Journal of Applied Bacteriology 1992,72, 134-138
Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas Debby Choi, R.S.W. Tsang and M.H. Ng Department ofMicrobiology, University of Hong Kong , Pathology Building, Queen Mary Hospital Compound, Hong Kong 3658/05/91: accepted 8 July 1991 D . C H O I , R . S . W . T S A N G A N D M . H . N G . 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard a n d 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of R a lipopolysaccharide, 106/ml of a smooth wild strain of Salm. typhimurium, a n d 1120 cells of Salm. heidelberg after enrichment culture for 4 h.
The most effective means of controlling salmonellosis is still early detection of the organisms and prevention of spread of the infections (Anon. 1988). This is because the large number of salmonella serotypes [ > 2000 serotypes identified (Le Minor & Popoff 1987)] that can cause infection in man also infect a wide variety of animals which may serve as a reservoir of the organisms. This large animal reservoir makes control of the infection difficult. Detection of salmonellas requires elaborate cultural methods of enrichment followed by isolation on selective differential agar media. T h e identification of suspicious colonies on agar media also requires a battery of biochemical tests and a cumbersome set of polyvalent and mono-specific anti-0 and -H sera for serological typing (Kauffmann 1966; Edwards & Ewing 1972). Over the last two decades much effort has been spent in research to improve methods for the detection of salmonellas. Enrichment serology has been the single most popular approach but many such procedures that use polyvalent antisera are suffering from the limitations of specificity due to the polyclonal nature of the antisera used (Sperber & Deibel 1969; Mohr et al. 1974; Sanborn et al. 1980; Robinson et al. 1983; Benge 1989). T o overcome this problem we have developed murine hybridoma monoclonal antibodies (MAbs) against common, but genus-specific, epitopes of salmonellas (Tsang et al. 1987, 1991a). With Correspondence t o :Dr Raymond Tsang, Department of Microbiology, Universip of Hona Kong, Pathology Building, Queen Mary Hospital Compound, Pokfulam Road, Hong Kong.
one such MAb T 6 (Tsang et al. 1987), we have developed a sandwich capture enzyme-linked immunosorbent assay (ELISA) that can detect whole cells and bacterial antigens of different serotypes of salmonellas that are most commonly encountered in human infections. In this report we describe the performance of such an assay and we provide results that show that such a method is likely to be useful in enrichment serology for the detection of common serotypes of salmonellas.
M A T E R I A L S AND METHODS Bacterial strains and culture conditions
These are described in Table 1 and included six salmonella and 21 non-salmonella standard strains obtained from American Type Culture Collection (Rockville, MD, USA) ; Salmonella senfenberg SH 1707, a gift from Prof. A.A. Lindberg (Department of Clinical Bacteriology, Karolinska Institute, Sweden); and 24 wild strains of salmonellas obtained from our clinical diagnostic bacteriology laboratory, Queen Mary Hospital, Hong Kong. Non-standard strains were identified in our laboratory by standard biochemical and serological methods (Kelly et al. 1985). The bacteria were grown in Brain Heart Infusion (BHI) broths (Oxoid) at 37°C under static incubation conditions. For assay by MAb T6, broth-grown bacteria were first killed by heat treatment at 100°C for 30 min in a boiling water bath.
CAPTURE E L I S A FOR S A L M O N E L L A B Y M A b 135
Table 1 Reactivity of 4 h broth cultures of Salmonella and overnight broth cultures of non-Salmonella in sandwich capture ELISA using
MAb T6 Mean* ELISA
Wild strains of salmonella
Mean ELISA O.D.
