Zbl. Bakt. 277, 345-356 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Monoclonal Antibodies Directed to the 0 Antigen of Salmonella Serogroup E Cross-React with Lipopolysaccharides of Salmonella Serogroups C, F and S IVAN MITOV, GEORGY GEORGIEV, RADKA IVANOVA, DRAGOMIR PETROV, VICTORIA LEVTEROVA, and DIM IT AR STRAHILOV Institute of Infectious and Parasitic Diseases, 1504 Sofia, Bulgaria

With 2 Figures· Received April 15, 1992 . Accepted June 11, 1992

Summary Four murine hybridoma clones secreting monoclonal antibodies (mAb) directed against lipopolysaccharide (LPS) antigen of S. newington, serogroup El, were obtained after a fusion of spleen cells of mice immunized with formaldehyde-killed bacteria and mouse myeloma cells of the X63-Ag8.653 line. Antigen binding properties and specificity of the mAb were studied in bacterial agglutination tests, passive hemolysis and its inhibition, passive hemagglutination tests, passive hemolysis and its inhibition, passive hemagglutination and immunoenzyme tests (ELISA and immunoblotting). Three of the mAb (24E6, 29E 1 and 45F6) were agglutinating and were active in all tests used, while mAb 31H12 did not agglutinate bacteria but revealed a high reactivity in the immunoenzyme reactions. It was found that the mAb reacted with LPS and Salmonella strains from serogroup E (E1, E2, E3 and E4) as well as from serogroups C (CI and C4), F and S thus showing that the 03 antigen possesses more than one epitope, one of which is represented on the LPS antigens of the serovars from the cross-reacting groups mentioned. According to the known chemical the most probable recognized epitope consits of mannose with ~-linkage to the next monosaccharide residue in the LPS chain.

Zusammenfassung Nach der Fusion von Milzzellen von Mausen, die mit Formaldehyd-abgetoteten Bakterien und Maus-Myelomzellen der Linie X63-Ag.8.653 immunisiert worden waren, wurden vier murine Hybridom-Klone gewonnen, die monoklonale Antikorper (mAb) abschieden, welche gegen das Lipopolysaccharid (LPS)-Antigen von S. newington, Serogruppe E1, gerichtet waren. Die Antigenbindungseigenschaften und die Spezifitat der mAb wurden mittels Tests zur bakteriellen Agglutination sowie mittels Immunenzymtests (ELISA und Immunoblotting) untersucht. Drei der mAb (24E6, 29E1 und 45F6) agglutinierten und waren in allen angewandten Tests aktiv, wogegen 31H12 keine Bakterien agglutinierte, in den Immunenzymtests aber eine hohe Reaktionsfahigkeit aufwies. Es wurde festgestellt, daIS

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1. Mitov et a1.

die mAb mit LPS und Salmonella-Stammen der Serogruppe E (E1, E2, E3 und E4) sowie der Serogruppen C (C1 und C4), Fund S reagierten, was zeigt, daIS das 03-Antigen mehrere Epitope aufweist, von denen eines in den LPS-Antigenen der Serovare der erwahnten Gruppen mit Kreuzreaktion vertreten ist. Nach den bekannten chemischen Strukturen der aus den mit den mAb reagierenden Serovaren gewonnenen LPS besteht das mit grolSter Wahrscheinlichkeit erkannte Epitop aus Mannose mit ~-Bindung an den nachsten Monosaccharidrest in der LPS-Kette. Introduction The serological classification and identification of Salmonella is based on their 0, H and Vi antigens according to the scheme of Kauffmann- White (6). Salmonella species are subdivided into 02-067 serological groups by their lipopolysaccharide (LPS) antigen specificities. A variety of 0 antigens had been determined by cross absorption of rabbit agglutinating antisera. Some of them, the group and some of the factor antigens, are used for serological identification and typing (6, 9). The conventional methods for the preparation of mono specific O-sera have certain shortcomings, which diminish their potential for studying the specificity of different antigen determinants. It is well known that immunization with a given serovar leads to the formation not only of antibodies specific for diagnostically important antigens but also of antibodies against other antigen determinants found on LPS of different Salmonella serogroups. The latter determinants often induce cross-reactive antibodies which must be discarded by cumbersome procedures of repetitive absorption (9). They are not included in the Kauffmann- White scheme and usually their chemical composition and structure as well as antigen specificity are poorly defined. Investigations of the cross-reacting antigen determinants, however, could be of a great importance in relation to the classification of the Salmonella serovars as well as the development of polyvalent antigen or antibody preparation for diagnostic purposes. In contrast, monoclonal antibodies (mAb) are strictly of monoepitopic specificity and may therefore be used for a precise definition of potentially important antigen epitopes. Furthermore they are easily standardizable which makes them suitable for diagnostic purposes. The preparation of diagnostically important anti-Salmonella mAb has already been reported (11, 13). In this investigation, we present data about the production and characterization of murine mAb directed to the 03 antigen of Salmonella serovars of group E that react with 0 antigens of salmonellae belonging to the Cl, C4, F, and S serological groups. The data about the reactive epitope recognized by the mAbs are also presented.

