be useful to isolate both of these SEs by the same immunoadsorbent coupled to the mAb

36 (Shinagawa et al., unpublished observation). The finding also suggests that mAbs with specificities for two or more than two serotypes of SEs may be more valuable to develop immunological methods to detect and/or screen different serotypes of SEs produced in foods. However, attempts to prepare mAbs with such specificities have not usually been made although mAbs reactive with three or more serotypes of SEs have been very rarely obtained [10,13]. Thus, this study was undertaken to prepare murine mAbs reactive with five serotypes of SEs to finally develop immunological methods for detection of SEs in foods involved in outbreaks of staphylococcal food poisoning,

3. MATERIALS AND METHODS

3.1. Purification of SEs SEs were purified by the methods described previously [5,14]. 3.2. Preparation of mAbs to SEs Male Balb/c mice (6-8 weeks old) were injected intraperitoneally with purified SE(s) with Freund's complete adjuvant (Difco Laboratories, Detroit, MI, U.S.A.). mAbs to SEs were prepared according to the procedures described previously [15-17]. 50 /tg of SEA was intraperitoneally injected into mice. Nine days after the initial injection, 50 /xg each of SEB, SEC2, and SED was given intraperitoneally. After 4 months, 100/tg of SEE was given intravenously without adjuvant. Mice were also immunized with SEA only by the same immunization schedule. Spleen cells were removed 4 days later. Cell hybridizations were performed by the methods described previously [15-17]. Hybrid cells were selected in HAT medium and hybridomas producing antibodies were cloned by the limiting dilution method, 3.3. Enzyme-linked immunosorbent assay (ELISA) Specificities of the mAbs to different serotypes of SEs were determined in triplicate in ELISA. Each well of polystyrene microtiter plates (Dynatech Lab. Inc., U.S.A.) was coated with 100/~1 of purified SEs (10 /~g/ml in 0.1 M carbonate buffer, pH 9.5) and incubated at 37°C for 3 h.

After coating, the wells were blocked with 1% bovine serum albumin in 0.02 M phosphate buffered saline (PBS, pH 7.2) at 4 o C overnight. After washing the plate 5 times with PBS containing 0.05% Tween 20, 100 /tl of purified mAb, which was isolated by affinity chromatography on protein A-Sepharose CL-4B (Pharmacia, Sweden), was added to each well and incubated at 37 o C for 2 h. After washing, 100 #1 of horseradish peroxidase-conjugated anti-mouse IgG (diluted to 1 : 1,000) was added to each well and incubated at 37 °C for 1 h. After washing, color development of the reaction mixture was carried out by the methods described previously [18]. For determination of neutralizing abilities of mAbs to antigenicities of SEs, a mAb (M3-6) and SEs (SEA, SEB, SEC2, SED, or SEE) were mixed to a final concentration of 200 /~g/ml and 100 /~g/ml, respectively. The mixture was incubated at 37 °C for 30 min. After incubation, 100 #1 of the mixture was added to each well coated with 100 ~tl of SEs at 10 # g / m l to determine the remaining antibody activity in the mixture.

3.4. Other methods Subclasses of the mAbs were determined by double gel diffusion using a rabbit anti-mouse IgG kit (Nordic Immunolab., Netherlands) and rabbit anti-mouse light chain. Inhibition tests for mitogenic and r-interferon (IFN-r)-inducing activities of SEA were carried out by the methods described previously [19-21].

4. RESULTS

4.1. Preparation and reactivity ofmAbs 456 hybrid cell lines were obtained by fusion of mouse spleen cells immunized with a combination of SEA, SEB, SEC2, SED, and SEE with myeloma cells. Of these hybrid cell lines tested, 15 cells were found to produce antibody activities in the supernatants. Four hybrid cell lines were found to produce antibodies reactive with SEA and SEE, whereas eight cell lines were found to produce antibodies reactive with SEB and SEC 2. A mAb produced by one cell line reacted with SEA, SED, and SEE. Two other hybrid cells were found to

37 Table 1

Table 2

Reactivity of purified M3-6 with staphylococcal enterotoxins (SEs)

Binding activities of rnAb M3-6 to SEA, SEB and SEC2 after mixing of SEA, SEB, SEC2, SED and SEE with M3-6

Amount of M3-4 a (ng/ml)

Absorbance at 492 nm SEA SEB SEC2

SED

SEE

SE mixed with M3-6

Percent binding (percent inhibition) a SEA SEB SEC2

10000 1000 100.0 10.0 1.0 0.1

> 2.0 1.348 0.411 0.126 0 0

1.811 1.116 0.488 0.147 0.026 0

1.398 0.557 0.167 0 0 0

None SEA SEB SEC2 SED SEE

100 15 18 56 14 18

a

> 2.0 1.172 0.404 0 0 0

> 2.0 1.013 0.217 0 0 0

M3-6 was purified with affinity chromatography on protein A Sepharose CL-4B.

