Journal of Applied Bacteriology 1992, 72, 302-308
Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of salmonella S. Kerr, H.J. Ball, D.P. Mackie, D.A. Pollock and D.A. Finlay Bacteriology Department, Veterinary Sciences Division, Stormont, Belfast, UK 3683/06/91: accepted 2 October 1991
s. K E R R , H.J. B A L L , D.P.MACKIE, D.A.POLLOCK A N D D.A. F I N L A Y . 1992. Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars a n d several related enterobacteria. T h o s e detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media b y conventional culture. O f these 58 were detected by t h e ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. T h e results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures a t a n early stage.
Salmonellas are ubiquitous enteric bacteria. Although asymptomatically carried by some animals they can cause important enteric and septicaemic diseases in man and other animals. T h e incidence of human infection is compounded by cross-contamination during intensive livestock rearing and food processing. Salmonella diagnosis in the majority of laboratories relies on costly and laborious culture screening with both non-selective and selective media. These culture procedures can take up to 5 d to confirm a sample as negative. ELISA-based techniques have the potential to simplify and accelerate detection. Such tests have been applied to salmonellas and include an antigen capture ELISA which uses affinity-purified polyclonal antisera raised to the flagellar common structural antigen (CSA-1) (Prusak-Sochaczewski & Luong 1989). There are others that are based on antigen capture using two monoclonal antibodies (MAb), one produced to a heat-extracted antigen and the other to a flagellar antigen (Mattingly 1984). These tests are commercially available but have limitations. T h e quantity of CSA-1 varies between Salmonella serovars (Smith et al. 1979; Ibrahim el al. 1985) and therefore there is variation in sensitivity. In addition, tests based on flagellins would be unable to detect non-flagellar serovars such as Salmonella pullorum or Salm. gallinarum, which cause pullorum disease and fowl typhoid. The MAb test produces many falsepositive results because of cross-reactivity with other enterobacteria (Curiale et al. 1990). Correspondence to : Dr S.Kerr, Bacteriology Department, I,iterinary Sciences Dizision, Stormont, Be(fast BT4 3SD, UK.
Salmonellas are antigenically complex ; over 2200 serovars have been differentiated by somatic lipopolysaccharide (LPS) or flagellar protein antigens (Le Minor 1984). They also share antigens with other enterobacteria. Genericallyspecific antigens have not been demonstrated. The aim of the present study was firstly to determine whether MAbs developed to salmonella outer membrane protein (OMP) contained any generic specificity, and subsequently to assess the use of selected MAbs in a microtitre antigen capture ELISA compared with conventional culture of clinical specimens. M A T E R I A L S AND M E T H O D S Antigens
Outer membrane (OM) fractions of Salm. enteritidis, Salm. typhimurium, Salm. virchow, Esrherichia coli, Klebsiella pneumoniae, Serratia rnarcescens and Citrobarter freundii were prepared by the standard sarcosine extraction method described by Filip et al. (1973). A 3-1 dextrose broth culture of each bacterium was incubated at 37°C for 16 h. The cells were pelleted by centrifugation at 15 000 g for 30 min, washed three times in saline and finally resuspended in 20 ml 10 mmol/l Tris HCl, 5 mmol/l EDTA, p H 7.8 (standard buffer). Cells were disrupted by five 1 min sonication pulses at 20 kcycles/s, each separated by a 1 min cooling interval. The cells were cooled in crushed ice before and during sonication. The sonicate was centrifuged at 15 000 g for 30 min to remove intact cells. The supernatant fluid was mixed with a quarter volume of 2 % (w/v) sodium n-lauroylsarcosine (Sigma) in standard buffer for 30 min at
S A L M 0 N E L L A M0 N OC LO N A L A N T I B O D I E S
room temperature followed by ultracentrifugation at 300000 g for 1 h. T h e pellet of O M was resuspended in 5 ml of standard buffer to which 1 volume of the detergent solution was added. After 1 h at room temperature the mixture was ultracentrifuged, washed once in saline and resuspended in 5 ml of standard buffer. Outer membrane protein was solubilized from the membrane fraction by the chaotropic reagent, guanidine thiocyanate (Sigma), which was added to give a 6 mol/l solution. The solution was mixed at room temperature for 1 h. Insoluble material was removed by ultracentrifugation at 300 000 g and the remaining solution dialysed against 100 volumes of 6 mol/l urea in standard buffer. Monoclonal antibodies