Materials and Methods Bacterial strains and antigen preparations. The Salmonella serovars used in this study were obtained from the National Microbiological Collection, Sofia, Bulgaria and confirmed by routine biochemical and serological methods. The bacteria were grown overnight on nutrient agar. Killing of bacteria used for immunization was carried out with 0.5% formaldehyde for 2 h at 37"C and overnight at room temperature followed by 3 washings with phosphate-buffered saline (PBS). LPS were extracted by the hot phenol-water method of Westphal et a1. (17) from S. newington (0: 3, 15), S. london (0: 3, 10), S. senftenberg (0: 1, 3, 19), S. aberdeen

Monoclonal Antibodies to 0 Antigen of Salmonella Serogroup E

347

(0: 11), S. oslo (0: 6, 7), S. kaduna (0: 6, 7, 14), S. paratyphi A (0: 1, 2, 12), S. typhimurium (0: 1,4,5, 12), S. uccle (0: 3, 54) and from S. typhi (0: 9,12). Lipopolysaccharide of S. minnesota R60 (Ra chemotype) was a kind gift from C. Galanos, Max-PlanckInstitute of Immunobiology, Freiburg, Germany. All LPS preparations were dissolved at a concentration 2 mg/ml in distilled water and neutralized with triethylamine when necessary. Production of monoclonal antibodies. BALB!c mice were immunized with 0.5 x 109 bacteria cells of formalin-killed S. newington in 0.85% saline on three occasions, 2 weeks apart, the last being 4 days before the fusion. The first and the second applications were made by the i.p. route and the third by the i.v. route. The splenocytes and murine myeloma cells of the X63-Ag8.653 line were fused according to the method of Kohler and Milstein (7) with 42% polyethylene glycol (MW 4000) and 15% DMSO in Dulbecco's Modified Eagle Medium (DMEM) for 4 min. The cells were suspended in selective hypoxanthine-aminopterin-thymidin medium in DMEM, containing 10% FCS and plated in 24-well plates. After the formation of hybridoma colonies, the cell supernatants were screened by the enzymelinked-immunosorbent assay (ELISA) for antibodies specific for the LPS antigens of S. newington. Cultures that proved positive were cloned twice by limiting dilution (1). Isotyping of the mAb was performed by ELISA using a monoclonal antibody typing kit from Calbiochem, San Diego, CA, USA, (Cat. No. 386445). Ascitic fluids or supernatants from cell cultures reaching a density of 1-1.5 x 10 6 cells!ml were used to determine the specific activity and to characterize the mAb. Ascitic fluid was produced in BALBIc mice primed i.p. with 0.5 ml pristance 7-30 days prior to the i.p. application of 0.5-1 x 10 7 hybridoma cells. Purification of the ascitic fluids was performed by precipitation with a saturated solution of ammonium sulphate, pH 7.0 (final concentration 50%). The precipitates were dissolved in PBS and dialyzed against PBS, pH 7.6. The final preparations were adjusted to a a concentration of 20 mg!ml protein and aliquots stored at -70°e. Bacterial agglutination tests. The slide agglutination tests were performed by mixing 20 [11 of antibody solution (undiluted or serially diluted) with bacteria grown on solid media and the results were read in two minutes. Microagglutination test (whole cell agglutination titre) was performed in V-shaped standard 96-wells microtitration plates. The antibody solution was serially diluted in 50 [11 PBS, and equal volumes of a suspension of live Salmonella bacteria (1.5 X 10 9 cells!ml in PBS) were then added. The plates were incubated at 37°C for 1 h and overnight at 4°C before estimation of the agglutination titre. The titre was expressed as the last dilution at which agglutination still took place. Passive hemolysis (PH) and passive hemagglutination (PHA) tests. A microtest version of the method described by Galanos et al. (3) was used to assess the complement-activating properties of the mAb. To 50 ~d of a twofold dilution of antibody in a standard microtitration 96-well plate, 50 [11 of O.s'X, suspension of sheep red blood cells (SRBC) coated with LPS (3, 12) and 25 [11 of complement (guinea pig serum, 1: 10) were added. Veronal buffer (VB), pH 7.2, was used as a diluent throughout. Hemolysis titres (the last dilution at which approximately 50% hemolysis occurred) were evaluated by visual observation after incubation of the plates for 1 h at 37°C and overnight at 4°C. For this test, 1 hemolytic unit was defined as the last dilution at which approximately 50% of the SRBC lysed. The passive hemagglutination test was performed in the same way as PH but without adding complement. The last dilution with hemagglutination was considered as a titre. Inhibition of PH (PHI). To a serial twofold dilution of LPS inhibitor in 25 [11 VB (starting concentration 10 [1g/well), two hemolytic units of the assessed mAb in 25 [11 VB were added. After 15 min at 37°C, 50 [11 of O.SOI;, SRBC coated with the homologous LPS antigen and 25 [11 complement solution, all in VB, were overlaid. The plates were incubated for 1 hat The inhibitory activity of LPS was expressed as the amount ([1g1well) corresponding to the last dilution at which inhibition of the lysis of coated SRBC still took place (4). ELISA. A sensitive ELISA technique as described by Freudenberg et al. (2) was used. Briefly, a stock solution of LPS antigens (2-5 [1g/ml in water)- was added to a chloroform! ethanol (1 : 9 v!v) mixture. For coating of polystyrene plates (Nunc-Immuno plates, Nunc,

