CA2078162A1 - Specific anti-salmonella monoclonal reagents, and unique serological approach for the detection of different common serotypes of salmonella and the like - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The detection and identification of Salmonella using murine hybridoma monoclonal antibodies is disclosed. A
panel of anti-Vi and anti-O specific monoclonal antibodies has been successfully applied for the serotyping of Salmonella while anti-lipopolysaacharide core specific monoclonal antibodies have been applied for the detection of different common serotypes of Salmonella. Using a combination of the anti-O and anti-core specific monoclonal antibodies in a sandwich capture ELISA, it is possible to accomplish simultaneous detection and serogrouping of Salmonella. The immunoassay approach for the detection of Salmonella uses multiple time point assay, which measures a rise in the level of the targeted microorganism or its antigens, thereby allowing better differentiation of true positives from non-specific reactions in the immunoassay.
This assay approach is therefore most suitable for immunoassay of infectious microorganisms as long as specific antisera or antibodies against the targeted microorganisms are available.

Description

2~7~ ~2 FIELD OF INVENTIO~
This invention relates to the use of a panel of murine hybridoma monoclonal antibodies that are directed against the Vi and O antigens of different common serogroups of Salmonella. These anti-Salmonella monoclonal antibodies can be used immediately after preparation without prior absorption as specific factor sera for serotyping of Salmonella.
A murine hybridoma cell line that produces monoclonal antibodies against the outer core region of the Salmonella lipopolysaccharide has also been developed for the detection of Salmonella To use such anti-Salmonella monoclonal antibodies in immunoassays for this diverse group of intestinal pathogens, a method has been developed that uses a combination of tnese reagents as well as a multiple time point assay that can detect and identify as well as differentiate between true positive findings from non-specific reactions that are prone to occur in immunoassays with biological specimens.

