DIAGN MICROBIOLINFECTDIS 1992;15:213-223

213

Characterization and Specificity Controls of Murine Monoclonal Antibodies Against Serogroup C1 Salmonella Raymond S.W. Tsang, Stojanka AleksiG Kwok H. Chan, Nelson W.H. Lau, and Mum H. Ng

Two IgG3 murine monoclonal antibodies, CI-1 and CI-2, that showed serologic specificities for the 0 antigens of serogroup C1 (0:6,7) Salmonella were established. The epitopes for the antibodies were found to reside on the repeating units of the serogroup C1 Salmonella lipopolysaccharide and were labile to sodium metaperiodate oxidation. Serologic reactivities of CI-1 and CI-2 were not inhibited by commercial monospecific antiserum to 0 antigen 7, but were inhibited to various degrees by anti-[O:6,7] serum. Both antibodies reacted strongly with all strains of serogroup C1 Salmonella that have either 0:61,7, 0:62,7, or 0:61,2,7 antigens. Reactivities of

Cl-1 and Cl-2 with the phage-14 lysogenized C1 strains that bear the phage-modified 0 antigen (0:6,7 --~ 0:6,7,14) were detected by slide agglutination method only and not by wholecell enzyme-linked immunosorbent assay. Both Cl-1 and C1-2 antibodies did not react with other 0 serogroups of salmonellae, nor with other Gram-negative or Gram-positive bacteria. The diagnostic value of these monoclonal antibodies together with a previously described monoclonal antibody against the serogroup C2 Salmonella was demonstrated using the slide agglutination method with monoclonal antibodies ascitic fluids.

INTRODUCTION

culture and identification of the isolate using biochemical and serologic methods (Ewing, 1986; Kauffmann, 1972). Serologic identification of Salmonella rests on the detection of specific O (cell-envelope lipopolysaccharide antigens) and H (flagellar proteins) antigens according to the Kauffmann-White scheme (Kauffmann, 1972; Le Minor and Popoff, 1987). O antigens are present in complex homo- or heteropolysaccharides, which constitute the repeating units of smooth lipopolysaccharide (LPS) of Gramnegative bacteria. Because of their complexity, many serologic specificities can be expected along the Opolysaccharide chain. The traditional method for raising antisera against Salmonella 0 antigens is by immunization of rabbits with Salmonella whole cells (Le Minor and Rohde, 1986). With the current knowledge that points to the numerous common antigens among the Enterobacteriaceae (Barber and Eylan, 1979), such polyclonal hyperimmune antisera against Salmonella naturally

Salmonellosis is a real or potential problem in all areas of the world. Although a large number of serotypes within the 46 O serogroups have been identified, most infections in humans are caused by organisms in the O serogroups of A (0:2) to E (0:3) (CDC, 1982; Gardner and Provine, 1987). A laboratory diagnosis of salmonellosis is confirmed by demonstrating the causative agent through From the Department of Microbiology(R.S.W.T., K.H.C., N.W.H.L., M.H.N.), Universityof Hong Kong, Hong Kong; and National Salmonella Center (S.A.), HygieneInstitute, Hamburg, Germany. Address reprint requests to Dr. R.S.W. Tsang, Department of Microbiology, Universityof Hong Kong, PathologyBuilding, Queen Mary Hospital Compound, PokfulamRoad, Hong Kong. Received 29 January 1991; revised and accepted 9 May 1991. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.O0

214

contain many cross-reacting antibodies. Although absorptions of the hyperimmune antisera with related bacteria have been used to improve the specificity of such polyclonal antisera, the quality of such absorbed antisera still depends on the antigenic characteristics of the bacteria used for absorption and the absorption techniques. Hence, serologic characterization of O antigens using conventional polyclonal monospecific antisera will leave many epitopes undefined. Also, serotyping of Salmonella based on polyclonal antisera suffers from the ill-defined nature of the antisera. Furthermore, batch-to-batch variations of different preparation of such antisera are difficult to control. With these facts in mind, we began to raise murine monoclonal antibodies (MAbs) against the O antigens of different serogroups of Salmonella that most commonly infect humans. In this report, we describe the characterization of two such MAbs that react specifically with O antigens of serogroup C1 Salmonella, and we demonstrate that these MAbs, together with a MAb against the serogroup Ca Salmonella (Luk et al., 1988), have practical applications in the serotyping of Salmonella isolates by using a simple slide agglutination technique.