Salmonella paratyphi A typhimurium choleraesuis newport enteritidis typhi T y 2 senftenberg Escherichia coli Klebsiella pneumoniae Enterobacter cloacae agglomerans Proteus mirabilis Providencia rettgeri Morganella morganii Shigella sonnei Jexneri Citrobacter freundii Serratia marcescens Yersinia enterocolitica Edwardsiella tarda Pseudomonas aeruginosa Acinetobacter anitratus lwoffii z41caligenesfaecalis .4eromonas hydrophila Flavobacterium meningosepticum Streptococcus faecalis Staphylococcus hemolyticus
ATCC 9150 ATCC 14028 ATCC 13312 ATCC 6962 ATCC 13076 ATCC 19430 SH 1707 ATCC 25922 ATCC 13883 ATCC 23355 ATCC 27155 ATCC 7002 ATCC 29944 ATCC 25830 ATCC 25931 ATCC 29903 ATCC 8090 ATCC 8 100 ATCC 9610 ATCC 15947 ATCC 27853 ATCC 19606 ATCC 15309 ATCC 8750 ATCC 7966 ATCC 13253
1.320 (+) 0.394 (+) 1.300 (+) 0.490 ( +) 0.469 ( +) 0.367 ( + ) 0.758 (+) 0.031 ( - ) 0.027 ( - ) 0.023 (-) 0.019 (-) 0.042 (-) 0.039 ( - ) 0.059 (-) 0.034 ( - ) 0.019 ( - ) 0.032 (-) 0.036 (-) 0.027 ( - ) 0.029 ( - ) 0.014 ( - ) 0.017 ( - ) 0.021 ( - ) 0.030 (-) 0.022 ( - ) 0.034 (-)
Salmonella heidelberg agona saintpaul derby stanley schwarzengrund
1.222 ( + ) 0.779 ( +) 0.868 (+) 0.597 ( + ) 1.223 ( +) 0.748 (+)
ATCC 29212 ATCC 29970
0.058 ( - ) 0.057 ( - )
infantis montevideo oranienburg braenderup thompson virchow mbandaka
0.982 ( +) 0.347 ( +) 0.159 ( + ) 0.977 (+) 0,565 ( + ) 0.610 (+) 0.509 (+)
muenchen hadar blockley
0.936 ( + ) 0.760 ( +) 0.314 (+)
panama napoli enterit idis
0.390 ( +) 1.211 ( + ) 0.786 ( + )
weltevreden london Portsmouth newlands anatum
1.487 (+) 1.452 ( + ) 1.454 (+) 0.962 ( +) 0.739 (+)
* Mean ELISA O.D. of duplicate determinations (reactivity).
Lipopolysaccharide (LPS) antigens
Purified Ra LPS from Salm. typhimurium his 386 was a gift of Dr S. Schlecht (Max-Planck-Institut fur Immunobiologie, Freiburg, Germany). Smooth LPS from Salm. typhimurium was purchased from Sigma.
Development of MAb T6 sandwich capture ELSA
Ascitic fluids of MAb T 6 were obtained as described by Tsang et al. (1987). MAb T 6 was conjugated with horse radish peroxidase enzyme Type VI (Sigma) by the method of Avrameas & Terynynck (1971). Optimal concentrations of MAb T 6 for coating ELISA wells as capture antibodies (1 : 5000 dilution of T 6 ascitic fluid) and enzyme conjugated MAb T 6 for detection of antigens (1 : 1000 dilution)
were determined by checker-board titrations. Coating of MAb T 6 as capture antibodies or coating of Salm. typhimurium Ra LPS antigens (5 pg/ml) for titrations of enzymeconjugated MAb T 6 were done in 0.05 mol/l carbonate-bicarbonate buffer, p H 9.6 at 4°C overnight and a volume of 100 p1 per well. Blocking of excess binding sites in the wells was done with 2 % bovine serum albumin (Sigma) in phosphate buffered saline pH 7.4 at 37°C for 1 h. ELISA plates were washed with 0.9% saline-0.05% Tween 20 (Sigma) four times with an interval of 2 min between each washing. All incubation steps were done at 37°C for 1 h, and all reactants were added in a volume of 100 p1 per well except where specified. Substrate used for the horse radish peroxidase enzyme was 0.1 mg/ml of 0phenylenediamine dihydrochloride (OPD) in a substrate buffer containing 0.05 mol/l Na,HPO, , 0.025 mol/l citric
136 DEBBY C HOl ET A L
acid, and 0.003% H z O z . Colour development was at 37°C for 30 min and the substrate-enzyme reaction was terminated with 25 p1 of 4 N HZSO4before it was measured with a Dynatech MR 710 microplate reader (Dynatech Laboratories, Chantilly, Virginia, USA). Statistical methods
Mean, standard deviations, and coefficients of variation were done according to methods described by Hill (1977).