BAC~GROUND ART
Salmonella comprises a diverse group of intestinal pathogens affecting both animals and man. Some serot~pes of Salmonella can cause invasivP systemic diseases such as enteric fever in man and fowl typhoid in chickens. Others are better known to be the most common cause of food-poisoning. Definite diagnosis of salmonellosis depends on the detection and isolation of the organism which is then identified by biochemical and serological means.
Serological identification of Salmonella is normally effected by slide and/or tube agglutination technique using polyclonal antisera prepared in experimental animals, e.g.
rabbit. Monospecific antisera for serotyping of Salmonella are prepared by exhaustive absorption with members that 2~78~ 62 share common antigenic factors. Despite the elaborate absorption procedures, which are timing consuming and have inherent problem of batch to batch variation that is difficult to control, the resulting antiserum is not truly mono-specific and still contains a mixture of antibodies.
Thus serotyping of Salmonella based on polyclonal antisera suffers ~rom the ill-defined nature of antisera, and there is a need to develop a more efficient approach to raise anti-Salmonella antibodies for serotyping.
The conventional approach for detection of Salmonella involves culture of the organism from specimens using standard bacteriological techniques which may involve inoculation of the specimens into a variety of media designed to enhance even small numbers of Sàlmonella to be detected. One commonly employed approach is to enrich the Salmonella in a broth medium followed by plating the incubated broth culture onto a solid agar medium which would allow any suspected Salmonella cells to grow up as visible colonies. The latter are then identified by a battery of biochemical and serological tests (Ewing W.H.
1986. Identification of Enterobacteriaceae, 4th edition.
New York: Elsevier). Because this elaborate protocol is both time consuming (taking as long as several days) and labour intensive (demanding a lot of experience from the technical personnel to recognize any suspected colonies from the final culture step on agar plate), there has always been a need to develop more rapid and less demanding method ~or the detection of Salmonella.
Hitherto, a number of alternative methods have been described, such as a DNA probe method (Fitts, R., Diamond, M., Hamilton, C., and Neri, M. 1983. DNA-DNA hybridization assay for detection of Salmonella spp. in foods. Applied and Environmental Microbiology, vol. 46: pp. 1146-1151;
Scholl, D.R., Kaufmann, C., Jollick, J.D., York, C.K., Goodrum G.R., and Charache, P. 1990. Clinicial Application ~7~2 of novel sample processing technology for the identification of Salmonellae by using DN~ probesO Journal of Clinical Microbiology, vol. 28: pp. 237-241), electrioal conductance (Smith, P.J., Boardman A., and Shutt, P.C. 1989. Detection of salmonellas in animal feeds by electrical conductance. Journal of Applied Bacteriology, vol. 67: pp. 575-588), and immunosensor (Luong, J.H.T., Prusak-Sochaczewski, E., and Guilbault, G.G. 1990. Development of a piezoimmunosensor for the detection of Salmonella tY~himurium. Annals of the New York Academy of Sciences, vol. 613: pp. 439-443). However these alterative approaches have not gained wide acceptance into the routine laboratory either because of problems in sensitivity or because of lack of specificity.
Since one of the key tests in the conventional approach for the identification of Salmonella rests on the serological typing based on the use of specific antiserum against Salmonella antigens, many alternative methods for Salmonella detection have been developed using ~0 immunological approaches with antisera developed for the serolo~ical typing of the bacteria (Sperber, W.H., and Deibel, R.H. 1969. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Applied Microbiology, vol. 17: pp. 533-539; Gibbs, P.A., Patterson, J.T., and Murray, J.G. 1972. The fluorescent an~ibody technique for the detection of Salmonella in routine use. Journal of ~pplied Bacteriology, vol. 35: pp. 405-413; Minnich, S.A., Hartman, P.A., and Heimsch, R.C. 1982. Enzyme immunoassay for detection of Salmonellae in foods. Applied and Environmental Microbiology, vol. ~3: pp. 877-883;
Cerqueira-Campos, M.-L., Peterkin, P.I., and Sharpe, A.N.
1986. Improved immunological membrane filter method for detection of food~borne Salmonella strains. Applied and Environmental Microbiology, vol. 52: pp. 124-127; Hassan, 2~7~2 J.O., Mockett, A.P.A., Mcleod, S., and Barrow, P.A. l9gl.
Indirect antigen-trap ELISAs using polyclonal antisera for detection of group B and D Salmonellas in chickens. Avian Pathology, vol. 20: pp. 271-281). Indeed over the years, there have been many immunoassays described for the detection of Salmonella (reviewed in Tsang, R.S.W. and Nielsen, K.H. 1992. Immunoassays for Salmonella. The Genetic Engineer and Biotechnologist, vol. 12: pp. 14-18~, but many still lack the specificity required due to the numerous common cross-reactive antigens present in the different members of the Family of Enterobacteriaceae and the inherent properties of polyclonal hyperimmunized e~perimental animal sera.
In the past decade, monoclonal antibodies against the common epitopes of Salmonella flagella antigen have also been applied in immunoassays for Salmonella (Robison, B.J., Pretzman, C.I., and Mattingly, J.A. 1983. Enzyme immunoassay in which a myeloma protein is used for detection of Salmonella. Applied and Environmental Microbiology, vol. 45: pp. 1816-1821; Mattingly, J.A., Robison, B.J., Boehm, A., and Gehle, W.D. 1985. Use of monoclonal antibodies for the detection of Salmon~lla in foods. Journal of Food Technology, vol. 39: pp. 90-94).
However not all salmonellae are detected by such monoclonal antibodies. Also false positive results due to cross-reactions with Citrobacter have been reported and results were obtained only after three days (Clayden, J.A., Alcock, S.J., and Stringer, M.F. 1987. Enzyme linked immunosorbent assays for the detection of Salmonella in Foods.
Immunological Techniques in Microbiology, pp. 217-229).
Therefore there is still a demand for a truly monospecific antiserum or antibody against the genus Salmonella that does not show cross reactions with non-Salmonella bacteria.
As mentioned above, although a number of alternative procedures for salmonellae detection have been proposed, ~137~2 the most commonly described alternative method is immunoassay. This is probably a re~lection o~ the practicality of such an approach in terms of both availability of reagents and familiarity of immunoassay technology to laboratory personnel. However like many other assays, the quality of the sample and the reagents employed primarily determine the accuracy of the assay.
Presence of no~ious substances in the sample may cause non-specific stickiness of reagents used in immunoassay leading to high background or false positive results. Although controls, such as inclusion of no-capture antibody or no-enzyme detector antibody molecule, may allow the detection of a non-specific reaction or high background, they do not necessarily provide an answer to differentiate between true positive results from non-specific reactions.
To develop an improved immunoassay for detection of Salmonella, there is the requirement of a good specific antiserum or antibody against the many di~ferent serotypes of Salmonella, as well as an immunoassay method that would minimize interferences from noxious substances which may be present in the many different types o~ possible samples that are known to or may contain Salmonella bacteria.