MATERIALS A N D METHODS Bacterial Strains, Culture Conditions, and Antigens All Salmonella strains used were either from the strain collection of National Salmonella Center, Hygiene Institute, (Hamburg, Germany) or were clinical isolates obtained from the clinical microbiology laboratory of Queen Mary Hospital, University of Hong Kong. All other bacteria were clinical isolates from the Queen Mary Hospital. Bacteria were identified by standard methods (Lennette et al., 1985) and were grown in either brain-heart infusion broths, nutrient agar plates, or chocolate blood agar plates (5% horse blood in Columbia blood agar base) for the fastidious organisms. All media were prepared from dehydrated powder purchased from Oxoid (London) according to the manufacturer's instructions. LPS antigens were extracted by the hot phenolwater method of Westphal and Jann (1965) and were purified by repeated ultracentrifugation.

R.S.W. Tsang et al.

isolate from stool specimen) using a dosage of 2.5 x 108 cells in 0.5-ml volume. The immunization was repeated four times with a 5-day interval between each immunization. Three days after the fifth injection, which was given intravenously with the same dosage, the mice were bled and their sera tested against LPS from the homologous strain of S. choleraesuis. Immune spleen cells from the mouse that gave the highest antibody response were fused with the myeloma cells NS1 using polyethylene glycol (PEG, molecular weight = 4000; BDH, Poole, England) as the fusing agent (KOhler and Milstein, 1975). Screening of hybridomas for the production of specific antibodies against S. choleraesuis LPS was done by enzyme-linked immunosorbent assay (ELISA), and those that produced specific antibodies were cloned twice by limiting dilution. Purified hybridomas were injected into mineral-oil-primed Balb/c mice for production of ascitic fluids. Ascitic fluids containing murine MAbs against serogroup C2 Salmonella were produced and obtained from our previous study (Luk et al., 1988).

Serologic Methods The immunoglobulin class and subclass of the MAbs were determined by double immunodiffusion using antimouse immunoglobulin antisera (Nordic Immunological Laboratories, Tillburg, The Netherlands). Whole-cell ELISAwas done according to the method described by Adams et al (1988). ELISA to measure antibodies to LPS antigens, competitive ELISAto determine the relationship of MAbs to commercial polyclonal antisera against the serogroups C1 (0:6,7) and C2 (0:8) Salmonella 0 antigens (Wellcome Research Laboratories, Beckenham, England; and Behringwerke, Marburg, Germany), and SDS-PAGE immunoblotting were done according to our previously described methods (Luk et al., 1987). Indirect immunofluorescence to examine the cross-reactivity of antiserogroup CI (0:6,7) antibody with different organisms was also done by our previously described method (Tsang et al., 1989). Testing of agglutination capacity and of the quality and specificity of these sera was made according to the recommendations of Le Minor and Rohde (1982 and 1986).

Production of MAbs

Periodate Oxidation

For the production of MAbs against the serogroup C1 Salmonella, Balb/c mice were immunized intraperitoneally with boiled whole cells (for 2.5 hr at 100°C) of Salmonella choleraesuis 6,7:c:1,5 (a clinical

Sodium metaperiodate oxidation of S. choleraesuis LPS was done on antigens coated onto ELISA microtiter plate according to the method of Woodward et al. (1985). Residual serologic activity with

215

MAbs to Salmonella Antigen 0-6

the MAbs after periodate oxidation was measured by ELISA.

RESULTS Serologic Reactivities of Anti-Salmonella Serogroup-C1-Specific MAbs with Different Bacteria From one single-cell fusion, two hybridomas (C1-1 and C1-2) that produced IgG3 MAbs reactive with S. choleraesuis LPS were obtained. By whole-cell ELISA, both MAbs reacted with 10 of 11 strains (or 9 of 10 serotypes) of serogroup C1 Salmonella tested. The positive serotypes included S. virchow, S. cholerae-

suis, S. paratyphi C, S. montevideo, S. georgia, S. infantis, S. oranienburg, S. potsdam, and S. braenderup [mean ELISA optical density (O.D.) given by these nine serotypes against the C1-1 and C1-2 MAbs were 0.858 and 1.075, respectively]. The only negative strain found was S. ohio, which had the O antigens 6, 7, and 14 (mean ELISAO.D. given by this strain against C1-1 and C1-2 were 0.005 and 0.021, respectively). Among the 48 other Salmonella strains tested and found negative were eight strains of S. paratyphi A (serogroup A), ten strains of serogroup B (S. typhi-

