JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 1899-1903
Vol. 29, No. 9
0095-1137/91/091899-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Characterization of Murine Monoclonal Antibodies against Serogroup B Salmonellae and Application as Serotyping Reagents RAYMOND S. W. TSANG,* K. H. CHAN, NELSON W. H. LAU, DEBBY K. W. CHOI, DENNIS K. S. LAW, AND M. H. NG Department of Microbiology, University of Hong Kong, Pathology Building,
Queen Mary Hospital Compound, Hong Kong Received 29 October 1990/Accepted 6 June 1991
Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other 0 serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.
Salmonellae are well-known bacterial pathogens that cause food poisoning (6). In a summary of food-borne disease outbreaks in the United States from 1983 to 1987, salmonellae were found to be responsible for more than 50% of all the identifiable bacterial causes (2). Figures reported from 1984 to 1986 have shown that 40 to 60% of the salmonellae isolated in the United States from humans belonged to serogroup B, and S. typhimurium is the single most commonly isolated salmonella (7). S. typhimurium, S. heidelberg, S. agona, S. saint-paul, and S. derby (all serogroup B) were among the 20 most commonly isolated serotypes reported in the United States during this period. In England and Wales, S. typhimurium was also the most commonly isolated salmonella from 1941 to 1987, with more than 30% of all isolates identified as this serotype (16). Similarly, S. typhimurium together with other serotypes of serogroup B salmonellae accounted for nearly half (4 of 10) to more than half (7 of 10) of the 10 most common serotypes that caused Salmonella food poisoning in England and Wales during a 13-year period, from 1966 to 1978 (6). Also, S of the 12 recently well-documented significant outbreaks of foodborne salmonellosis were due to group B organisms (20). These reports signify the importance and the prevalence of the serogroup B organisms, which together with organisms in the 0 serogroups A, C, D, and E are responsible for >95% of Salmonella infections in humans (3, 5). In this report, we describe the production and characterization of murine monoclonal antibodies (MAbs) reactive with and specific for serogroup B salmonellae. We also evaluate the use of these murine MAbs for the serotyping of salmonellae by the slide agglutination method in a routine diagnostic bacteriology laboratory.
*
MATERIALS AND METHODS Bacterial strains, culture conditions, and antigens. Bacterial strains used in this study either were purchased from the American Type Culture Collection, Rockville, Md., or were clinical isolates from our clinical bacteriology laboratory at Queen Mary Hospital, Hong Kong (see Table 2). Bacteria obtained from our clinical diagnostic bacteriology laboratory were identified by standard methods (13). Bacteria for extraction of lipopolysaccharide (LPS) were grown in brain heart infusion broth (Oxoid Ltd., London, England) under static incubation conditions at 37°C for 16 to 18 h. Bacteria for colony dot blot enzyme immunoassay and slide agglutination tests were grown on nutrient agar (Oxoid Ltd.) plates or chocolate blood agar (5% horse blood in Columbia blood agar base [Oxoid Ltd.]) plates when growth demanded more-enriched media. All agar plates were incubated at 37°C for 16 to 18 h with 5% carbon dioxide when required. Purified LPS antigens from S. typhimurium and S. typhi were purchased from Sigma Chemical Company (St. Louis, Mo.). LPS from S. paratyphi A, S. montevideo, S. blockley, and S. newlands was extracted from formalin-inactivated and washed bacterial cells by the hot phenol-water method of Westphal and Jann (19) and was purified by repeated
ultracentrifugation. Production of MAbs. BALB/c mice were immunized intraperitoneally with boiled (2.5 h at 100°C) whole cells of S. typhimurium at a dosage of 2.5 x 108 cells in a 0.5-ml volume. The immunization was repeated four times, with an interval of 5 days between immunizations. An intravenous booster injection of the same number of cells was given 1 week after the last intraperitoneal injection. Three days after the intravenous injection, the mice were bled from their orbital veins and their sera were tested against LPS antigens from S. typhimurium. Immune spleen cells from the mouse that had the highest antibody response were fused with NS1 myeloma cells by using polyethylene glycol (molecular
Corresponding author. 1899
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J. CLIN. MICROBIOL.
TSANG ET AL.
TABLE 1. Reactivities of anti-Salmonella serogroup B MAbs with purified Salmonella LPS from 0 serogroups A to E Mean ELISA OD at 492 nm for MAbsa: Serotype (serogroup)
None
None (antigen control) S. paratyphi A (A) S. typhimurium (B) S. montevideo(C1) S. blockley (C2) S. typhi (D1) S. newlands (E1)
(control) 0.021 0.020 0.021 0.019 0.015 0.010 0.005
B1-1
M04-2
M04-3
M044
M04-5
M04-8
0.015 0.036 0.754 0.007 0.007 0.006 0.005
0.031 0.046 0.855 0.021 0.006 0.017 0.018
0.033
0.010
0.016
0.021
1.114 0.845 0.018 0.018
0.007 0.690 0.006 0.001
0.008 0.777 0.005 0.000
0.178 0.805 0.010 0.015
0.022 0.012
0.005 0.002
0.006 0.004
0.020 0.008
a The titers of the MAbs were determined by ELISA and defined as the dilutions that yielded OD values of about 0.100 and at least two times the background control OD values. The MAbs were used at the following dilutions: B1-1 (titer = 1:500,000), 1:5,000; M04-2 (titer = 1:500,000), 1:10,000; M04-3, M04-4, and M04-5 (titer = >1:1,000,000), 1:100,000; and M04-8 (titer = 1:50,000), 1:5,000.