p r o d u c e a n t i b o d i e s reactive with five serotypes of SEA, SEB, SEC 2, SED, a n d SEE. By s u b c l o n i n g a n d s u b c u l t u r i n g of these two h y b r i d cell lines p r o d u c i n g a n t i b o d i e s reactive with five serotypes of SEs, we finally isolated three clones p r o d u c i n g antibodies highly reactive with five serotypes of SEs. They were designated M I - 1 , M2-1, a n d M3-6 which were I g G 1 (k). O n the other h a n d , two clones (A3-3 a n d A5-5) were finally o b t a i n e d b y fusion of mouse spleen cells i m m u n i z e d with SEA o n l y with m y e l o m a cells, The reactivities of affinity-purified m A b s such as M I - 1 , M2-1, a n d M3-6 with different serotypes of SEs were d e t e r m i n e d b y ELISA. M3-6 was most reactive with SED. It was more reactive with SEA t h a n SEB, SEC2, or SEE (Table 1). A l t h o u g h similar results were also o b t a i n e d with M I - 1 a n d M2-1, there were slight differences a m o n g their b i n d i n g to SEs such as SEB, SEC 2, a n d SEE.

100 18 19 62 15 22

100 22 26 56 19 26

a 100 #i of the M3-6 (200 #g/ml) and SE (100 #g/ml) mixture were added to each well of ELISA plate coated with SEA SEB and SEC2 to determine the remaing antibody activity. Absorbance of the M3-6 to SEA, SEB, or SEC2 without preincubation at 492 nm was taken as 100% binding.

4.2. Neutralization tests After m A b M3-6 was mixed with five serotypes of SEs, the r e m a i n i n g a n t i b o d y activity i n the m i x t u r e was d e t e r m i n e d b y ELISA. W h e n M3-6 a n d either SEA or SEB were mixed at 37 ° C for 30 min, the r e m a i n i n g a n t i b o d y activity i n the mixture was f o u n d to decrease (Table 2). W i t h S E D a n d SEE, similar results were o b t a i n e d as f o u n d for SEA a n d SEB. Similar findings were also o b t a i n e d with M I - 1 a n d M2-1. O n the other h a n d , the r e m a i n i n g a n t i b o d y activity i n the m i x t u r e was n o t significantly decreased b y m i x i n g M3-6 with SEC 2 (Table 2). N e i t h e r m i t o g e n i c i t y of SEA to lymphocytes n o r I F N - r p r o d u c t i o n i n d u c e d b y SEA were effectively i n h i b i t e d b y a n y of M I - 1 , M2-1, M3-6, a n d A5-5 at the highest c o n c e n t r a t i o n s used, whereas they were completely i n h i b i t e d b y A 3 - 3 (Table 3).

Table 3 Neutralization activities of mAbs to mitogenic and interferon-producing activities of SEA MAB

Percent inhibition

Name

Reactivity

[3H]thymidine incorporation

A3-3 A5-5 MI-1 M2-1 M3-6

SEA and SEE SEA SEA, SEB, SEC2, SED and SEE SEA, SEB, SEC2, SED and SEE SEA, SEB, SEC2, SED and SEE

100 0 0 0 0

IFN--t production 100 0 0 0 0

38 5. D I S C U S S I O N Several attempts have been made to prepare mAbs to SEs [9-13]. The mAbs obtained have been used to analyze type-specific and cross-reacting epitopes on SEs [9-12], to isolate SEs [11,13], and to screen SEs produced in foods [19,20]. To detect SEs in incriminated foods, mAbs with reactivities with all serotypes of SEs are more valuable than those with specificity for one or two serotypes of SEs. However, such mAbs have not usually been obtained since only one or two SEs have generally been used as an immunogen to prepare mAbs [9-13]. To prepare mAbs with specificites for all serotypes of SEs, five different serotypes of SEs (SEA, SEB, SEC 2, SED, and SEE) were used as immunogens in this study. We finally obtained three mAbs reactive with five serotypes of SEs in addition to 13 mAbs reactive with two or three serotypes of SEs. All three mAbs (MI-1, M2-1, and M3-6) with specificties for five serotypes of SEs were found to be similarly reactive with different serotypes of SEs: they were most reactive with SED, reactive with SEA, and slightly less reactive with SEB, SEC 2, and SEE. On the other hand, Meyer et al. [13] have obtained mAbs which reacted with SEB and SEC 1 more strongly than SED and SEE, suggesting that the specificity of the mAbs may be different from those of mAbs obtained in this study. The difference between the previous [10,13] and present results may be accounted for by the difference in immunization schedule. Either SEA only or both SEA and SED have been used as immunogens in the previous studies [10,13], whereas all of SEA, SEB, SEC 2, SED, and SEE were used as immunogens in this study. When both SEA and SED were used as immunogens, a mAb has been reactive with both SEB and SEC 1 more strongly than SEA, SED, or SEE althouh it bound to all serotypes of SEs [13]. When SEA only was used as an immunogen, on the other hand, the mAbs were reactive with both SEA and SEE more strongly than SEB, SEE1, or SED [10]. Thus, specificities of mAbs for SEs obtained may be dependent upon the combination of different SEs used as immunogens. The present findings suggest that mAbs capable