A BALB/c mouse was immunized subcutaneously with the solubilized OMPs of Salm. enteritidis mixed with the adjuvant, Quil A (Superfos, DK-Vedbaek, Denmark). Three days after the final intrasplenic inoculation with the O M P fraction the mouse spleen cells were fused with NSO myeloma cells (Galfre & Milstein 1981) and the hybridomas maintained in RPMI 1640 medium (Gibco, Paisley, U K ) supplemented with 20% gamma-globulin-free horse serum (Gibco). Culture fluids from actively growing hybridomas were screened by ELISA after coating polystyrene microtitre plate wells (Dynatech, Virginia, USA) with OMPs from the three salmonella serovars and four related enterobacteria listed above. Those hybridomas exhibiting salmonella specificity were cloned twice by limiting dilution. Monoclonal antibody screening was extended to plates coated with solubilized bacteria including Proteus, Pasteurella, Campylobacter, Pseudomonas, Yersinia, Strefltococcus and Staphylococcus species and Salmonella serovars. These bacteria were obtained from blood agar cultures suspended in saline and solubilized by heating at 56°C for 20 min with sodium dodecylsulphate (0.01 % w/v). The most promising cell lines were grown in dialysis sac culture (Sjorgren-Jansson & Jeansson 1985) or inoculated into mice to produce ascites fluid. Ascites production was initiated by intraperitoneal priming of BALB/c mice with Freund’s incomplete adjuvant 3 d prior to cell line inoculation (Mueller et at. 1986). IgG was purified from the ascites or sac fluid by the caprylic acid and ammonium sulphate precipitation method (McKinney & Parkinson 1986) and stored’at - 70°C. lmmunoblots
Sodium dodecylsulphate-poiyacrylamide gel electrophoresis (SDS-PAGE) of the Salm. enteritidis O M P extract was performed on a 12.5% running gel with a 5% stacking gel, and transferred to nitrocellulose by overnight blotting at 17 mA.
Strips from the nitrocellulose blots were incubated sequentially with the following: 2% (w/v) bovine serum albumin in 0.01 mol/l phosphate-buffered saline (PBS; p H 7.2) with 1 mmol/l E D T A and 0.5% (v/v) Tween 80; MAb dilutions, except for the negative control strip; and affinitypurified goat antimouse peroxidase (Jackson, Westgrove, PA, USA). Incubation was at 37°C for 1 h and strips were washed with PBS before the addition of each new reagent. The peroxidase substrate used was 0.5 mg 3,3’diaminobenzidine tetrahydrochloride/ml (Sigma) in 0.02 mol/l Tris/HCl buffer, p H 7-2, with 0.3 pl/ml 30% (v/v) H z O z . After incubation at room temperature for 10 min the reaction was stopped by washing with distilled water. ELSA
T h e antigen capture ELISA was performed on polystyrene microtitre plates (Dynatech) with 100 pl volumes of each reagent per well. T h e optimum reagent dilutions were established by titration. Purified MAb in 0.05 mol/l carbonate buffer, p H 9.5, was used to coat the wells at 4°C overnight. Thereafter plates were incubated at 37”C, and after each incubation the wells were washed with six changes of 0.01 mol/l PBS, p H 7.2, containing 0.05% (v/v) Tween 20. The incubation steps after coating of the plates were: test sample broth for 1 h ; the second MAb, which had been biotinylated (Hofmann et al. 1982), for 1 h ; streptavidin-peroxidase (Sigma) for 1 h ; the substrate, 0.1 mg 3,3’,5,5’tetramethyl benzidine hydrochloride (Sigma) dissolved in 10 p1 of dimethyl sulphoxide/ml citrate phosphate buffer, p H 5.5, with 1 pl/ml 30% (v/v) H,Oz’ for 25 min at 37°C. T h e substrate reaction was terminated with 50 pl 2.5 mol/l HZSO4 per well. The absorbance was measured at 450 nm with an ELISA plate reader (Titretek, Multiskan). T h e negative and positive controls consisted respectively of an uninoculated broth and a boiled salmonella culture containing 106 cfu/ml. Positives were recorded for values above twice that of the average negative control reading. Sensitivity
Test sensitivity was determined from cultures in selenite, Rappaport-Vassiliadis and dextrose broth of Salm. enteritidis, Salm. typhimurium, Salm. choleraesuis, Salm. dublin and Salm. mbandaka incubated at 37°C for 16 h. Decimal dilutions were then prepared in dextrose broth. Viable counts were determined on blood agar (Miles & Misra 1938) after incubation at 37°C for 16 h. Clinical samples
Clinical specimens were tested for salmonellas by standard culture methods in parallel with the antigen capture assay.