weight, 4,000; BDH, Poole, England) as the fusing agent (11). Hybridomas were screened for the production of specific antibodies against S. typhimurium LPS antigens by enzyme-linked immunosorbent assay (ELISA), and hybridomas that produced specific antibodies were cloned twice by the limiting-dilution method. Purified hybridomas were injected intraperitoneally into mineral oil-primed mice for the production of ascitic fluids. Serological methods. The immunoglobulin classes and subclasses of the MAbs were determined by double immunodiffusion with anti-mouse immunoglobulin antisera purchased from Nordic Immunological Laboratories (Tillburg, The Netherlands). The ELISA (4) to measure antibodies to LPS antigens was done as described previously (14). The whole-cell ELISA was done as described by Adams et al. (1). The colony dot blot enzyme immunoassay was done by spotting boiled (100°C, 30 min) whole bacterial cells onto nitrocellulose paper (0.45-,um pore diameter; Schleicher & Schuell, Dassel, Germany) and subsequently processing according to the instruction manual of the Bio-Rad Immuno-blot assay kit (Bio-Rad Laboratories, Richmond, Calif.). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Salmonella LPS and its visualization by silver staining (18) or immunoblotting with the MAbs were done as described previously (14). Serotyping of the salmonellae with the MAbs was done by standard procedures (10). Sodium meta-periodate oxidation of S. typhimurium LPS was done on antigens coated onto an ELISA microtiter plate by the method of Woodward et al. (21), and the residual serological activity of the LPS antigens after periodate oxidation was measured by ELISA against the MAbs. RESULTS Production of murine MAbs against serogroup B salmonellae. From two separate fusion experiments with spleen cells from two mice immunized with boiled whole cells of S. typhimurium, six hybridomas that produced high levels of antibodies reactive with S. typhimurium LPS antigens were obtained. Cells from these six hybridoma lines have been deposited at the European Collection of Animal Cell Cultures of Public Health Laboratory Service Centre for Applied Microbiology and Research, Salisbury, United Kingdom, and have been given the accession numbers 91031425 to 91031430. Three antibodies were of the immunoglobulin G3 isotype (MO4-2, M04-3, and M04-8), two were immunoglobulin Gl antibodies (MO4-4 and M04-5), and one belonged to the immunoglobulin M class (B1-1). Ascitic
TABLE 2. Salmonella serotypes and standard strains of other bacterial species that tested negative with MAbs M04-2, M04-4, and M04-5 Strain used
Serogroup C1 S. virchow S. lille S. choleraesuis S. paratyphi C S. montevideo S. georgia S. infantis S. mbandaka S. ohio S. oranienburg S. potsdam S. braenderup S. thompson S. singapore S. isangi
Serogroup C2 S. S. S. S. S. S. S. S. S. S. S.
hadar
hiduddify manhattan newport kottbus
blockley chailey muenchen
litchfield herston bardo
Serogroup DI S. typhi S. dublin S. enteritidis S. S. S. S.
panama sendai berta durban
Serogroup E1 S. S. S. S. S.
anatum
london newlands
meleagridis weltevreden
Serogroup E4 S. krefeld S. senftenberg
Strain used
Standard strains S. paratyphi A ATCC 9150 S. choleraesuis ATCC 13312 S. newport ATCC 6962 S. typhi Ty2 ATCC 19430 S. enteritidis ATCC 13076 Escherichia coli ATCC 35218 Shigella flexneri ATCC 29903 Shigella sonnei ATCC 25931 Klebsiella pneumoniae ATCC 13883 Enterobacter agglomerans ATCC 27155 Enterobacter cloacae ATCC 23355 Proteus mirabilis ATCC 7002 Providencia rettgeri ATCC 29944 Morganella morganii ATCC 25803 Citrobacterfreundii ATCC 8090 Serratia marcescens ATCC 8100 Edwardsiella tarda ATCC 15947 Yersinia enterocolitica ATCC 9610 Pseudomonas aeruginosa ATCC 27853 Acinetobacter anitratus ATCC 19606 Acinetobacter lwoffii ATCC 15309 Flavobacterium meningosepticum ATCC 13253 Alcaligenes faecalis ATCC 8750 Moraxella lacunata ATCC 17967 Aeromonas hydrophila ATCC 7966 Vibrio parahaemolyticus ATCC 66740 Haemophilus influenzae ATCC 14035 Staphylococcus epidermidis ATCC 51825 Streptococcus pyogenes ATCC 19615 Streptococcus pneumoniae ATCC 27336
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SALMONELLA SEROGROUP B-SPECIFIC MAbs
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TABLE 3. Susceptibility of S. typhimurium LPS epitopes to sodium meta-periodate oxidation measured by ELISA
with anti-Salmonella serogroup B-specific MAbs Mean ELISA OD (% residual activity) for MAba:
Na meta-periodate concn (mM)
None
0 1 2 5 10 20 50 100
(control) 0.014 0.013 0.020 0.017 0.016 0.011 0.008 0.013
B1-1 1.054 1.006 0.951 0.909 0.899 0.776 0.804 0.886
(95.4) (90.2) (86.2) (85.3) (73.6) (76.3) (84.1)
M04-2 1.029 0.725 0.486 0.444 0.148 0.056 0.064 0.061
(70.5) (47.2) (43.2) (14.4) (5.4) (6.2) (5.9)
M04-3 0.832 0.779 (93.6) 0.711 (85.5) 0.565 (67.9) 0.311 (37.4) 0.181 (21.8) 0.120 (14.4) 0.156 (18.8)
M04-4 0.981 0.291 0.165 0.125 0.060 0.012 0.004 0.011
(29.7) (16.8) (12.7) (6.1) (1.2) (0.4) (1.1)
M04-5 1.050 0.390 (37.1) 0.243 (23.1) 0.145 (13.8) 0.070 (6.7) 0.007 (0.7) 0.003 (0.3) 0.010 (1.0)
M04-8 1.088 1.054 1.012 0.808 0.514 0.134 0.027 0.085
(96.9) (93.0) (74.3) (47.2) (12.3) (2.5) (7.8)
a Residual serological activity of the S. typhimurium LPS epitopes after sodium meta-periodate treatment is expressed as a percentage of original activity without treatment. The MAbs were used at the following dilutions: B1-1, M04-2, and M04-8, 1:5,000; M04-4 and M04-5, 1:50,000; and M04-3, from tissue culture fluid, was used undiluted.