3re.

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1. Mitov et al.

Roskilde, Denmark), 100 ftl containing 1 ftg LPS were placed in each well and the solvent was evaporated overnight at room temperature. The coated plates were stored at the same temperature and were used when necessary in a standard ELISA procedure. Briefly, the plates were washed twice with PBS, pH 7.2, supplemented with 0.05% Tween 20 (PBS/ Tween). Non-specific binding sites were blocked with 200 ftl of 3% fetal calf serum in PBS for 2:: 1 h at 3rc and the plates were washed twice. Antibody dilutions in the blocking buffer were added and the plates incubated for 2 h at 37°C followed by five more washings. One hundred microliters of a peroxidase-conjugated goat anti-mouse (H +L chains) antibody diluted 1 : 1000 were added and the plates were incubated again for 2 h at 37°C. After five washes, the enzyme reaction was developed for 30 min at room temperature with 0.04% H 2 0 z and 0.04% o-phenylendiamine (Sigma) in phosphate-citrate buffer, pH 5.0. The reaction was stopped with 25 ftl4N H 2 S0 4 and the optical density was measured at 492 nm using a Uniskan microtest plate photometer (Labsysem, Finland). Polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE was carried out in a discontinuous buffer system with 4% and 12.5% acrylamide in the stacking and the separating gels, respectively, as described in (8). The LPS preparations were mixed 1: 1 with sample buffer containing 4% sodium dodecyl sulphate (SDS), 10% 2-mercaptoethanol, 20% glycerol and 0.01 % bromophenol blue in 0.1 M Tris-glycin buffer, pH 6.8. Aliquots of 2 ftl (5 ftg LPS) were applied to the gels and were run at 18 rnA in the stacking and at 35 rnA in the separating gel. SDS-fractionated LPS were stained by the silver staining method of Tsai and Frash (15). Immunoblotting of the fractionated LPS was performed by the method of Towbin et a1. (14). LPS were transferred from separation gels to nitrocellulose membranes (pores of 0.45 ftm, Schleicher and Schuell, Dassel, Germany) overnight at 90 rnA. After blocking with 3% bovine serum albumin in PBSlTween, the membranes were incubated overnight at 4°C with supernatants of the cultivated hybridomas. The mAb bound to the LPS bands were visualized using peroxidase-conjugated goat anti-mouse antibodies diluted 1 : 500 in the blocking buffer for 2 h at room temperature. The colour was developed with 0.06% 4-chloro-lnaphtol, 20% methanol (vol/vol) and 0.04% H 2 0 z in Tris-buffered saline.