murium, S. agona, S. derby, S. saintpaul, S. stanely, S. heidelberg, S. paratyphi B, S. schwarzengrund, and S. san-diego), ten strains of serogroup C2 (S. blockley, S. harda, S. hiduddify, S. manhattan, S. newport, S. kottbus, S. chailey, S. muenchen, S. litchfield, and S. herston), ten strains of serogroup D (S. typhi, S. enteritidis, S. sendai, S. panama, S. berta, and S. durban ), seven strains of serogroup E~ ( S. newlands, S. london, S. vejle, S. give, S. meleagridis, S. westhampton, and S. weltevreden), and two strains of serogroup E4 (S. krefeld and S. senftenberg). The negative strains all have ELISA O.D. < 0.050. Also negative were 20 species of non-Salmonella Enterobacteriaceae (Escherichia coli,

Shigella flexneri, S. sonnei, S. boydii, Klebsiella pneumoniae, K. oxytoca, Enterobacter aerogenes, E. cloacae, E. agglomerans, Proteus mirabilis, P. vulgaris, Providencia stuartii, P. alcalifaciens, P. rettgeri, Morganella morganella, Edwardsiella tarda, Citrobacter freundii, C. diversus, Serratia marcescens, and Yersinia enterocolitica), 20 species of other Gram-negative bacteria (Pasteruella multocida, Acinetobacter anitratus, A. lwoffii, AIcaligenes faecalis, Moraxella osloensis, Flavobacterium meningosepticum, Branhamella catarrhalis, Pseudomonas aeruginosa, P. putida, P. maltophilia, P. mendocina, P. acidovorans, P. fluorescens, Aeromonas hydrophila, A. sobria, A. caviae, Plesiomonas shigelloides, Vibrio cholerae Eltor, V. parahaemolyticus, and Haemophilus influenzae), and seven species of Gram-positive bacteria (Staphylococcus epidermidis, Bacillus species, diphtheroid bacillus, Streptococcus facealis, S. pyogenes, cx-hemolytic streptococci, and nonhemolytic streptococci).

FIGURE 1 Reactivity of SDS-PAGE separated and electrotransferred lipopolysaccharide antigens with the monoc|onal antibodies C1-1 and CI-2.

Immunochemical Characterization to Determine the Epitope Specificity of the MAbs Cl-1 and CI-2 By SDS-PAGE immunoblot, both MAbs Cl-1 and C12 reacted with LPS from S. montevideo (serogroup C1 with O antigens 6 and 7) to give a ladder-type of reaction pattern (Figure 1). Both antibodies did not react with LPS from S. paratyphi A (serogroup A), S. typhimurium (serogroup B), S. blockley (serogroup C2), S. typhi (serogroup D1), and S. newlands (serogroup El), thus confirming the whole-cell ELISA results. Furthermore, the reactivities of C1-1 and C1-2 with the S. montevideo LPS was abolished by Na metaperiodate treatment of the LPS antigens (Table 1), with the epitope for C1-1 being more susceptible to the oxidation reaction. Two sets of experiments were used to relate the serologic specificities of the MAbs C1-1 and C1-2 to the Salmonella 0 factors 6 and 7 characteristic of serogroup C1. One was an ELISA inhibition test using commercial anti-Salmonella O-factor-specific antisera. Another experiment examined the reactivities of the MAbs with microorganisms that share crossreacting antigens with the serogroup C1 (0:6,7) Salmonella, namely, Candida species, which have a mannose-rich cell wall that cross-reacts with the Salmonella 0 antigen 7. Commercial anti-[O:6,7], but not

216

R.S.W. Tsang et al.