fluids produced from these six hybridoma cell lines gave high titers of antibodies to the S. typhimurium LPS antigens in an ELISA. The titers ranged from 1:50,000 to >1:1,000,000, and the highest titers were given by the M04-3, M04-4, and M04-5 hybridoma MAbs (Table 1). Immunochemical characterization of MAbs. In a preliminary study, the six MAbs were allowed to react with purified LPS antigens from Salmonella serotypes that represented the 0 serogroups A, B, C1, C2, D1, and E1 (Table 1). Four antibodies (MO4-2, M04-4, M04-5, and B1-1) were specific for LPS antigens from S. typhimurium, while the remaining two antibodies (MO4-3 and M04-8) were found to crossreact with S. paratyphi A LPS. To confirm the serogroup specificity of the MAbs, a whole-cell ELISA with Salmonella serotypes of 0 serogroups A to E was done. Results from the whole-cell ELISA experiments confirmed the ELISA results obtained by using purified LPS antigens. MAbs M04-3 and M04-8 were found to react with whole cells of S. paratyphi A (12 strains were tested, and mean ELISA optical density [OD] values of 0.659 and 0.394 were obtained with M04-3 and M04-8, respectively). These two antibodies also reacted with all 10 strains of serogroup B salmonellae, including S. typhimurium, S. agona, S. derby, S. saint-paul, S. stanley, S. heidelberg, S. paratyphi B, S. schwarzengrund, and S. sandiego (mean ELISA OD values were 1.270 and 1.264 for MAbs M04-3 and M04-8, respectively). The remaining four MAbs, M04-2, M04-4, M04-5, and B1-1, were specific for serogroup B salmonellae, and they reacted with all nine serotypes of serogroup B Salmonella, with mean ELISA OD values of 1.057, 0.815, 0.948, and 0.516, respectively. None of the six antibodies reacted with any of the Salmonella serotypes from serogroups C1, C2, D1, E1, and E4 (Table 2). To ascertain the specificities of MAbs M04-2, M04-4, and M04-5, 30 standard strains from the American Type Culture Collection were tested against the MAbs by a colony dot blot enzyme immunoassay. All three serogroup B-specific MAbs reacted with S. typhimurium ATCC 14028 but did not react with the non-group B Salmonella serotypes or with any of the other standard bacterial strains listed in Table 2. The antigens that reacted with the MAbs were studied by SDS-PAGE immunoblot. S. typhimurium LPS antigens were separated by SDS-PAGE and electrotransferred to a nitrocellulose paper, and the separated and transferred LPS components were reacted with the MAbs. All six MAbs reacted with the 0 antigens of the S. typhimurium LPSs, since each gave a ladder-type reactive pattern consistent
with the 0-antigen ladder pattern observed by silver staining. The somatic 0 antigens of serogroup B salmonellae may consist of 0 factors 1, 4, 5, 12, and 27. 0 antigens 4 and 12 are regarded as the basic 0 factors present in all serogroup B salmonellae. 0 antigen 4 is the specific somatic antigen for all serogroup B salmonellae and hence is diagnostic for this O serogroup of organisms. 0 antigen 12 can be found in a number of 0 serogroups, such as A, B, and D, and is a minor 0 factor since its presence is not diagnostic for any particular 0 serogroup. Other 0 factors, 1, 5, and 27, that may be present in serogroup B salmonellae are a result of chemical modification of the basic 0 antigen. For example, 0 antigens 1 and 27 result from modification by the presence of lysogenic phages which add into the basic structures additional specificities. 0 antigen 5 results from 0 acetylation of 0-antigen 4 and is therefore present only in strains of serogroup B Salmonella. To find out whether the MAbs were reacting with 0 antigen 5, strains of S. derby without 0 antigen 5 were selected for either direct reaction with the MAbs (slide agglutination with ascitic fluids) or specific removal of the MAbs by absorption studies followed by an ELISA with S. typhimurium LPS antigens. All six MAbs strongly agglutinated with four strains of S. derby without 0 antigen 5. Similarly, strains of S. derby without 0 antigen 5 removed all six MAbs as effectively as a strain of S. typhimurium with 0 antigen 5. Both studies therefore indicated that the MAbs were reacting with an antigen unrelated to the conventional 0 antigen 5. The sensitivities of the different MAb epitopes to sodium meta-periodate oxidation were studied by testing treated and untreated S. typhimurium LPS with the MAbs by ELISA. The results presented in Table 3 show that two serogroup B-specific MAbs (MO4-4 and M04-5) were highly sensitive to meta-periodate oxidation whereas the serogroup A crossreactive MAbs (MO4-3 and M04-8) were relatively resistant to this treatment. For the serogroup B-specific MAbs M04-4 and M04-5, as little as 1 mM periodate was enough to reduce the reactivity of S. typhimurium LPS with the MAbs by >50%. The epitope for MAb M04-2 was also very labile to the meta-periodate treatment, since a 50% decrease in reactivity of the S. typhimurium LPS with the MAb was observed with 2 to 5 mM periodate treatment. The epitopes for MAbs M04-3, M04-8, and B1-1 were relatively more resistant to periodate oxidation. MAbs B1-1, M04-3, and M04-8 also appeared to be different from one another and from the rest of the serogroup B-specific MAbs in the following ways: the epitope for MAb B1-1 appeared to be
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J. CLIN. MICROBIOL.
TSANG ET AL.
highly resistant to meta-periodate oxidation and was stable to treatment with up to 100 mM periodate, and the epitopes for MAbs M04-3 and M04-8 were less stable to the periodate treatment than that of MAb B1-1 but were still more resistant to such treatment than epitopes for the serogroup B-specific MAbs. Serotyping of salmonellae with MAbs M04-4, M04-5, and M04-8. Forty-eight strains of salmonellae from 0 serogroups A to E were tested against the MAbs by the slide agglutination method. Two strains of S. paratyphi A (serogroup A) and eight serotypes of group C1, nine serotypes of group C2, seven serotypes of group D1, six serotypes of group E1, and two serotypes of group E4 Salmonella tested did not react with the MAbs. On the other hand, all 14 strains (including eight serotypes) of group B Salmonella tested were all strongly and rapidly agglutinated by the MAbs, even when the ascitic fluids were diluted to 1:16 (Table 4).
TABLE 4. Serotyping of salmonella strains by slide agglutination with anti-Salmonella serogroup B-specific MAbs Serogroup and
No. of strains
se etested
No. giving withpositive MAba: results M04-4
M04-5
M04-8
2
0
0
0
S. typhimurium S. derby S. stanley S. saint-paul S. agona S. paratyphi B S. sandiego S. heidelberg
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
infantis montevideo virchow choleraesuis braenderup lille
1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
S. S. S. S. S. S. S. S. S.
newport blockley chailey hiduddify hadar bardo bovismorbificans litchfield manhattan