RESULTS A standard dose-response curve for the detection of Salm. typhimurium his 386 Ra LPS was established. Mean ELISA 0.D.s together with their standard deviations obtained from 10 assays are shown in Fig. 1. The low values of coefficient of variation obtained in the sandwich capture ELISA indicated that the MAb T 6 used in the present format of assay has good reproducibility for the detection of salmonella LPS antigens. In deciding an ELISA O.D. value for reporting a test as positive, we chose a value greater than the mean ELISA O.D. obtained from no antigen control experiments plus three times their standard deviations which was 0.058 + 3 x 0.012 = 0.094. When 25
--8 ..-E c
Concentration of LPS (ng/ml)
Fig. 1 Standard dose-response curves for the detection of Salmonella typhimurium Ra and smooth LPS. m, Ra LPS; smooth LPS ; *, coefficient of variation
such a cut-off value was used, MAb T 6 could detect at least 1 ng/ml of salmonella Ra LPS which gave a mean ELISA O.D. value of 0.130. However, when Salm. typhimurium smooth LPS was measured, MAb T 6 could detect only 62.5 ng/ml of it in the present sandwich capture ELISA. MAb T 6 in the present sandwich capture ELISA can also detect wild type smooth Salm. typhimurium cells at a concentration of at least 106/ml. Presence of Escherichaa coli at a level of 109/ml did not seem to interfere with the assay of Salm. typhimurium although lower ELISA O.D. values were obtained when the assay was done in the presence of E. coli. T o evaluate the specificity of the present assay, overnight broth cultures of 2 1 non-salmonella standard strains obtained from ATCC (Table 1, including 13 species of Enterobacteriaceae, six species of other Gram-negative bacteria and two of Gram-positive bacteria) were tested. None of these 21 non-salmonella bacteria reacted in the capture ELISA with MAb T 6 and the mean ELISA O.D. given by these 21 non-salmonella bacteria was 0.032 with a range of 0.014 to 0.059. In contrast to the overnight broth cultures of non-salmonella bacteria, 4 h broth cultures of seven standard salmonella strains and 24 wild salmonella strains from the common 0 serogroups of A to E were all detected by the capture ELISA test. The mean ELISA O.D. obtained with these 31 salmonella strains was 0.812 with a range of 0.159 to 1.487. T o study the possibility of using the present MAb sandwich capture ELISA test in enrichment serology, different numbers of a smooth wild strain of Salm. heidelberg (serogroup 0 : 4 [B]) were seeded into BHI broths for incubation at 37°C. After incubation for 2-4 h samples were removed for assay in the capture ELISA. BHI broths seeded with 24480 cfu of Salm. heidelberg were detected only after 4 h but not 2 h incubation. This level of sensitivity was confirmed when 1120 Salm. heidelberg was seeded into BHI broths for incubation of 4 h and assayed after concentrating fourfold through centrifugation of 2 ml broths and resuspending the pelleted cells in 0.5 ml of the medium (Table 2). DISCUSSION
Previously (Tsang et al. 1987, 1991a) we have shown that MAbs against the outer core epitopes of salmonella LPS are specific for salmonellas and we have used such MAbs for the characterizations of the LPS outer core structures of a wide variety of salmonella strains (Tsang et al. 1991b). Another potential appliqation of such MAbs would be for the detection of salmonella organisms in clinical or food specimens. The advantages of using such MAbs are twofold: (1) they have been shown to be specific for sal-
C A P T U R E ELISA FOR S A L M O N E L L A B Y M A b 137
Table 2 Effect of inoculum size on the ELISA detection of Salmonella heidelberg in broth cultures
Mean* ELISA O.D. for Inocufum size (cell no.)