TABLE 1 Effect of Na Metaperiodate Oxidation of the Salmonella montevideo Lipopolysaccharide on Its Reaction with the MAbs CI-1 and C1-2 Mean EL1SAO.D. a at 492 nm for MAbsb

Concentration (mM) of Metaperiodate Used

C1-1

0 1 2 5 10 20

0.613 0.070 (11)c 0.037 (6) 0.043 (7) 0.036 (5) 0.021 (3)

C1-2 0.919 0.334 0.184 0.164 0.111 0.073

No Ab Control

(36) (20) (18) (12) (8)

0.008 0.003 0.002 0.001 0.001 0.000

aELISAresults were mean of triplicate determinations. bMAbs CI-1 and C1-2were used at a concentrationof 1:5000. cpercentage of residual activitycompared with untreated controls.

the anti-[O:7], anti-[O:8], and anti-[Vi] antisera, inhibited the binding of C1-1 and C1-2 to the S. montevideo LPS antigens (Table 2). Also, the MAb CI-2 was found to be less affected by inhibition with the anti-[O:6,7] antiserum. MAbs CI-1 and C1-2 did not react with Candida albicans (seven strains), Candida krusei (one strain), Candida parasilopsis (two strains), and Candida tropicalis (two strains) by slide agglutination and immunofluorescence.

Specificity Controls of MAbs Against Serogroup C1 and C2 Salmonella Ascitic fluids of MAbs CI-1 and CI-2 both gave a slide agglutinating titer of 1:64 against the serogroup C1 Salmonella having O antigens 6 and 7. Using a 1:10 dilution of the ascitic fluids, 172 strains of salmonellae recovered from our clinical diagnostic bacteriology laboratory were tested (Table 3). Among the 36 strains of serogroup C1 (0:6,7) Salmonella tested, all were strongly and rapidly agglutinated by the serogroup-Cl-specific MAbs. None of the 136 strains of other serogroups of salmonellae tested was found to be agglutinated by the MAbs. Ascitic fluids of MAbs CI-1, C1-2, and MO8 (specific for serogroup C1 Salmonella; Luk et al., 1988) were then tested against 48 reference strains of serogroups C1 and C2 Salmonella together with 20 strains of other related Salmonella 0 serogroups in the National Reference Center for Salmonella in Germany, and the results are presented in Table 4. MAbs CI1 and CI-2 agglutinated with all the serogroup C1 Salmonella tested regardless of their O antigen 6 composition, that is, O:61, 0:62, or O:61,2. Presence of O antigen 14 in the group C1 strains also did not appear to interfere with their agglutination by the group C1 specific MAbs, although such strains did not agglu-

tinate so strongly in the MAbs. Furthermore, strains of group C1 Salmonella belonging to the subspecies I, II, III, IV, and VI were also positive, as well as strains of 0:6,14,18 belonging to the O serogroup K (O:18). MAb MO8 agglutinated with all group C2 Salmonella except for the previously designated group C3 strains, which lack the O antigen 6. More important was the observation that the group-C1- and C2-specific MAbs agglutinated with strains of their homologous group only, with no cross reactions between the C1 Salmonella and MO8 MAbs or between the C2 Salmonella and CI-1 and C1-2 MAbs; that is, they could be used right after preparation without prior absorption as specific factor sera for serotyping of Salmonella.

DISCUSSION The MAbs C1-1 and C1-2 recognized easily accessible epitopes on the surface of serogroup C1 (0:6,7) Salmonella since both antibodies reacted with these Salmonella by whole-cell ELISA and by slide agglutination test (Tables 3 and 4). The reactive determinants for these two MAbs were found to be present on the O side chains of the LPS molecules (Figure 1). The structure of the O antigen of serogroup Cl Salmonella has been determined only recently (Lindberg et al., 1988) and is found to have an N-acetyl-glucosamine residue linked to four linear mannose residues, one of which may carry a terminal glucose substitution. O antigen 7 is believed to involve the linear mannose backbone of the O-antigenic side chain since there is reciprocal cross reaction between serogroup C1 Salmonella and the yeast Candida albicans (Lindberg and Le Minor, 1984). O antigen 6 is believed to involve the terminal glucose substituted

MAbs to Salmonella A n t i g e n 0 - 6

TABLE 2

217

Inhibition of the Binding of the M A b s C1-1 a n d C1-2 to the Salmonella montevideo L i p o p o l y s a c c h a r i d e (LPS) b y C o m m e r c i a l Antisera to the Salmonella 0 antigens: [6,7], [7], [8], a n d Vi Antigen Mean a ELISAO.D. at 492 nm for MAbs b CI-1 C1-2

Commercial Antisera for Inhibition c

No Antigen Control

LPS

No inhibitor

0.017

Anti-0:6, 7 1:10 1:100 1:500 1:1000 Anti-0:7 1:10 1:100 1:500 1:1000 Anti-0:8 1:10 1:100 1:500 1:1000 Anti-Vi 1:10 1:100