1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
S. S. S. S. S. S. S.
dublin berta enteritidis panama durban sendai
1 1 1 1 1 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
weltevreden newlands
1 1 1 1 1 1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
E4 S. krefeld S. senftenberg
1 1
0 0
0 0
0 0
48
14
14
14
A, S. paratyphiA B
Cl
DISCUSSION This paper describes six murine MAbs reactive with S. typhimurium LPS antigens. Two were found to cross-react with S. paratyphi A (serogroup A) (Table 1) and may therefore be directed against one or both of the common and minor 0 factors 1 and 12 (12). Further tests to elucidate the epitope specificities of these two MAbs, M04-3 and M04-8, were not carried out, since they did not have the immediate application of detection and identification of serogroup B Salmonella strains. The other four MAbs appeared to be specific for serogroup B salmonellae, and at least two had been shown to react with the most common serotypes found in both Hong Kong (unpublished observation) and the United States (7), which included S. agona, S. derby, S. heidelberg, S. saint-paul, and S. typhimurium. The serogroup B-specific MAbs were reacting with the 0 antigens, because SDS-PAGE immunoblots have shown that they were reacting with the repeating units of the LPS antigens which bear the 0-antigenic factors (9). Since they were specific for serogroup B, the likely epitope that they recognized was 0 antigen 4, which is the 3,6-dideoxyhexose abequose linked oxl,3 to D-mannose (8). Other immunochemical studies have shown that the epitopes recognized by these serogroup B-specific MAbs were sensitive to sodium meta-periodate oxidation (Table 3), which implied that the 6-deoxyhexose rhamnose was most probably also involved in the reactive epitope for these MAbs. This is because it is the only sugar in the basic tetrasaccharide unit of serogroup B Salmonella 0 antigen, i.e., abequose aol - 3 mannose al -> 4 rhamnose al -- 3 galactose, which contains neighboring hydroxy groups (9). These results therefore suggested that the MAbs bind to regions of the serogroup B Salmonella 0 antigens extending over three sugar residues of abequose al -- 3 mannose otl -4 rhamnose. These results, coupled with the finding that the present MAbs were able to react with purified LPS antigens from an S. typhimurium semirough mutant (SH 777) (data not shown), which has only one 0-antigen unit in its LPS, indicated that the antigen combining sites of the mouse antibodies were of sizes of three to four sugar residues, with a minimum of three sugars involved. Besides providing some basic information on the mouse immunoglobulin molecules, the present murine MAbs have practical applications in the routine identification of salmonellae. It was demonstrated in this report that two of the serogroup B-specific MAbs (M04-4 and M04-5) were an efficient agglutination reagent for the serotyping of salmo-
S. S. S. S. S. S. S. S.
C2
ohio thompson
D
El
S. S. S. S. S. S.
typhi anatum
westhampton london meleagridis
Total
The agglutinating titers of the MAb ascitic fluids were 1:64 for M04-4 and M04-8 and 1:32 for M04-5. Slide agglutination tests were performed with a 1:16 dilution of each ascitic fluid. a
nellae (Table 4) by the simple and routine slide agglutination test, which is familiar to most technologists working in the bacteriology laboratory. In summary, we have produced and characterized murine MAbs against the serogroup B salmonellae and have dem-
VOL. 29, 1991
onstrated their practical applications in the identification of these intestinal pathogens by the slide agglutination method. Currently, we are developing ELISA methods that utilize these MAbs for the rapid detection of serogroup B salmonellae through enrichment serology (15, 17). ACKNOWLEDGMENTS This work was supported by a Strategic Research Grant from the University of Hong Kong and by a grant from the Royal Hong Kong Jockey Club to the Institute of Applied Molecular Biology. REFERENCES 1. Adams, L. B., M. C. Henk, and R. J. Siebeling. 1988. Detection of Vibrio cholerae with monoclonal antibodies specific for serovar 01 lipopolysaccharide. J. Clin. Microbiol. 26:18011809. 2. Bean, N. H., P. M. Griffin, J. S. Goulding, and C. B. Ivey. 1990. Foodborne disease outbreaks, 5-year summary, 1983-1987. Morbid. Mortal. Weekly Rep. 39(SS-1):15-57. 3. Centers for Disease Control. 1982. Salmonella surveillance annual summary, 1980. Centers for Disease Control, Atlanta. 4. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-135. 5. Gardner, P., and H. T. Provine. 1987. Manual of acute infection. Early diagnosis and treatment, 2nd ed, p. 352. Little, Brown & Co., Boston. 6. Gilbert, R. J., D. Roberts, and G. Smith. 1984. Food-borne diseases and botulism, p. 477-514. In G. R. Smith (ed.), Topley and Wilson's principles of bacteriology, virology and immunity, 7th ed., vol. 3. Edward Arnold, London. 7. Hargrett-Bean, N. T., A. T. Pavia, and R. V. Tauxe. 1988. Salmonella isolates from humans in the United States, 19841986. Morbid. Mortal. Weekly Rep. 37(SS-2):25-31. 8. Hellerqvist, C. G., B. Lindberg, S. Svensson, T. Holme, and A. A. Lindberg. 1969. Structural studies on the 0-specific side chains of the cell wall lipopolysaccharides from Salmonella typhimurium LT2. Carbohydr. Res. 9:237-241.
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9. Jann, K., and B. Jann. 1984. Structure and biosynthesis of 0-antigens, p. 138-186. In E. T. Rietschel (ed.), Chemistry of endotoxin, vol. 1. Elsevier Science Publishers B. V., Amsterdam. 10. Kelly, M. T., D. J. Brenner, and J. J. Farmer III. 1985. Enterobacteriaceae, p. 263-277. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 11. Kohler, G., and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 12. Le Minor, L., and M. Y. Popoff. 1987. Antigenic formulas of the Salmonella serovars, 5th ed. Institut Pasteur, Paris. 13. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). 1985. Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 14. Luk, M. C., R. S. W. Tsang, and M. H. Ng. 1987. Murine monoclonal antibody specific for lipopolysaccharide of Salmonella serogroup A. J. Clin. Microbiol. 25:2140-2144. 15. Mikhail, I. A., W. R. Sanborn, and J. E. Sippel. 1983. Rapid, economical diagnosis of enteric fever by a blood clot culture coagglutination procedure. J. Clin. Microbiol. 17:564-565. 16. PHLS Communicable Diseases Surveillance Centre. 1988. Communicable disease report July to September 1987. Community Med. 10:66-72. 17. Sperber, W. H., and R. H. Deibel. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17: 533-539. 18. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119. 19. Westphal, O., and K. Jann. 1965. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-90. 20. WHO Expert Committee. 1988. Salmonellosis control: the role of animal and product hygiene. WHO Tech. Rep. Ser. 774:16. 21. Woodward, M. P., W. W. Young, Jr., and R. A. Bloodgood. 1985. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78:143-153.