Table 1. Activity of mAbs a in bacterial agglutination tests, PH, PHA and ELISA mAb b

24E6 29El 45F6 3IH12

Bacterial agglutination C Slide

Micro

320 5120

1280 20480 640 0

RO

0

PHd

PHA d

ELISA d

25600 204800 6400 3200

6400 2800 400 6400

6400 51200 3200 1638400

End reciprocal titres (lIT). Monoclonal antibodies precipitated with ammonium sulphate from ascitic fluids. C Agglutination of S.lanka (3,15 :r:z6). d SRBC and polystyrene plates coated with 03,15 LPS. a

b

Monoclonal Antibodies to 0 Antigen of Salmonella Serogroup E

349

Results

Production of mAb against 03,15 antigen of S. newington Following immunization with killed S. newington bacteria, mice were pretested and splenocytes from those animals exhibiting a high titre of serum antibodies were selected for fusion with X63-AgS.653 myeloma cells. The culture supernatants were screened by ELISA after two separate fusions and the hybridoma clones 24E6, 29El, 45F6 and 31H12 were found to secrete mAb specific for the LPS 0 : 3, 15 of the strain used for immunization. The first three of the mAb were of the IgM class while 31H12 was of IgG2a isotype. Reactivity of the mAb from ascitic fluids to the homologous antigen was assessed by agglutination tests, PH, PHA and ELISA. The results are presented in Table 1. It was found that mAb 24E6, 29El, 45F6 agglutinated the S. lanka serovar with the 0 : 3, 15 antigen. All four antibodies reacted to a various degree with LPS 03, 15 both in PH, PHA and ELISA. Antibody 31H12 showed no activity for agglutinating bacteria, neither in the slide nor in the more sensitive microagglutination Table 2. Specificity of mAbs a as determined by slide agglutination with different strains of Salmonella serovars belonging to serogroups OA to 054 Serogroup (0 antigens) E1 E2 E3 E4 0:54 F S Cl C4 Other

(0:3,10) (0:3,15) (0:3,15,34) (0:1,3,19) (0:3,54) (0:11) (0:41) (0:6,7) (0:6,7,14) from A to 0:54

Monoclonal antibodya 24E6

29E1

45F6

10/10 b 4/4 3/3 2/2 111 3/3 4/4 6/6 3/3

101l0b 4/4 3/3 2/2 111 3/3 4/4 6/6 3/3

101l0 b 4/4 3/3 2/2 111 3/3 4/4 6/6 3/3

0/61

0/61

0/61

a Ascitic fluids diluted 1: 1O. b Number of agglutinated strains / Number of tested strains. Table 3. Endpoint tit res (lIT) obtained in ELISA between mAbs and LPS extracted from strains of different serogroups mAB a

24E6 29E1 45F6 31H12 a

LPS 0:3,15

0:3,10

0:3,54

011

041

06,7

3200 25600 1600 1638400

1600 6400 1600 409600

3200 51200 1600 3200

3200 25600 1600 25600

3200 51200 3200 12800

800 1600 800 1600

Monoclonal antibodies precipitated with ammonium sulphate from ascitic fluids.

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I. Mitov et al.