% Inhibition

No Antigen Control

LPS

% Inhibition

0.461

NA

0.019

0.790

NA

0.014 0.016 0.013 0.016

0.086 0.193 0.322 0.329

81 58 30 29

0.017 0.017 0.025 0.021

0.435 0.635 0.744 0.771

45 20 6 2

0.022 0.026 0.040 0.016

0.452 0.410 0.495 0.427

2 11 0 7

0.021 0.020 0.020 0.040

0.797 0.790 0.804 0.787

0 0 0 0

0.019 0.019 0.015 0.043

0.495 0.447 0.533 0.420

0 3 0 9

0.022 0.048 0.016 0.042

0.796 0.769 0.791 0.766

0 3 0 3

0.021 0.027

0.543 0.465

0 0

0.017 0.176

0.821 0.764

0 3

aEuSA results were mean of triplicate determinations. bMAbs CI-1 and C1-2 were used at a dilution of 1:10,000. ~Titers of commercial antisera were: anti-0:6,7 = 1:160; anti-0:8 = 1:160; anti-Vi 1:160; and anti0:7 = unknown. Anti-[0:6,7], anti-[O:8], and anti-[Vi] sera were from the Wellcome Research Laboratories, UK; anti-[0:7] serum was from Behringwerke AG, Germany. NA, not applicable.

onto the third m a n n o s e residue f o u n d in the m i d d l e of the p e n t a s a c c h a r i d e b a c k b o n e structure of the g r o u p C 1 0 antigen (Lindberg a n d Le Minor, 1984). To d e t e r m i n e w h i c h antigenic d e t e r m i n a n t s of the s e r o g r o u p C1 Salmonella LPS (for e x a m p l e , O antigen 6, 7, or others) are r e s p o n s i b l e for the reaction w i t h the MAbs C1-1 a n d C1-2, cross reactions of the M A b s with different Candida species w e r e sought. N e i t h e r M A b s reacted w i t h a n y of the s e v e n strains of C. albicans tested n o r did t h e y react w i t h a n y of the five strains of C. tropicalis, C. krusei, a n d C. parasilopsis. In a g r e e m e n t w i t h this finding w a s the lack of inhibition of b i n d i n g of M A b s C1-1 a n d CI-2 to the S. montevideo LPS b y factor-specific a n t i s e r u m to O antigen 7 (Table 2). In contrast, an a n t i s e r u m containing antibodies to b o t h O antigens 6 a n d 7 inhibited the b i n d i n g of b o t h C1-1 a n d C1-2 to the S. montevideo LPS, with C1-1 b e i n g m o r e sensitive to the inhibition t h a n C1-2. This indicated that b o t h M A b s C1-1 a n d C1-2 w e r e reacting w i t h at least part of the c o n v e n -

tional O antigen 6 as r e c o g n i z e d b y polyclonal antisera. This c o n t e n t i o n is further s u b s t a n t i a t e d b y the finding that b o t h the C1-1 a n d C1-2 e p i t o p e s w e r e sensitive to s o d i u m m e t a p e r i o d a t e oxidation. The slight difference b e t w e e n the t w o M A b s , C1-1 a n d CI-2, in t e r m s of their sensitivity to inhibition b y the anti-[O:6,7] s e r u m w a s also reflected in their e p i t o p e sensitive to periodate oxidation. The epitope for MAb C1-1 w a s m o r e sensitive to inhibition b y the anti[0:6,7] s e r u m a n d w a s m o r e sensitive to p e r i o d a t e oxidation. This m a y indicate that the terminal glucose et-linked to the m a n n o s e in the O polysaccharide of s e r o g r o u p C1 Salmonella is likely i n v o l v e d in the e p i t o p e r e c o g n i z e d b y M A b C1-1. The e p i t o p e for M A b CI-2 w a s not c o m p l e t e l y inhibited b y the anti-[O:6,7] s e r u m a n d w a s m u c h less sensitive to destruction b y p e r i o d a t e oxidation. This m a y indicate that the e p i t o p e for M A b C1-2 m a y involve the backbone polysaccharide structure m o r e than the sideb r a n c h terminal glucose residue.

TABLE 3

S e r o t y p i n g of Salmonellae by Slide Agglutination Test with the C11 a n d C1-2 Ascitic Fluids in the Diagnostic Bacteriology Laboratory of Q u e e n Mary Hospital, H o n g K o n g ~

Serogroups B

Serotypes S. S. S. S. S. S.