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0095-1137/91/091899-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Characterization of Murine Monoclonal Antibodies against Serogroup B Salmonellae and Application as Serotyping Reagents RAYMOND S. W. TSANG,* K. H. CHAN, NELSON W. H. LAU, DEBBY K. W. CHOI, DENNIS K. S. LAW, AND M. H. NG Department of Microbiology, University of Hong Kong, Pathology Building,
Queen Mary Hospital Compound, Hong Kong Received 29 October 1990/Accepted 6 June 1991
Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other 0 serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.
Salmonellae are well-known bacterial pathogens that cause food poisoning (6). In a summary of food-borne disease outbreaks in the United States from 1983 to 1987, salmonellae were found to be responsible for more than 50% of all the identifiable bacterial causes (2). Figures reported from 1984 to 1986 have shown that 40 to 60% of the salmonellae isolated in the United States from humans belonged to serogroup B, and S. typhimurium is the single most commonly isolated salmonella (7). S. typhimurium, S. heidelberg, S. agona, S. saint-paul, and S. derby (all serogroup B) were among the 20 most commonly isolated serotypes reported in the United States during this period. In England and Wales, S. typhimurium was also the most commonly isolated salmonella from 1941 to 1987, with more than 30% of all isolates identified as this serotype (16). Similarly, S. typhimurium together with other serotypes of serogroup B salmonellae accounted for nearly half (4 of 10) to more than half (7 of 10) of the 10 most common serotypes that caused Salmonella food poisoning in England and Wales during a 13-year period, from 1966 to 1978 (6). Also, S of the 12 recently well-documented significant outbreaks of foodborne salmonellosis were due to group B organisms (20). These reports signify the importance and the prevalence of the serogroup B organisms, which together with organisms in the 0 serogroups A, C, D, and E are responsible for >95% of Salmonella infections in humans (3, 5). In this report, we describe the production and characterization of murine monoclonal antibodies (MAbs) reactive with and specific for serogroup B salmonellae. We also evaluate the use of these murine MAbs for the serotyping of salmonellae by the slide agglutination method in a routine diagnostic bacteriology laboratory.
*
MATERIALS AND METHODS Bacterial strains, culture conditions, and antigens. Bacterial strains used in this study either were purchased from the American Type Culture Collection, Rockville, Md., or were clinical isolates from our clinical bacteriology laboratory at Queen Mary Hospital, Hong Kong (see Table 2). Bacteria obtained from our clinical diagnostic bacteriology laboratory were identified by standard methods (13). Bacteria for extraction of lipopolysaccharide (LPS) were grown in brain heart infusion broth (Oxoid Ltd., London, England) under static incubation conditions at 37°C for 16 to 18 h. Bacteria for colony dot blot enzyme immunoassay and slide agglutination tests were grown on nutrient agar (Oxoid Ltd.) plates or chocolate blood agar (5% horse blood in Columbia blood agar base [Oxoid Ltd.]) plates when growth demanded more-enriched media. All agar plates were incubated at 37°C for 16 to 18 h with 5% carbon dioxide when required. Purified LPS antigens from S. typhimurium and S. typhi were purchased from Sigma Chemical Company (St. Louis, Mo.). LPS from S. paratyphi A, S. montevideo, S. blockley, and S. newlands was extracted from formalin-inactivated and washed bacterial cells by the hot phenol-water method of Westphal and Jann (19) and was purified by repeated
ultracentrifugation. Production of MAbs. BALB/c mice were immunized intraperitoneally with boiled (2.5 h at 100°C) whole cells of S. typhimurium at a dosage of 2.5 x 108 cells in a 0.5-ml volume. The immunization was repeated four times, with an interval of 5 days between immunizations. An intravenous booster injection of the same number of cells was given 1 week after the last intraperitoneal injection. Three days after the intravenous injection, the mice were bled from their orbital veins and their sera were tested against LPS antigens from S. typhimurium. Immune spleen cells from the mouse that had the highest antibody response were fused with NS1 myeloma cells by using polyethylene glycol (molecular
Corresponding author. 1899
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TABLE 1. Reactivities of anti-Salmonella serogroup B MAbs with purified Salmonella LPS from 0 serogroups A to E Mean ELISA OD at 492 nm for MAbsa: Serotype (serogroup)
None
None (antigen control) S. paratyphi A (A) S. typhimurium (B) S. montevideo(C1) S. blockley (C2) S. typhi (D1) S. newlands (E1)
(control) 0.021 0.020 0.021 0.019 0.015 0.010 0.005
B1-1
M04-2
M04-3
M044
M04-5
M04-8
0.015 0.036 0.754 0.007 0.007 0.006 0.005
0.031 0.046 0.855 0.021 0.006 0.017 0.018
0.033
0.010
0.016
0.021
1.114 0.845 0.018 0.018
0.007 0.690 0.006 0.001
0.008 0.777 0.005 0.000
0.178 0.805 0.010 0.015
0.022 0.012
0.005 0.002
0.006 0.004
0.020 0.008
a The titers of the MAbs were determined by ELISA and defined as the dilutions that yielded OD values of about 0.100 and at least two times the background control OD values. The MAbs were used at the following dilutions: B1-1 (titer = 1:500,000), 1:5,000; M04-2 (titer = 1:500,000), 1:10,000; M04-3, M04-4, and M04-5 (titer = >1:1,000,000), 1:100,000; and M04-8 (titer = 1:50,000), 1:5,000.