test. It also exhibited the lowest titre in PH. However in ELISA, its actlVlty was considerably higher than that of the agglutinating antibodies 24E6, 29E1 and 45F6. The hybridoma clones 24E6 and 29E1, secreting mAbs that reacted with highest titres in all serological assays have been deposited at the National Collection of Microorganisms and Cell Cultures, Sofia, Bulgaria, under the numbers 1870 and 1871. Specificity of mAb The specificity of the antibodies 24E6, 29El, 45F6 and 31H12 was initially defined by the slide agglutination test using strains of 97 serovars from Salmonella serogroups A to 0: 54 (Table 2). The first three antibodies agglutinated strains of group E, that express different combinations of 0 antigen factors: 0: 3, 10; 0: 3, 15; 0: 3, 15,34 and 0: 1, 3, 19 and an S. uccle strain with the 0: 3, 54 antigen. Thus the presumption that the epitope(s) recognized by the antibodies is represented on the 03 group antigen seems to be justified. Besides it was found that the three agglutinating mAb crossreacted with strains belonging to groups F(O: 11), 5(0: 41), Cl(O: 6, 7) and C4 (0: 6, 7, 14). Antibody 31H12 did not agglutinate any of the strains used. In a different set of experiments, microagglutination was performed. Varying titres were obtained, the highest ones with the strains of groups E, F and 5 and the lowest, with the strains from groups Cl and C4. The remaining serogroups did not react. The specificity of the mAb for LP5 of the serogroups reacting in the slide agglutination test wa, established by ELISA (Table 3). The agglutinating mAb 24E6, 29El and 45F6 bound to all these LP5 with comparable titres, while the titres of MAb 31H12 for LPS of groups E2 (0: 1, 15) and El (0: 3, 10) were considerably higher. The mAb did not react with LPS from serogroups A, B, 0 and S. minnesota R60. Some of the antigens that reacted with the mAb were assessed by immunoblotting. Lipopolysaccharides 0:3, 10; 0:3,15; 0:1, 3,19; 0:11 and 0:41 analyzed by 50S-PAGE showed rhe characteristic ladder-like pattern after silver staining (Fig.1). After transfer to nitrocellulose and subsequent immunoblotting, it was found that the mAb reacted with the bands corresponding to the 0 antigen. The agglutinating antibodies 29E1, 45F6 (Figs.2a and 2b) and 24E6 (not shown) reacted in a similar way with the faster movmg bands (having smaller O-specific chains) of LP5 from groups El, E2 and E4 and with most of the bands of LPS from groups 011 and 041. Antibody 31 H 12 bound to all bands of the LPS tested (Fig.2c). The PHI test supplied additional information regarding the specificity of the mAb. The hemolysis of O' 3, 15 LPS-coated SRBC by mAb 24E6 and 45F6 was inhibited to a comparable degree by LP5 from groups El, E2, E4, F, S, Cl and S. uccle (0: 3, 54) while for inhibition of hemolysis by mAb 29El considerably more LPS from groups S and Cl had to be employed thus showing a distinct epitope specificity. The reaction with MAb 31H12 was blocked by a comparable amount of LPS from groups E compared to the agglutinating MAbs, but considerably greater quantities of LPS were necessary from the other cross-reacting LPS. Inhibition of hemolysis was achieved also by means of O-mannose being the immunodominant sugar in the 03 antigen, while amounts 4-16 times wert necessary when mAb 31H12 was applied (Table 4). Other monosaccharides such as glucose showed no inhibition. The reaction was negative when serologically unrelated LP5 from serogroups A, B, D, and S. minnesota R60 were used as inhibitors.

Monoclonal Antibodies to 0 Antigen of Salmonella Serogroup E

1

2

3

4

351

5

Fig. 1. Silver staining of SOS-PAGE of purified LPS from Salmonella strains (see Materials and Methods) of serogroups E1, E2, E4, Sand F. Lines: 1. LPS 0: 3, 10 (El); 2. LPS 0: 3,15 (E2); 3. LPS 0: 1, 3, 19 (E4)1 4. LPS 0: 41(S) and 5. LPS 0: 11 (F).

1

2

345 A

1

2

3 B

4

5

1

2

3

4

5

c

Fig. 2. Immunoblotting of LPS from Salmonella strains of serogroups El, E2, E4, Sand F (see Materials and Methods) with mAb 29El (A), 45F6 (B) and 31H12 (C). Lines 1: LPS 0: 3, 10 (E1); 2. LPS 0: 1, 15 (E2); 3. LPS 0: 1, 3,19 (E4); 4. LPS 0: 41 (S) and 5. LPS 0: 11 (F). 23

Zbl. Bakr. 27m

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I. Mitov et al.

Discussion This paper deals with four mAb against the 03 antigen of Salmonella serovars of serogroups EI-E4 that cross-react with 0 antigens of other groups of the same genus. Three of the hybridoma clones prepared, 24E6, 29E1, and 45F6 produced agglutinating IgM mAb, reacting with the homologous LPS antigen also in PH, PHA and ELISA. A fourth hybridoma, 31H12, produced IgG2a mAb that did not agglutinate any of the strains. This antibody, however, was highly active in ELISA (Table 1). By slide and microagglutination assays using bacteria from 97 strains of serogroups A to 0: 64 it was found that the agglutinating MAbs 24E6, 29El and 45F6 crossreacted with strains from serogroups F(O: 11), S(O: 41), C1(0: 6, 7) and C4(0: 6,7, 14) (Table 2). The results from PH, PHI, PHA, ELISA and immunoblots confirmed those obtained by agglutination. The nonagglutinating mAb 31H12 seemed to exhibit similar specificity but contrary to the other mAb, its reaction with 0: 3, 10 and 0: 3, 15 LPS was much stronger in ELISA (Table 3). These data show that the antibodies recognize an epitope on the 03 antigen of salmonellae from group E, which is presented also in the LPS antigens of serogroups F, S, Cl and C4. Comparison of the mAb in PHI (Table 4) provides additional support for assuming that the antibodies partially differ in their specificity: 24E6 and 45F6 were inhibited by comparable quantities of LPS; 29E1 differed since it required considerably more LPS 0: 41 and 0: 6, 7 for inhibition. Antibody 31H12 was inhibited most effectively by LPS of strains from group E and to a lesser degree, by the other cross-reactive LPS. In