BROAD DESCRIPTION OF THE I~3VENTIOld An object of the invention is to conduct serotyping of Salmonella by employing monoclonal antibodies which can be used without prior absorption as factor specific sera.
The existing serotyping sera are produced in ~xperimental animals by injection with Salmonella whole cells and absorptions with related bacteria are undertaken on the resulting antiserum to remove cross-reacting antibodies and hence rendering the antisera monospeci~ic.
The anti-Salmonella monoclonal antibodies developed according to the invention are truly monospecific and hence can bs used without prior absorption. In contrast to the 2~7~2 conventional anti-Salmonella serotyping sera, the monoclonal antibodies have much less batch~to-batch variation in the process of production. Being truly monospecific in kheir serological specificity, and having less inherent variations from batch-to-batch preparation, the monoclonal antibodies are therefore superior to the conventional sera produced in experimental animals.
Another object of the invention is to provide an improved laboratory diagnostic method for the detection of salmonellosis. The advantage of the diagnostic method according to the invention which uses enrichment serology includes reduction of costs and time required as compared to the conventional approach which relies on the isolation and identification of Salmonella growing up as colonies on agar media. The present method also has advantages over other enrichment serology methods for the detection of Salmonella in that:
(a) it is more specific than those described in the literature since it uses a monoclonal antibody that is directed against a genus-specific epitope present in the outer core region of the Salmonella lipopolysaccharide structure; and (b) it has advantage over both the conventional culture approach and the existing enrichment serology mPthod in that it can also be adapted to carry out serogrouping of the Salmonella at the same time as it is detected.
Therefore it can provide additional information as to the type of Salmonella that is being detected and the serogrouping step adds confirmation to the presence of Salmonella in the sample.
A further object of the invention is to provide comparative results obtained for the sample both before and after suitable incubation in a medium that would allow the target microorganism to multiply and hence the amount of antigen to be detected to increase over time. As a result, 7 2~7~