Broth cultures 2h
280 560 1120 2240 4480 8960 17920
0*039(-) 0.034( -) 0.031( -) 0.033( - ) 0.032(-) 0.035(-) 0.039( -)
0.055( -) 0.055( -) 0.060( -) 0.070( - ) 0.111(+) 0.179( +) 0.365( +)
0.039( -) 0.038( -) 0.034( -) 0.033( - ) 0.037(-) 0.042( -) 0.045( -)
0.075( -) 0.084( --) 0.094( +) 0.180( ) 0.271(+) 0.446( +) 0.716( +)
* Mean ELISA O.D. of duplicate determinations (reactivity).
t 2 ml of broth culture was concentrated four-fold through.centrifugation and resuspension of pelleted cells in 0.5 ml of medium. monellas and do not cross-react with other bacteria (Tsang et al. 1987); (2) a single antibody can detect a large number of different serotypes of salmonellas from the common 0serogroups of A (0: 2) to E (0: 3) (Tsang et al. 1987, 1991b). T h e MAb T 6 was found to have good affinity for salmonella antigens and whole cells, and can therefore be used as a capture antibody in sandwich ELISA. T h e method described is reproducible (Fig. 1) and is as sensitive as other ELISA assays described for measurement of soluble antigens (Yolken 1985). However, the nature of the antigen that the MAb T 6 detects may make it less favourable for the detection of antigens or bacterial cells of the smooth wild phenotype. Also we have observed that detection of LPS epitopes is most sensitive if whole cell suspension is used to measure both the cell-associated and soluble or released cell free LPS antigens together as has been done in the present experiments. This is because it is well known that Gram-negative bacteria release substantial amounts of LPS as membrane blebs during growth (De Voe & GilChrist 1973; Hoekstra et a l . 1976). I n experiments in which cells were centrifuged to remove the soluble released antigens the sensitivity of the assay was reduced by a magnitude of almost 10-fold (results not shown). T o compensate for the small numbers and also less exposed epitopes that the T6 antibody has to detect, we have shown previously (Tsang et al. 1987) that treatment of salmonella cells with Tris-EDTA buffer may make the cryptic core LPS epitopes more accessible for detection. In experiments in which cells removed from the soluble released antigens were treated with Tris-EDTA, the 10-fold loss in sensitivity due to removal of soluble antigens was recovered from making more subtle epitopes accessible for detection due to the chemical treatment. I t seems possible therefore
that Tris-EDTA buffer treatment of whole cell suspension could further increase the sensitivity of the present method as it would expose both the cell-associated and soluble free cryptic core LPS epitopes available for reaction with the MAb T6. Despite the limitation that the present MAb T 6 is not directed against a pronounced epitope in terms of its surface accessibility and its prevalence, we have shown that small numbers (1 120) of salmonella cells after 4 h culture in enriched broth can become detectable (Table 2). Also the presence of large numbers of other bacteria did not seem to interfere with the MAb T 6 capture ELISA for the detection of salmonella organisms. In view of the present results, it seems most probable that MAb T 6 is suitable for use in enrichment serology for the detection of salmonella. Presently we are trying this approach for the detection of salmonellas in clinical specimens after growth in selective enrichment media. ACKNOWLEDGEMENTS 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 to the Institute of Applied Molecular Biology, University of Hong Kong. We also wish to thank Prof. A.A. Lindberg (Department of Clinical Bacteriology, Karolinska Institute, Sweden) for the supply of Salm. senfenberg SH 1707 strain, and D r S. Schlecht (Max-Planck-Institut fur Immunobiologie, Germany) for the supply of purified LPS from Salm. typkimurium his
386. REFERENCES A N O N . (1988) Salmonellosis control: the role of animal and product hygiene. Report of a WHO Expert Committee. Technical Report Series 774. Geneva : World Health Organization. A V R A M E ASS. , & T E R N Y N C T.K(1971) , Peroxidase labelled
antibody and Fab conjugate with enhanced intracellular penetration. Immunochemistry 8, 1175-1 179. BENGE, G .R . (1989) Detection of Salmonella species in faeces by latex agglutination in enrichment broth. European Journal of Clinical Microbiology G Infectious Disease 8, 29+298. D E VOE, I.W. & G I L C H R I S T J .,E . (1973) Release of endotoxin in the form of cell wall blebs during in vitro growth of Neisseria meningitidis. Journal of Experimental Medicine 138, 1156-1167. E D W A R DP.R. S , & E W I N G W.H. , (1972) IdentiJcation of the Enterobarteriaceae pp. 146258. Minnesota Burgess. H I L L , A.B. (1977) A Short Textbook of Medrcal Statistics pp. 61-95. London: Hodder & Stoughton. D., HOEKSTRA , V A N D E R L A A N ,J . W . , L E I J , L . D . & W I T H O L T LB., (1976) Release of outer membrane fragments from normally growing Eshertchia coli. Biochemical and Biophysical .4cta 455, 8889-8899.