of reacting with epitopes shared in common by all serotypes of SEs could be used as the first antibody in a double sandwich ELISA procedure to screen samples for the presence of any of the serotypes of SEs if type-specific mAbs can be used as the second antibody. Thus, the mAbs with such specificities are potentially useful for the development of rapid, sensitive, and specific immunological methods such as ELISA for detection of all serotypes of SEs.

ACKNOWLEDGEMENT This study was supported in part by a Grantin-Aid from the Ministry of Education, Science, and Culture of Japan.

REFERENCES [1] Bergdoll, M.S. (1977) in Immunological Aspects of Foods (Catsmpoolas, N., Ed.), pp. 195-220, AVI publishing Co. Inc., Westport, CT. [2] Bergdoll, M.S. (1979) (Riemann, H. and Bryan, F.L., Eds.) in Food-Borne Infections and Intoxications, 2nd edn. Academic Press Inc., New York. [3] BergdoU, M.S., Borja, C.R., Robbins, R.N. and Weiss, K.F. (1971)Infect. Immun. 4, 593-595. [4] Lee, A.C-M., Robbins, R.N., and Bergdoll, M.S. (1978) Infect. Immun. 21,387-391. [5] Shinagawa, K., Kunita, N. and Sakaguchi, G. (1977) Jpn. J. Bacteriol. 32, 829-835 (in Japanese with English summary). [61 Johnson, H.M., Bukovic, J.A. and Kauffman, P.E. (1972) Infect. Immun. 5, 645-647. [71 Spero, L. and Morlock, B.A. (1979) J. Immunol. 122, 1285-1289. [8] Spero, L., Morlock, B.A. and Metzger, J.F. (1978) J. Immunol. 120, 86-89. [9] Thompson, N.E., Ketterhagen, M.J. and BergdoU, M.S. (1984) Infect. Immun. 45, 281-285. [10] Edwin, C., Tatini, S.R. and Maheswaran, S.K. (1986~ Appl. Environ. Microbiol. 52, 1253-1257. [11] Lapeyre, C., Kaveri, S.V., Jartin, F. and Stronsberg, A.D. (1987) Mol. Immunol. 24, 1243-1254. [12] Thompson, N.E., BergdoU, M.S., Metzger, R.F., Bennett, R.W., Miller, L. and Macmillan, J.D. (1986) in Monoclonal Antibodies Against Bacteria (Macario, A.T.J. and C.E., de Macario, Eds.), pp. 23-59, Academic Press Inc., London. [13] Meyer, R.F., Miller, L., Bennett, R.W. and Macmillan, J.D. (1984) Appl. Environ. Microbiol. 47, 283-287.

39 [14] Shinagawa, K., Kunita, N. and Sakaguchi, G. (1975) Japan. J. Bacteriol. 30, 683-692 (in Japanese with English summary). [15] de St Groth, S.F. and Sheidegger, D. (1980) J. Immunol. Methods 35, 1-21. [16] Galfre, G. and Milstein, C. (1981) Methods Enzymol. 73, 3-46. [17] Goding, J.W. (1980) J. Immunol. Methods 39, 285-308. [18] Shinagawa, K., Watanabe, K., Matsusaka, N., Konuma, H. and Sugii, S. (1990) Jpn. J. Vet. Sci. 52, 847-850.

[19] Kawaguchi-Nagata, K., Okamura, H., Shoji, K., Kanagawa, H., Semma, M. and Shinagawa, K. (1985) Microbiol. Immunol. 29, 183-193. [20] Peary, D.L., Adler, W.H. and Smith, R.T. (1990) J. Immunol. 105, 1453-1458. [21] Langford, M.P., Stanton, G.L. and Johnson, H.M. (1978) Infect. Immun. 22, 62-68.

Murine monoclonal antibodies reactive with staphylococcal enterotoxins A, B, C2, D, and E.

By fusion of mouse spleen cells immunized with five different staphylococcal enterotoxins (SEA, SEB, SEC2, SED, and SEE) with myeloma cells, we obtain...
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