Table 4. Passive hemolysis inhibition of mAbs by LPS extracted from serovars of different serogroups Inhibitor

Serogroup

El

E2 E4 0:54 F S C1 A B D S. minnesota R60

a b

Minimal inhibitory concentration a for mAb: 24E6

29E1

45F6

3IH12

LPS 0:3,10 0:3,15 0: 1,3,19 0:3,54 0:11 0:41 0:6,7

0,039 0,019 0,078 0,001 0,001 0,001 1,250

0,039 0,039 0,019 0,019 0,001 0,156 10

0,019 0,019 0,039 0,039 0,039 0,039 0,312

0,156 0,078 0,078 1,250 1,250 5 2,5

0:1,2,12 0:1,4,5,12 0:9,12

>10 b >10 >10

>10 >10 >10

>10 >10 >10

>10 >10 >10

Ra

>10

>10

>10

>10

D-mannose D-glucose

o,no

1,560 >25

0,390 >25

6,250 >25

>25

The amount (f!glwell) corresponding to the last dilution at which inhibition of lysis of SRBC coated with 03,15 LPS still takes place. > 10, >25 - maximal used amount in the first well.

Monoclonal Antibodies to 0 Antigen of Salmonella Serogroup E

353

contrast to ELISA and PHI, immunoblotting revealed no differences among the agglutinating mAb. They yielded similar patterns of reaction with the LPS used. The mAb 31H12 differed in its reaction with both high and low molecular weight forms of the LPS tested, while mAbs 24E6, 29E1 and 45F6 bound preferentially to the lower molecular weight forms of LPS. Mannose is the immunodominant sugar in the 03 antigen (9). In our study, this was confirmed by the ability of mannose, but not of other monosaccharides, to inhibit the hemolysis of SRBC coated with LPS 0: 3, 15. The immunodominant structure is believed to consist of a monosaccharide residue and the linkage to the adjacent monosaccharide (9). The disaccharide D-mannose-p ~ L-rhamnose, which is part of the trisaccharide unit with the sequence mannose-rhamnose-galactose, forming the 0specific side chains of LPS of serogroup E, is the immunodominant structure of 03 antigen. The linkage between D-galactose and the D-mannose residues in the repeating units is al ~6 in groups E1 and E4 and ~1 ~6 in groups E2 and E3 (Table 5) which obviously does not influence the specificity of the 03 antigen (9). Tsang et al. (16) obtained mAb directed against the epitope in which a1~6 linkage that joins the 0 repeating units of serogroups Eland E4 is involved. The mAb presented here not only reacted with Salmonella from serogroups E1 and E4 but also with E2 and E3. They also bound LPS of groups C 1 and C4 that contained D-mannose residues in the backbone chain, joined together by a ~1 ~2 linkage but not with C2 and C3 in which groups mannose residues were connected to the next unit by a-linkages. This finding seems to indicate that the minimal structure recognized by the antibodies is build up at least of D-mannose linked by a ~-bond to the next monosaccharide residue, D-rhamnose in serogroup E and D-mannose in serogroup C. The activity of the mAb with strains and LPS from serogroup C in the agglutination assays and ELISA was the lowest compared to that of serogroup E. In PHI, significantly higher quantities of 06, 7 LPS were necessary to inhibit the hemolysis. It is possible that the ~l ~ 2 linkage to the neighbounng D-mannose residue (instead of ~1 ~4) partially altered the homologous epitope on the LPS of group c:. The other cross-reacting groups F and S have been shown to contain mannose as a constituent of their LPS (10). The investigated strains belonging to serogroups A, B, D and E have similar backbone chains differing in the anomeric position of the constituent sugars, the linkage between the galactose and the mannose residues, and the presence of branched and 0acetylated sugars. The LPS from salmonellae of serogroups Band D2 have a Dmannose-~l ~4-L-rhamnose In the backbone chain but with branched abequose or tyvelose on a mannose unit (5, 9). Despite the similarity in the LPS structure, the antibodies did not react with serovars from serogroups Band D2. Most probably, the monosaccharide residues linked to D-mannose in the backbone chain alter considerably the epitope(s) recognized by the mAb. Bearing in mind that mannose frequently occurs as a component of the polysaccharide antigens of Salmonella and other microbial species, the mAb we have developed can be used for detection and confirmation of similar epitopes in the investigated microorganisms. Our investigations in this direction are in a progress. Although these mAb cannot be used directly in the routine serological identification of the Salmonella strains, their cross-reactivity allows an application in polyvalent diagnostic preparations as agglutiantion reagents or in ELISA methods through enrichment cultivation techniques. Other highly productive hybridoma lines secreting agglutinating mAb to 0 and H antigens of Salmonella (04[, 042., 05, 07, 08, 09, 010, 027, Hh, Hi, He, HI and H6) were currently developed by our group. These mAb have been