weight, 4,000; BDH, Poole, England) as the fusing agent (11). Hybridomas were screened for the production of specific antibodies against S. typhimurium LPS antigens by enzyme-linked immunosorbent assay (ELISA), and hybridomas that produced specific antibodies were cloned twice by the limiting-dilution method. Purified hybridomas were injected intraperitoneally into mineral oil-primed mice for the production of ascitic fluids. Serological methods. The immunoglobulin classes and subclasses of the MAbs were determined by double immunodiffusion with anti-mouse immunoglobulin antisera purchased from Nordic Immunological Laboratories (Tillburg, The Netherlands). The ELISA (4) to measure antibodies to LPS antigens was done as described previously (14). The whole-cell ELISA was done as described by Adams et al. (1). The colony dot blot enzyme immunoassay was done by spotting boiled (100°C, 30 min) whole bacterial cells onto nitrocellulose paper (0.45-,um pore diameter; Schleicher & Schuell, Dassel, Germany) and subsequently processing according to the instruction manual of the Bio-Rad Immuno-blot assay kit (Bio-Rad Laboratories, Richmond, Calif.). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Salmonella LPS and its visualization by silver staining (18) or immunoblotting with the MAbs were done as described previously (14). Serotyping of the salmonellae with the MAbs was done by standard procedures (10). Sodium meta-periodate oxidation of S. typhimurium LPS was done on antigens coated onto an ELISA microtiter plate by the method of Woodward et al. (21), and the residual serological activity of the LPS antigens after periodate oxidation was measured by ELISA against the MAbs. RESULTS Production of murine MAbs against serogroup B salmonellae. From two separate fusion experiments with spleen cells from two mice immunized with boiled whole cells of S. typhimurium, six hybridomas that produced high levels of antibodies reactive with S. typhimurium LPS antigens were obtained. Cells from these six hybridoma lines have been deposited at the European Collection of Animal Cell Cultures of Public Health Laboratory Service Centre for Applied Microbiology and Research, Salisbury, United Kingdom, and have been given the accession numbers 91031425 to 91031430. Three antibodies were of the immunoglobulin G3 isotype (MO4-2, M04-3, and M04-8), two were immunoglobulin Gl antibodies (MO4-4 and M04-5), and one belonged to the immunoglobulin M class (B1-1). Ascitic
TABLE 2. Salmonella serotypes and standard strains of other bacterial species that tested negative with MAbs M04-2, M04-4, and M04-5 Strain used
Serogroup C1 S. virchow S. lille S. choleraesuis S. paratyphi C S. montevideo S. georgia S. infantis S. mbandaka S. ohio S. oranienburg S. potsdam S. braenderup S. thompson S. singapore S. isangi
Serogroup C2 S. S. S. S. S. S. S. S. S. S. S.
hadar
hiduddify manhattan newport kottbus
blockley chailey muenchen
litchfield herston bardo
Serogroup DI S. typhi S. dublin S. enteritidis S. S. S. S.
panama sendai berta durban
Serogroup E1 S. S. S. S. S.
anatum
london newlands
meleagridis weltevreden
Serogroup E4 S. krefeld S. senftenberg
Strain used
Standard strains S. paratyphi A ATCC 9150 S. choleraesuis ATCC 13312 S. newport ATCC 6962 S. typhi Ty2 ATCC 19430 S. enteritidis ATCC 13076 Escherichia coli ATCC 35218 Shigella flexneri ATCC 29903 Shigella sonnei ATCC 25931 Klebsiella pneumoniae ATCC 13883 Enterobacter agglomerans ATCC 27155 Enterobacter cloacae ATCC 23355 Proteus mirabilis ATCC 7002 Providencia rettgeri ATCC 29944 Morganella morganii ATCC 25803 Citrobacterfreundii ATCC 8090 Serratia marcescens ATCC 8100 Edwardsiella tarda ATCC 15947 Yersinia enterocolitica ATCC 9610 Pseudomonas aeruginosa ATCC 27853 Acinetobacter anitratus ATCC 19606 Acinetobacter lwoffii ATCC 15309 Flavobacterium meningosepticum ATCC 13253 Alcaligenes faecalis ATCC 8750 Moraxella lacunata ATCC 17967 Aeromonas hydrophila ATCC 7966 Vibrio parahaemolyticus ATCC 66740 Haemophilus influenzae ATCC 14035 Staphylococcus epidermidis ATCC 51825 Streptococcus pyogenes ATCC 19615 Streptococcus pneumoniae ATCC 27336
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SALMONELLA SEROGROUP B-SPECIFIC MAbs
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TABLE 3. Susceptibility of S. typhimurium LPS epitopes to sodium meta-periodate oxidation measured by ELISA
with anti-Salmonella serogroup B-specific MAbs Mean ELISA OD (% residual activity) for MAba:
Na meta-periodate concn (mM)
None
0 1 2 5 10 20 50 100
(control) 0.014 0.013 0.020 0.017 0.016 0.011 0.008 0.013
B1-1 1.054 1.006 0.951 0.909 0.899 0.776 0.804 0.886
(95.4) (90.2) (86.2) (85.3) (73.6) (76.3) (84.1)
M04-2 1.029 0.725 0.486 0.444 0.148 0.056 0.064 0.061
(70.5) (47.2) (43.2) (14.4) (5.4) (6.2) (5.9)
M04-3 0.832 0.779 (93.6) 0.711 (85.5) 0.565 (67.9) 0.311 (37.4) 0.181 (21.8) 0.120 (14.4) 0.156 (18.8)
M04-4 0.981 0.291 0.165 0.125 0.060 0.012 0.004 0.011
(29.7) (16.8) (12.7) (6.1) (1.2) (0.4) (1.1)
M04-5 1.050 0.390 (37.1) 0.243 (23.1) 0.145 (13.8) 0.070 (6.7) 0.007 (0.7) 0.003 (0.3) 0.010 (1.0)
M04-8 1.088 1.054 1.012 0.808 0.514 0.134 0.027 0.085
(96.9) (93.0) (74.3) (47.2) (12.3) (2.5) (7.8)
a Residual serological activity of the S. typhimurium LPS epitopes after sodium meta-periodate treatment is expressed as a percentage of original activity without treatment. The MAbs were used at the following dilutions: B1-1, M04-2, and M04-8, 1:5,000; M04-4 and M04-5, 1:50,000; and M04-3, from tissue culture fluid, was used undiluted.