Number Tested (total)

Number Positive

17 1 13 2 4 1

0 0 0 0 0 0

38

0

2 1 20 3 1 1 1 1 1 1 1 1 2

2 1 20 3 1 1 1 1 1 1 1 1 2

36

36

33 7 1 10 4 10 3 1 1 1 1

0 0 0 0 0 0 0 0 0 0 0

72

0

2 1 2 6 0

0 0 0 0 0

11

0

8 3 0 2

0 0 0 0

13

0

2

0

2

0

172

36

typhimurium paratyphi B derby agona saint-paul bradford

Total C1

S. S. S. S. S. S. S. S. S. S. S. S. S.

braenderup thompson infantis virchow choleraesuis paratyphi C montevideo georgia oranienburg potsdam mbandaka ohio lille

Total S. S. S. S. S. S. S. S. S. S. S.

C2

manhattan litchfield bovis-morbificans newport chailey blockley hadar hiduddify kottbus muenchen herston

Total S. panama S. durban S. typhi S. enteritidis Untyped group D organisms

D

Total S. S. S. S.

E1

anatum weltevreden muenster meleagridis

Total S. krefeld

E4

Total AtoE

aCl-1 and C1-2 ascitic fluids were used at a dilution of 1:10.

M A b s to Salmonella A n t i g e n 0 - 6

219

L y s o g e n i z a t i o n of s e r o g r o u p C1 Salmonella b y p h a g e 14 r e s u l t s in c h e m i c a l m o d i f i c a t i o n of t h e O - p o l y s a c c h a r i d e s t r u c t u r e ( F u l l e r a n d S t a u b , 1968) a n d t h e a p p e a r a n c e of t h e O a n t i g e n 14. T h e t e r m i n a l g l u c o s e s u b s t i t u t i o n of t h e t h i r d m a n n o s e r e s i d u e is n o w c h a n g e d to i n v o l v e t h e f o u r t h m a n n o s e i n s t e a d . This m i n o r m o d i f i c a t i o n of t h e O - p o l y s a c c h a r i d e s t r u c t u r e is m o s t p r o b a b l y r e s p o n s i b l e for t h e dim i n i s h e d r e a c t i v i t i e s w i t h M A b s CI-1 a n d C1-2. T h u s f o u r s t r a i n s of p h a g e - 1 4 - m o d i f i e d C1 Salmonella ( t w o S. ohio 6 , 7 , 1 4 : b : 1 , w a n d t w o S. lille 6,7,14:z38:-) d i d n o t s h o w r e a c t i v i t i e s w i t h t h e M A b s C1-1 a n d CI-2

TABLE

b y w h o l e cell ELISA. A l s o w i t h t h e p h a g e - 1 4 - m o d i f i e d C1 Salmonella s t r a i n s , t h e i r a g g l u t i n a t i o n i n t h e M A b s w e r e n o t a s s t r o n g as o t h e r C1 Salmonella s t r a i n s w i t h o u t t h e p r e s e n c e of p h a g e 14. T h i s w a s e v i d e n t w h e n d i f f e r e n t d i l u t i o n s of t h e M A b s ascitic f l u i d s w e r e u s e d for a g g l u t i n a t i o n w i t h C1 Salmonella strains w i t h o r w i t h o u t O a n t i g e n 14. S t r a i n s of C1 Salmonella w i t h o u t a n t i g e n O:14 w e r e a g g l u t i n a t e d b y t h e M A b s at d i l u t i o n s of at l e a s t 1:20 to 1:30, w h e r e a s s t r a i n s with phage-14 modification were only agglutinated w i t h l o w e r d i l u t i o n s of M A b s ascitic f l u i d s ( t h a t is, 1:10-1:20).

S e r o t y p i n g of G r o u p s C1 a n d C2 a n d O t h e r R e l a t e d O S e r o g r o u p s of Salmonella b y M o n o c l o n a l A n t i b o d i e s C1-1/CI-2 ( S p e c i f c for G r o u p C1) a n d M O 8 (Specific for G r o u p C2) in t h e N a t i o n a l R e f e r e n c e C e n t e r for Salmonella, G e r m a n y

4

Agglutinating Results with Different Dilutions of MAbsa C1-1 C1-2 MO8 Test Strains

1:10

1:20

1:30

1:10

1:20

1:30

+ + + +

+ + + +

+ + +

+ + + +

+ + + +

+ + (+)