354

L Mitov et a!.

Table 5. Chemical structure of 0 repeating units of Salmonella serogroups A, B, C, D z and Ea

Par

D-Gle

11

A

(0:1,2,12)

a!3 -+2 D-Man

11

0!4 1~4

a

D-Gle

11

a!3 (0:1,4,5,12) -+2 D-Man

H

L-Rha 1--1>3 D-Gall~ a a

OAc-Abe B

Agglutinationb

Structure

Serogroup

11

0!4 1~4

L-Rha 1--1>3 D-Gall~

~

(-)

a

~

D-Gle U

C

(0:6,7) (0:6,7,14)

11 J3

-->2 D-Man 1-+2D-Man 1-+2D-man 1--1>2D-Man 1--1>3 D-GleNAc 1--1> (+) ~

~

~

D-Gle

/' C

(0:6,7,14)

.c'

~

11

11

B

J3

-+2 D-Man 1-+2 D-Man 1--1>2 D-man 1--1>2 D-Man 1--1>3 D-GleNAc 1--1> (+) af~

af~

Tvv

-+6 D-Man

af~

af~

~

D-Gle

'II at3

D2 (0:9,46)

~

D-Gle

11

uJ4 1~4

~

L-Rha 1-+3 a

D-Gal1~

(-)

a OAc

E1

(0:3,10)

16

--1>6 D-Man 1--1>4 L-Rha 1--1>3 D-Gal1~ ~

£2

(0:3,15)

a.

(+)

a.

-+6 D-Man 1-+4 L-Rha 1--1>3 D-Gal1~ ~ a ~

(+)

G-Glc

11

aJ4 E3

(0:3,15,34) --1>6 D-Man

1~4

~

L-Rha 1--1>3 a.

D-Gal1~

(+)

~

G-Glc

11

at6 £4

(0:1,3,19)

>6D-Man1-->4L-Rhal-+3D-Gall--1> ~ a a.

(+)

For the LPS structure see reference 5 and 9. Slide agglutination of the strains tested in each serogroup whose number is shown in Table 2. Abbreviations: Abe - abequose, Gal- galactose, Gle - glucose, Man - mannose, NAe - N-acetyl, OAc - O-acetyl, Par - paratose, Rha - rhamnose, Tyv - tyvelose. a

b

Monoclonal Antibodies to 0 Antigen of Salmonella Serogroup E

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proved to be a useful rool for replacement of the polyclonal factor sera in the laboratory identification of Salmonella. In summary, the results of this study show that the 03 antigen consists of more than one epitope, and one of them is represented in the LPS of Salmonella serogroups C, F and S. According to the known structures of LPS obtained from serovars reacting with the mAb, the most probably recognized epitope consists of D-mannose with ~-linkage to the next unit in the LPS chain. Acknowledgments. We thank Dr. C. Galanos and Dr. O. Liideritz, Max-Planck-Institute of Immunobiology, Freiburg, Germany, for their critical review of the manuscript. The excellent technical assistence of V. Pasco va and L. Petrova was of substantial significance in carrying out this study.

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Assosiated Professor I. Mitov, M. D., Ph. D., Institute of Infectious and Parasitic Diseases, Blvd. Y. Sakazov 26, 1504 Sofia, Bulgaria