fluids produced from these six hybridoma cell lines gave high titers of antibodies to the S. typhimurium LPS antigens in an ELISA. The titers ranged from 1:50,000 to >1:1,000,000, and the highest titers were given by the M04-3, M04-4, and M04-5 hybridoma MAbs (Table 1). Immunochemical characterization of MAbs. In a preliminary study, the six MAbs were allowed to react with purified LPS antigens from Salmonella serotypes that represented the 0 serogroups A, B, C1, C2, D1, and E1 (Table 1). Four antibodies (MO4-2, M04-4, M04-5, and B1-1) were specific for LPS antigens from S. typhimurium, while the remaining two antibodies (MO4-3 and M04-8) were found to crossreact with S. paratyphi A LPS. To confirm the serogroup specificity of the MAbs, a whole-cell ELISA with Salmonella serotypes of 0 serogroups A to E was done. Results from the whole-cell ELISA experiments confirmed the ELISA results obtained by using purified LPS antigens. MAbs M04-3 and M04-8 were found to react with whole cells of S. paratyphi A (12 strains were tested, and mean ELISA optical density [OD] values of 0.659 and 0.394 were obtained with M04-3 and M04-8, respectively). These two antibodies also reacted with all 10 strains of serogroup B salmonellae, including S. typhimurium, S. agona, S. derby, S. saint-paul, S. stanley, S. heidelberg, S. paratyphi B, S. schwarzengrund, and S. sandiego (mean ELISA OD values were 1.270 and 1.264 for MAbs M04-3 and M04-8, respectively). The remaining four MAbs, M04-2, M04-4, M04-5, and B1-1, were specific for serogroup B salmonellae, and they reacted with all nine serotypes of serogroup B Salmonella, with mean ELISA OD values of 1.057, 0.815, 0.948, and 0.516, respectively. None of the six antibodies reacted with any of the Salmonella serotypes from serogroups C1, C2, D1, E1, and E4 (Table 2). To ascertain the specificities of MAbs M04-2, M04-4, and M04-5, 30 standard strains from the American Type Culture Collection were tested against the MAbs by a colony dot blot enzyme immunoassay. All three serogroup B-specific MAbs reacted with S. typhimurium ATCC 14028 but did not react with the non-group B Salmonella serotypes or with any of the other standard bacterial strains listed in Table 2. The antigens that reacted with the MAbs were studied by SDS-PAGE immunoblot. S. typhimurium LPS antigens were separated by SDS-PAGE and electrotransferred to a nitrocellulose paper, and the separated and transferred LPS components were reacted with the MAbs. All six MAbs reacted with the 0 antigens of the S. typhimurium LPSs, since each gave a ladder-type reactive pattern consistent
with the 0-antigen ladder pattern observed by silver staining. The somatic 0 antigens of serogroup B salmonellae may consist of 0 factors 1, 4, 5, 12, and 27. 0 antigens 4 and 12 are regarded as the basic 0 factors present in all serogroup B salmonellae. 0 antigen 4 is the specific somatic antigen for all serogroup B salmonellae and hence is diagnostic for this O serogroup of organisms. 0 antigen 12 can be found in a number of 0 serogroups, such as A, B, and D, and is a minor 0 factor since its presence is not diagnostic for any particular 0 serogroup. Other 0 factors, 1, 5, and 27, that may be present in serogroup B salmonellae are a result of chemical modification of the basic 0 antigen. For example, 0 antigens 1 and 27 result from modification by the presence of lysogenic phages which add into the basic structures additional specificities. 0 antigen 5 results from 0 acetylation of 0-antigen 4 and is therefore present only in strains of serogroup B Salmonella. To find out whether the MAbs were reacting with 0 antigen 5, strains of S. derby without 0 antigen 5 were selected for either direct reaction with the MAbs (slide agglutination with ascitic fluids) or specific removal of the MAbs by absorption studies followed by an ELISA with S. typhimurium LPS antigens. All six MAbs strongly agglutinated with four strains of S. derby without 0 antigen 5. Similarly, strains of S. derby without 0 antigen 5 removed all six MAbs as effectively as a strain of S. typhimurium with 0 antigen 5. Both studies therefore indicated that the MAbs were reacting with an antigen unrelated to the conventional 0 antigen 5. The sensitivities of the different MAb epitopes to sodium meta-periodate oxidation were studied by testing treated and untreated S. typhimurium LPS with the MAbs by ELISA. The results presented in Table 3 show that two serogroup B-specific MAbs (MO4-4 and M04-5) were highly sensitive to meta-periodate oxidation whereas the serogroup A crossreactive MAbs (MO4-3 and M04-8) were relatively resistant to this treatment. For the serogroup B-specific MAbs M04-4 and M04-5, as little as 1 mM periodate was enough to reduce the reactivity of S. typhimurium LPS with the MAbs by >50%. The epitope for MAb M04-2 was also very labile to the meta-periodate treatment, since a 50% decrease in reactivity of the S. typhimurium LPS with the MAb was observed with 2 to 5 mM periodate treatment. The epitopes for MAbs M04-3, M04-8, and B1-1 were relatively more resistant to periodate oxidation. MAbs B1-1, M04-3, and M04-8 also appeared to be different from one another and from the rest of the serogroup B-specific MAbs in the following ways: the epitope for MAb B1-1 appeared to be
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highly resistant to meta-periodate oxidation and was stable to treatment with up to 100 mM periodate, and the epitopes for MAbs M04-3 and M04-8 were less stable to the periodate treatment than that of MAb B1-1 but were still more resistant to such treatment than epitopes for the serogroup B-specific MAbs. Serotyping of salmonellae with MAbs M04-4, M04-5, and M04-8. Forty-eight strains of salmonellae from 0 serogroups A to E were tested against the MAbs by the slide agglutination method. Two strains of S. paratyphi A (serogroup A) and eight serotypes of group C1, nine serotypes of group C2, seven serotypes of group D1, six serotypes of group E1, and two serotypes of group E4 Salmonella tested did not react with the MAbs. On the other hand, all 14 strains (including eight serotypes) of group B Salmonella tested were all strongly and rapidly agglutinated by the MAbs, even when the ascitic fluids were diluted to 1:16 (Table 4).
TABLE 4. Serotyping of salmonella strains by slide agglutination with anti-Salmonella serogroup B-specific MAbs Serogroup and
No. of strains
se etested
No. giving withpositive MAba: results M04-4
M04-5
M04-8
2
0
0
0
S. typhimurium S. derby S. stanley S. saint-paul S. agona S. paratyphi B S. sandiego S. heidelberg
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
3 2 2 2 2 1 1 1
infantis montevideo virchow choleraesuis braenderup lille
1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
S. S. S. S. S. S. S. S. S.
newport blockley chailey hiduddify hadar bardo bovismorbificans litchfield manhattan