138 D E B B Y CHOl E T A L .
KAUFFMAN F ,. (1966) The Bacteriology of Enterobacteriaceae pp. 55-304. Baltimore: Williams & Wilkins. K E L L Y ,M . T . , B R E N N E R ,D . J . & F A R M E RJ,. J . (1985) Enterobacteriaceae. In Manual of Clinical Microbiology, 4th edn. ed. Lennette, E.H., Balows, A., Hausler Jr., W.J. & Shadomy, J.S. pp. 263-277. Washington : American Society for Microbiology. L E M I N O R ,L. & POPOFF,M.Y. (1987) Antigenic Formulas of the Salmonella Serovars 5th edn. Paris : Institute Pasteur. M O H R , H . K . , T R E N KH.L. , & Y E T E R I A N M. , (1974) Comparison of fluorescent-antibody methods and enrichment serology for the detection of Salmonella. Applied Microbiology 27,324-328. ROBISON,B.J., P R E T Z M A NC, . I . & M A T T I N G L YJ ,. A . (1983) Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae. .4pplied and Environmental Microbiology 45, 1816-1821. S A N B O R NW, . R . , L E S M A N AM, . & E D W A R D SE,. A . (1980) Enrichment culture coagglutination test for rapid, low-cost diagnosis of salmonellosis. Journal of Clinical Microbiology 12, 151-1 55. S P E R B E RW , . H . & D E I B E L ,R . H . (1969) Accelerated procedure for Salmonella detection in dried foods and feeds involv-
ing only broth cultures and serological reactions. .4p#lied Microbiology 17, 533-539. T S A N GR, . S . W . , C H A N , K . H . , C H A U , P.Y., W A N , K . C . , N G , M . H . & S C H L E C H T S. , (1987) A murine monoclonal antibody specific for the outer core oligosaccharide of Salmonella lipopolysaccharide. Infection and Immunity 55, 21 1-2 16. T S A N GR.S. , W., NIELSEN, K . , H E N N I N G ,M . D . , SCHLECHT, S. & A L E K S I ~ ,S. (1991a) A murine monoclonal antibody that recognizes a genus-specific epitope in the Salmonella lipopolysaccharide outer core. Zentralblatt fur Bakteriologie 274, 446455. T S A N GR.S.W., , S C H L E C H TS, . , A L E K S I ~ s., , CHAN, K . H . & C H A U ,P . Y . (1991b) Lack of the a-1, 2-linked Nacetyl-D-glucosamine epitope in the outer core structures of lipopolysaccharides from certain 0 serogroups and subspecies of Salmonella enterica. Research in Microbiology Institut Pasteur 142, 521-530. Y O L KE N , R .H . ( 1985) Solid phase enzyme immunoassays for the detection of microbial antigens in body fluids. In Manual of Clinical Microbiology, 4th edn. ed. Lennette, E.H., Blows, A., Hausler, W.J. Jr & Shadomy, H.J. pp. 949-957. Washington: American Society for Microbiology.