304 S. K E R R E T A L
Fig. 1 Outer membrane protein extracts
of salmonella and related enterobacteria, electrophoresed as described in Materials and Methods. Samples were run in duplicate. A, Salmonella enteritidis; B, Salm. typhirnurium; C, Salm. virchow; D, Escherichia coli; E, Klebsiella pneumoniae; F, Serratia marcesens and G , Citrobacter freundii. The molecular weight markers were: 1,66 kDa; 2,45 kDa; 3, 36 kDa; 4, 29 kDa; 5,24 kDa; 6, 20 kDa and 7, 14.2
Samples from a variety of animal sources, including tissues and faeces and environmental samples (dust and fluff from chicken-houses), were tested. In a preliminary trial 200 samples were tested after incubation for 48 h and, in the main investigation, 2100 samples were tested after incubation for 24 h in the salmonella selective media Rappaport-Vassiliadis and/or selenite and/or brilliant green broths. Environmental samples were pre-enriched in peptone water at 37°C for 16 h. Samples were sterilized by boiling for 15 min before testing with the ELISA. Specimens which were positive by ELISA but negative for salmonellas by culture were re-investigated by the standard culture procedure. Individual bacterial colonies were separately incubated in dextrose broth at 37°C for. 16 h. These cultures were then tested by ELISA and those reacting with the MAbs were identified with an analytical profile index (20 E, API, Bio Merieux, Basingstoke, UK).
cal trial. Immunoblot analysis (Fig. 2) showed that MAbs 6 C l l and 3D8 targeted a polypeptide of molecular weight 36 kDa. (a)
4 36 kDa
RESULTS The SDS-PAGE of the Salm. enteritidis O M P used to immunize the mouse for the hybridoma fusion and the other O M P fractions used in MAb screening are shown in Fig. 1. From the hybridoma fusion the seven MAbs were selected, each of which was unreactive with the nonsalmonella O M P or SDS-treated antigens but was reactive for all 57 of the salmonella serovars tested (Table 1). When applied to the antigen capture format, each MAb could be used for both capture and biotinylated development in one test or in combination with one of the other MAbs. T h e combination of MAbs 6 C l l and 3D8 used respectively for capture and biotinylated development gave the highest ELISA absorbance readings and so was used for the clini-
Fig. 2 The nitrocellulose blot of Salmonella enteritidis outer membrane proteins. (a) Stained with Coomassie blue. (b) Probed with monoclonal antibodies 3D8 and 6C11. The polypeptide targeted and its molecular weight are indicated
SALMONELLA MONOCLONAL ANTIBODIES
Table 1 Salmonella serovars positive with the antigen capture ELISA based on monoclonal antibodies targeting an outer membrane protein. Serovars are classified according to the
lipopolysaccharide ‘0’ group Group A paratyphi A
Group E bedford binza
lexington london meleagridis muenster newhaw senftenberg
brandenburg bredeney derby heidelberg indiana paratyphi B sandiego sarajane typhimurium
Group F parera
poona havana Wichita
braenderup cho1era esu is
Group I gaminara
edinburg gilbert goldcoast kentuck y mbandaka montevideo
Group 0 alachua
newport Ohio oranien burg
Atypical group arizonae
Group J kinodoni
rissen tennessee thompson virchow roterberg Group D berta dublin enteritidis gallinarum
panama pullorum rostock typhi
Salmonellas solubilized with 0.01 YO(w/v) SDS were successfully used as coating antigen during the hybridoma screening ELISA and found to give higher readings than with boiled antigen. In the antigen capture format this SDS concentration inactivated the capture antibody, and so boiled antigen was used.