+ +

+ +

+

+ +

+ +

(+)

+ +

+ +

+ +

+ +

+

+

+ +

+ +

+ +

+ +

+ +

+

+ +

+ +

+ +

+ +

+ +

+

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

+

+ +

+ +

+ +

+ +

+ +

+

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

+ + + + + +

+ + + + + +

+ + + + +

+ + + + + +

+ + + + + +

+ (+) +

++

+

-

++

(+)

++

++

--

++

+

1:10

S. choleraesuis 61,7:-:1,5 62,7:-:1,5

S. lomita 62,7:e,h:1,5

S. thompson 61,2,7:k:1,5

S. montevideo 6~,2,7:g,m,s:-

S. oranienburg 61,2,7:m,t:-

S. bareilly 61,2,7:y:1,5

S. infantis 6,12,7:r:1,5

S. virchow 61,2,7:r:1,2

S. oslo 6~,2,7:a:e,n,x

S. paratyphi C 61,2,7,Vi:c:1,5

S. georgia 6L2,7:b:e,n,z15

S. potsdam 61,2,7:1,v:z6

S. braenderup 61,2,7:e,h:e,n,z15 S. II 61,2,7:b:z39 S. II 61,2,7:g,m,s,t:Group C1

O:14+

S. ohio 6,7,14:b:1,w

S. eimsbuttel 6,7,14:d:1,w

m

w

220

R.S.W. T s a n g et al.

TABLE 4

Continued Agglutinating Results with Different Dilutions of MAbs a C1-1 C1-2 MO8

Test Strains

1:10

S. livingstone 6,7,14:d:1,w S. nieukerk 6,7,14:d:z6 S. montevideo 6,7,14:g,m,s:S. oranienburg 6,7,14:m,t:S. thompson 6,7,14:k:1,5 S. infantis 6,7,14:r:1,5 S. lille 6,7,14:z38:S. II 6,7,14:g,m,s,t:Arizona 0:6,7 S. IIIb 6,7:l,v:zs3 S. IV

1:20

1:30

1:10

1:20

++

+

-

++

+

++

+

-

++

(+)

++

+

-

++

+

++

+

--

++

+

++

+

--

++

+

++

+

--

++

+

++

+

--

++

(+)

++

++

--

+ +

+

++

+

--

++

+

6,7:z4,z23:-

++

+

(+)

++

+

S. VI 6,7:z41:1,7

++

+

(+)

++

+

++

+

(+)

++

(+)

++

+

(+)

++

(+)

++

+

(+)

++

(+)

1:30

1:10

Group O:18

S. rawash 6,14,18:c:e,n,x S. usumbura 6,I4,18:d:1,7 S. cerro 6,14,18:z4,z23:-

Cl-1

CI-2

1:10

1:10

MO8 1:10

1:20

Group O:H

S. carrau 6,14,24:y:1,7 S. bahrenfeld 6,14,24:e,h,:1,5 S. albuquerque 1,6,14,24:d:z6 S. onderstepoort 1,6,14,24:e,h:1,5 S. boecker 1,6,14,25:1,v:1,7 S. fischerkietz 1,6,14,25:y:e,n,x S. uzaramo 1, 6,14,25:z4,z23:S. IIlb 6,14:b:e,n,x

Not done Not done Not done Not done Not done Not done Not done Not done

1:30

M A b s t o Salmonella A n t i g e n

TABLE 4

221

0-6

Continued A g g l u t i n a t i n g Results with Different Dilutions of M A b s a C1-1 CI-2 MO8 1:10

1:10

1:10

1:20

1:30

S. IV

6,14:z4,z23:-

Not done

S. II 1,6,14:Zlo:1,5

Not done

Group 0:8 (=

C2)

S. muenchen 6,8:d:1,2

++

++

(+

++

+

-

++

+

-

++

+

(+

++

+

-

++

+

-

++

+

-

++

+

(+

++

+

-

++

+

(+

++

+

(+

++

+

-

++

+

(+)

++

+

(+)

++

+

(+)