1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
S. S. S. S. S. S. S.
dublin berta enteritidis panama durban sendai
1 1 1 1 1 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
weltevreden newlands
1 1 1 1 1 1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
E4 S. krefeld S. senftenberg
1 1
0 0
0 0
0 0
48
14
14
14
A, S. paratyphiA B
Cl
DISCUSSION This paper describes six murine MAbs reactive with S. typhimurium LPS antigens. Two were found to cross-react with S. paratyphi A (serogroup A) (Table 1) and may therefore be directed against one or both of the common and minor 0 factors 1 and 12 (12). Further tests to elucidate the epitope specificities of these two MAbs, M04-3 and M04-8, were not carried out, since they did not have the immediate application of detection and identification of serogroup B Salmonella strains. The other four MAbs appeared to be specific for serogroup B salmonellae, and at least two had been shown to react with the most common serotypes found in both Hong Kong (unpublished observation) and the United States (7), which included S. agona, S. derby, S. heidelberg, S. saint-paul, and S. typhimurium. The serogroup B-specific MAbs were reacting with the 0 antigens, because SDS-PAGE immunoblots have shown that they were reacting with the repeating units of the LPS antigens which bear the 0-antigenic factors (9). Since they were specific for serogroup B, the likely epitope that they recognized was 0 antigen 4, which is the 3,6-dideoxyhexose abequose linked oxl,3 to D-mannose (8). Other immunochemical studies have shown that the epitopes recognized by these serogroup B-specific MAbs were sensitive to sodium meta-periodate oxidation (Table 3), which implied that the 6-deoxyhexose rhamnose was most probably also involved in the reactive epitope for these MAbs. This is because it is the only sugar in the basic tetrasaccharide unit of serogroup B Salmonella 0 antigen, i.e., abequose aol - 3 mannose al -> 4 rhamnose al -- 3 galactose, which contains neighboring hydroxy groups (9). These results therefore suggested that the MAbs bind to regions of the serogroup B Salmonella 0 antigens extending over three sugar residues of abequose al -- 3 mannose otl -4 rhamnose. These results, coupled with the finding that the present MAbs were able to react with purified LPS antigens from an S. typhimurium semirough mutant (SH 777) (data not shown), which has only one 0-antigen unit in its LPS, indicated that the antigen combining sites of the mouse antibodies were of sizes of three to four sugar residues, with a minimum of three sugars involved. Besides providing some basic information on the mouse immunoglobulin molecules, the present murine MAbs have practical applications in the routine identification of salmonellae. It was demonstrated in this report that two of the serogroup B-specific MAbs (M04-4 and M04-5) were an efficient agglutination reagent for the serotyping of salmo-
S. S. S. S. S. S. S. S.
C2
ohio thompson
D
El
S. S. S. S. S. S.
typhi anatum
westhampton london meleagridis
Total
The agglutinating titers of the MAb ascitic fluids were 1:64 for M04-4 and M04-8 and 1:32 for M04-5. Slide agglutination tests were performed with a 1:16 dilution of each ascitic fluid. a
nellae (Table 4) by the simple and routine slide agglutination test, which is familiar to most technologists working in the bacteriology laboratory. In summary, we have produced and characterized murine MAbs against the serogroup B salmonellae and have dem-
VOL. 29, 1991
onstrated their practical applications in the identification of these intestinal pathogens by the slide agglutination method. Currently, we are developing ELISA methods that utilize these MAbs for the rapid detection of serogroup B salmonellae through enrichment serology (15, 17). ACKNOWLEDGMENTS This work was supported by a Strategic Research Grant from the University of Hong Kong and by a grant from the Royal Hong Kong Jockey Club to the Institute of Applied Molecular Biology. REFERENCES 1. Adams, L. B., M. C. Henk, and R. J. Siebeling. 1988. Detection of Vibrio cholerae with monoclonal antibodies specific for serovar 01 lipopolysaccharide. J. Clin. Microbiol. 26:18011809. 2. Bean, N. H., P. M. Griffin, J. S. Goulding, and C. B. Ivey. 1990. Foodborne disease outbreaks, 5-year summary, 1983-1987. Morbid. Mortal. Weekly Rep. 39(SS-1):15-57. 3. Centers for Disease Control. 1982. Salmonella surveillance annual summary, 1980. Centers for Disease Control, Atlanta. 4. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-135. 5. Gardner, P., and H. T. Provine. 1987. Manual of acute infection. Early diagnosis and treatment, 2nd ed, p. 352. Little, Brown & Co., Boston. 6. Gilbert, R. J., D. Roberts, and G. Smith. 1984. Food-borne diseases and botulism, p. 477-514. In G. R. Smith (ed.), Topley and Wilson's principles of bacteriology, virology and immunity, 7th ed., vol. 3. Edward Arnold, London. 7. Hargrett-Bean, N. T., A. T. Pavia, and R. V. Tauxe. 1988. Salmonella isolates from humans in the United States, 19841986. Morbid. Mortal. Weekly Rep. 37(SS-2):25-31. 8. Hellerqvist, C. G., B. Lindberg, S. Svensson, T. Holme, and A. A. Lindberg. 1969. Structural studies on the 0-specific side chains of the cell wall lipopolysaccharides from Salmonella typhimurium LT2. Carbohydr. Res. 9:237-241.
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9. Jann, K., and B. Jann. 1984. Structure and biosynthesis of 0-antigens, p. 138-186. In E. T. Rietschel (ed.), Chemistry of endotoxin, vol. 1. Elsevier Science Publishers B. V., Amsterdam. 10. Kelly, M. T., D. J. Brenner, and J. J. Farmer III. 1985. Enterobacteriaceae, p. 263-277. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 11. Kohler, G., and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 12. Le Minor, L., and M. Y. Popoff. 1987. Antigenic formulas of the Salmonella serovars, 5th ed. Institut Pasteur, Paris. 13. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). 1985. Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 14. Luk, M. C., R. S. W. Tsang, and M. H. Ng. 1987. Murine monoclonal antibody specific for lipopolysaccharide of Salmonella serogroup A. J. Clin. Microbiol. 25:2140-2144. 15. Mikhail, I. A., W. R. Sanborn, and J. E. Sippel. 1983. Rapid, economical diagnosis of enteric fever by a blood clot culture coagglutination procedure. J. Clin. Microbiol. 17:564-565. 16. PHLS Communicable Diseases Surveillance Centre. 1988. Communicable disease report July to September 1987. Community Med. 10:66-72. 17. Sperber, W. H., and R. H. Deibel. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17: 533-539. 18. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119. 19. Westphal, O., and K. Jann. 1965. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-90. 20. WHO Expert Committee. 1988. Salmonellosis control: the role of animal and product hygiene. WHO Tech. Rep. Ser. 774:16. 21. Woodward, M. P., W. W. Young, Jr., and R. A. Bloodgood. 1985. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78:143-153.