T h e sensitivity of the antigen capture ELISA for the five salmonellas investigated was lo8 cfu,/ml, when the coating antibody was obtained from dialysis sac fluid. Ascitespurified MAb used as coating antibody gave a capture sensitivity of lo6 cfu/ml regardless of the medium used. Ascites fluid was shown to have a 50-fold higher antibody titre than sac fluid. T h e antigen capture ELISA in the preliminary trial of the 200 specimens tested after incubation for 48 h in selective media yielded 24 false-positive results compared with standard culture. These results were caused by strains of Enterobacter amnigenus (18) and Ent. cloacae (6). The main investigation involved incubation in selective media for 24 h. T h e results from the 2100 samples are given in Table 2. A total of 58 (2.7%) salmonella field strains comprising 10 serovars were detected by ELISA and confirmed by standard culture. T h e 65 (3.1%) false-positive ELISA results were attributable to the enterobacter species, mostly isolated from small intestine specimens. T h e cross-reactivity of Enterobacter spp. was markedly reduced when cultures were boiled for more than 30 min. T h e same extended boiling treatment had no effect on the activity with Salm. enteritidis or Salm. typhimurium. In a trial 100 samples were boiled for 1 h until a false-negative result was obtained for Salm. newport. This result was shown to be due to the prolonged boiling period and as a precaution in the main trial of 2100 test samples the boiling was restricted to 15 min. The three (0.14%) false-negative results obtained with the ELISA were shown in culture to be Salm. dublin present in small numbers isolated from bovine lung and small intestine. Two strains of Salm. arzzonae initially detected by ELISA only were confirmed with API 20E tests. DISCUSSION
This is the first report of MAbs produced to salmonella OMP. A wide variety of salmonellas, including nonflagellate serovars, have been detected with MAbs, with greater generic specificity than previously described. Antigen capture ELISA has proved to be a highly specific diagnostic tool for biological substances that have relatively few antigens, e.g. viruses and toxins. The antigenic complexity of the large Salmonella genus is problematic, in that attempts to uncover common and exclusive antigens must overcome two main obstacles. Firstly the salmonella serovar-specific antigens, particularly the outer membrane LPS ‘0’ chains, are highly immunogenic. This was shown in a previous unpublished study in our laboratory where MAbs produced to a sarcosine-extracted outer membrane fraction exhibited specificity to the serovar OMP. Secondly, many antigens are shared with other Enterobacte-
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Specimen Bovine Ovine Porcine Canine Equine Caprine Avian Miscellaneous Total
ELISA positives confirmed by culture* 22 14 6 -
ELISA False positives
Culture false negatives detected by ELISA
Table 2 Results of 2100 clinical samples cultured in selective media for 24 h and screened by antigen capture ELISA
8 7 1 1
* Salmonella dublin (34), Salm. choleraesuis (6), Salm. typhimuriun (3), Salm. derby ( 3 ) , Salm. mbandaka (3), Salm. colorado (2), Salm. tennessee (2), Salm. enteritidis (2), Salm. arizonae (2), Salm. indiana (1). riaceae, such as the glycolipid enterobacterial common antigen (Kunin 1963). This was evident in the present study as normal mouse sera would bind to salmonella and other enterobacteria O M P coatings used in ELISAs. As a precaution, antibody for biotinylation was obtained from dialysis fluid rather than ascites fluid, to avoid biotinylation of contaminating native mouse antibodies which might produce . false-positive results in the antigen capture ELISA. T o overcome L P S contamination of the sarcosineextracted OM, the OMPs were solubilized with guanidine thiocyanate. This chaotropic agent is highly effective at protein solubilization. In bacterial membrane purification (Moldow et al. 1972) this has been shown to remove most LPS. After dialysis against 6 mol/l urea the O M P remained in solution. Solubility in urea was selected to avoid interference with SDS-PAGE and to reduce toxicity to the mouse. The hybridomas produced to the Salm. enteritidis O M P were screened against OMP prepared from that serovar. Since the main aim of the study was to look for salmonella generically-specific antigens, two other common serovars, Salm. typhimurium and Salm. virchow, belonging to different LPS ‘0’ groups were also included in the initial screening. Outer membrane proteins of Citrobacter, Klebsiella, Serratia and’Escherichia strains were also screened because they are representative of the most closely related enterobacteria to salmonella (Brenner 1985). Despite this screening at an early stage, the final developed test revealed cross-reactivity with the genus Enterobacter, another member of the Enterobacteriaceae family. Although this antigen capture ELISA described has detected every salmonella serovar examined to date, it is open to speculation