S. manhattan 6,8:d:1,5

S. newport 6,8:e,h:l,2 S. II 6,8:m,t:1,5 S. blockley 6,8:k:1,5

S. Iitchfield 6,8:1,v:1,2

S. manchester 6,8:1,v:1,7

S. bovismorbificans 6,8:r:1,5

S. goldcoast 6,8:r:1,w

S. hadar 6,8:zl0:e,n,x

S. hiduddify 6,8:1,z13,z2s:1,5

S. kottbus 6,8:e,h:1,5

S. chailey 6,8:z4,z23:eln,zi5

S. herston 6,8:d:e,n,z15 S. II 6,8:z29:e,n,x

S. haardt 8:k:1,5

Not done

S. kentucky 8,20:i:z6

Not done

S. amherstiana 8:1,v:1,6

Not done

S. emek 8,20:g,m,s:-

Not done

S. enteritidis 9,12:g,m: -

Not done

S. senftenberg 1,3,19:g,s,t: -

Not done

S. aberdeen 11:i:1,2

Not done

S. london 3,10:1,v:1,6

Not done

S. poona 1,13,22:z:1,6

Not done

222

TABLE 4

R.S.W. Tsang et al.

Continued Agglutinating Results with Different Dilutions of MAbsa C1-1 C1-2 MO8 1:10

1:10

1:10

1:20

1:30

S. waycross 41:Z4,Z23: .

.

.

Not done

.

S. uccle

54:g,s,t: .

.

.

.

Not done

"+ +, quick and strong positive reaction; +, positive agglutination; (+), late positive agglutination; and - , negative agglutination.

The discrepancy between whole-cell ELISAand slide agglutination results observed in the phage-14-modified C2 Salmonella strains was not surprising because whole-cell E L I S A procedures involved multiple washing steps and can hence detect only strong antigenantibody reactions, whereas slide agglutination is known to favor the detection of low-affinity antigenantibody reactions. Infection by phage 14 in the C1 Salmonella leads to slight modification of the O-polysaccharide chains, which probably leads to a lesser best fit of the CI-1 and C1-2 MAbs with the phage14-modified LPS structures, which can still be detected by slide agglutination but not by the EUSA method. The diminished reactivities of the MAbs C11 and C1-2 with the phage-modified serogroup C1 strains also suggested that the epitopes for MAbs C1-1 and CI-2 may involve further residue(s) other than the glucose --+ mannose disaccharide. Indeed, our studies with other anti-Salmonella serogroupspecific MAbs have also provided evidence that the antigen-combining sites of mouse MAbs bind to polysaccharide antigens with epitope sizes of 3-4 sugar residues (authors' unpublished observations). The exquisite specificity of MAbs CI-1 and C1-2 was reflected in their lack of reactivity with 47 species of other bacteria and 136 strains of nonserogroup C1 Salmonella tested. Ascitic fluids of these MAbs, together with our previously described MAb against the serogroup C2 Salmonella, have separately been demonstrated (Luk et al., 1988; this study) in a routine hospital diagnostic bacteriology laboratory in Hong Kong to have strong agglutinating power, and hence are good serotyping reagents against their homologous group of Salmonella. In the present study, we have further extended this observation by per-

forming serotyping studies of different serotypes of group C1 and C2 Salmonella using ascitic fluids of these MAbs in a Salmonella Reference Laboratory in Germany. Such findings confirmed that these MAbs are useful serotyping reagents for the identification of Salmonella. The results have also revealed that MAbs C1-1 and C1-2 are specific for factor 0-6 of serogroup C~ (0:6,7 and O:6,7,14 of Salmonella subspecies I, II, III, IV, and VI in the Kauffmann-White scheme; Le Minor and Popoff, 1987), which in detail may possess the factors 61, 61,2, and 62, according to Le Minor and Rohde (1982). However, factor 0-6 of serogroup C2 (6,8), as well as factor 0-6 of serogroup H (6,14; 6,14,24; and 1,6,14,25), which differ from factor 0-6 of serogroup C1, do not react with the MAbs. In contrast, ascitic fluids of MAb MO8 showed strong agglutination with serogroup C2 (0:6,8) Salmonella only and did not react with Salmonella of the previously designated serogroup C3, which may have antigens 0:8 or 0:8,20. Cross reactions of MAb MO8 with the serogroup C1 Salmonella was also not observed. In summary, we have produced and characterized extensively two murine MAbs specific for serogroup C1 Salmonella and provided evidence that these MAbs together with MAb against serogroup C2 Salmonella are group-specific and useful diagnostic reagents for the identification of these two common O serogroups of Salmonella.

This work was supported by a strategic research grant from the University of Hong Kong, and a grant from the Royal Hong Kong Jockey Club.

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