about whether there is a generically-specific salmonella antigen. Immunoblot analysis revealed that MAbs 6C11 and 3D8 used in the trial were targeted towards a 36 kDa polypeptide (Fig. 2). This molecular weight corresponds approximately to that of the porins and OmpA which migrate closely together and both of which are highly immunogenic (Brown & Hormaeche 1989). Polypeptides of this molecular weight were also evident in the SDS-PAGE of the salmonella-related enterobacteria O M P used in screening (Fig. 1). On the basis of the trial 58 (2.7%) of the samples were salmonella-positive by the ELISA and substantiated by standard culture. Despite 65 (3.1 %) false-positive results obtained with the ELISA, the remaining 94.2% of screened cultures could have been eliminated after 24 h, offering potentially considerable financial savings. Furthermore, when ELISA-positive samples are cultured the lactosepositive Enterobacter spp., the false-positive reactants, are easily distinguishable from the usually lactose-negative salmonellas. T h e ELISA test is negative for the nonpathogenic Citrobarter spp. which can frequently cause confusion in standard culture procedures, delaying diagnosis due to biochemical similarities with salmonellas (Van der Walt & Steyn 1989). Enterobacters are common gut commensals. During this trial most ELISA false positives were of intestinal origin. The ELISA might give fewer false positives if food rather than clinical samples was tested, where enterobacters would be comparatively infrequent. If Enterobarter remains the only cross-reacting genus there could be selective methods to reduce its multiplication in culture relative to salmonellas. In addition, the antigen capture ELISA should detect
SA L M 0 N E LLA M 0 N 0 C L O N A L A N T I BOD I ES 307
salmonellas amongst abundant bacteria particularly Proteus and Klebsiella spp. which on agar plates can mask small numbers of salmonella. T h e commercially available Salmonella-Tek ELISA (Organon Teknika Corp., NC, USA) employs two M A b s which, although they d o not cross-react with enterobacters, do cross-react with other Enterobacteriaceae including Cztrobacter strains (D’Aoust & Sewell 1988). In trials, the Salmonella-Tek E L I S A has given false-positive and falsenegative results respectively of 6% and 0.4% (Bailey et al.) and 43.2% and 8.4% (St Clair & Klenk 1989). T h e sensitivity of the Salmonella-Tek ELISA at lo6 cfu/ml (Eckner et al. 1986) is comparable with the test developed in this study. Salmonella numbers can remain below this value in cultures heavily contaminated with non-salmonellas (D’Aoust 1981). Therefore selective culture methods remain important for pre-enrichment prior to testing. In the present study three bovine samples containing Salm. dublin in small numbers isolated from brain and lung tissue were not detected by our ELISA. Positive ELISA results were obtained for intestine and liver specimens from this clinical case which contained large numbers of salmonella in culture. T w o strains of Salm. arizonae isolated from snakes were initially detected by ELISA. These atypical salmonellas were missed by standard culture because they were lactose-positive. I n conclusion, the MAb ELISA test developed in this study. has the potential for making a presumptive salmonella-positive diagnosis within 24 h of culture in selective enrichment broth. Work is continuing to investigate whether pre-enrichment in non-selective medium would eliminate false negatives, and on cultural and serological methods of eliminating the false positives. ,
ACKNOWLEDGEMENTS W e thank Ross Breeders Ltd, Midlothian, Scotland for funding this project. Appreciation is extended to J. Donaghy, Food Microbiology Division, Newforge, Belfast, D r J. Barr and G.M. Hogg of the Bacteriology Department, Royal Victoria Hospital, Belfast and D r T. Wilson of the Bacteriology Department, Belfast City Hospital for providing salmonella serovars.
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