the immunological method according to the invention permits better discrimination of true positive findings from false positive results which are common in immunoassays due to non-specific stickiness of immunological reagents in the test, especially when the test is done on biological or food samples.
Accordingly, one aspect of the invention provides a reagent for use in the serotyping of Salmonella, comprising a panel of murine hybridoma anti-Salmonella monoclonal antibodies against the Vi and the somatic O-antigens of common serogroups of Salmonella which can be used without prior absorption as factor specific sera, said panel comprising one or more monoclonal antibodies selected from the group consisting of M02, M04, C1-1, MO8, M09, E1-1, E2-l and Vi; and said common serogroups including A, B, C1, C2,D1, E1, E4 and phage modified E1 Salmonella.
Another aspect of the invention provides a reagent for specific detection of different common serotypes of Salmonalla which comprises a murine hybridoma monoclonal antibody directed against the genus-specific epitope of the Salmonella lipopolysaccharide outer core region, said monoclonal antibody consists of T6 murine hybridoma monoclonal antibody.
A further aspect of the invention provides a method for specific detection of different common serotypes of Salmonella, which comprises, (a) treating a test sample in an enrichment culture medium for incubation; and (b) applying the resulting enriched test sample to a sandwich capture ELISA assay with murine hybridoma monoclonal antibodies directed against the genus-specific epitope of the Salmonella lipopolysaccharide outer core region, said monoclonal antibodies consisting of T6 murine hybridoma monoclonal antibodies.
A further aspect of the invention provides a method for simultaneously detecting and serogrouping Salmonella 8 ~7~
serotypes, which comprises applying sandwich capture ELISA
assay to an enriched test sample using unlabelled monoclonal antibody T~ as capture antibody, and enzyme labelled monoclonal antibodies that are directed against the Vi or 0-antigens of serogroups A, B, C1, C2, D1, E1, E4 and phage modified E1 Salmonella as detector antibodies.
A further aspect of the invention provides a quantitative immunoassay method for the detection of Salmonella, which comprises: (a) treating a test sample in an enrichment culture medium for incubation; (b) applying the resulting enriched test sample to a sandwich capture ELISA assay with a panel of murine hybridoma anti-Salmonellalipopolysaccharidemonoclonal antibodies, either against the Vi antigen or the somatic 0-antigens of common serogroups of Salmonella and/or the genus-specific epitope present in the Salmonella lipopolysaccharide outer core region, as capture and detector antibodies, said panel comprising one or more monoclonal antibodies selected from the group consisting of Vi, M02, ~04, Cl~ 08, M09, El-l, E2-l and T6; said common serogroups including A, B, C1, C2, D~, E1, E4 and phage modified E1 Salmonella; and (c) taking multiple time point ELISA measurements on the enrichment culture at 0 hour of incubation and at subsequent time : during the Salmonella culture step.
Yet a further aspect of the invention provides a quantitative immunoassay method Por the ~etection of infectious agents, which comprises taking multiple time point ELISA measurements of a test sample undergoing enrichment serology testing .in the presence of a specific antiserum against the individual infectious microorganism at 0 hour of incubation and at subsequent time during a culture step.

2 ~

DETAILED DESCRIPTION OF T~E INVENTION
8eroloyiaal R~agents :
A panel of murine hybridoma cell lines that produce specific and unique monoclonal antibodies against different Salmonella antigens has been developed for improving the laboratory diagnosis of salmonellosis. The specific anti-Salmonella monoclonal antibodies include those directed against the somatic O-antigens of the most common O
serogroups (namely those from groups A to E) as well as the Vi antigen of Salmonella. Such a panel of anti-Salmonella monoclonal antibodies is capable of use without prior absorption as typing antisera for Salmonella.
The panel of murine hybridoma monoclonal antibodies specific for different parts of the Salmonella somatic or lipopolysaccharide (LPS) antigen has been developed for use in immunoassays for Salmonella detection and/or typing.
These murine hybridoma monoclonal antibodies include: (i) T6 which is directed against the common outer core of Salmonella LPS; and (ii) those which are directed against the Vi and the O-antigens of the most common O-serogroups of A, B, C1, C2, D1, E1, E4 and phage modified E1 (Vi, MO2, MO4, Cl-1, MO8, MO9, El-l, and E2-1). These hybridoma cell lines have been deposited in the European collection of Animal Cell Cultures, PHLS Centre for Applied Microbiology & Research, Public Health Laboratory Service, Porton Down, Salisbury, England (Cell Line Deposit ~ccession Numbers: T6 - 91070323, MO2 - 91070318, MO4 - 91071820, Cl-l 91070319, MO8 - 91070320, MO9 - 91070321, El-l - 91070322, Vi6a - 91071816, and E2-1 - not yet deposited). These murine monoclonal antibodies can be used without prior absorption as either genus-specific or factor specific sera for the detection and/or serotyping of Salmonella.
Mouse ascitic fluids induced by these murine hybridoma cell lines can be used directly without absorption for slide agglutination test to identify Salmonella.

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The mouse ascitic fluid induced by the murine hybridoma cell lines containing the monoclonal antibodies should be titrated before use to find out the most optimal amount or dilution for use in the slide agglutination test using Salmonella organisms that are expected to give either positive and negative slide agglutination reaction. The diluted, ascitic fluid may be stabilized in diluent containing preservative and may be stored at either -200 C
for long term storage or at 40 C. Positive and negative control Salmonella microorganism should be used to test the monoclonal antibodies at the beginning of each working day or at least weekly for quality assurance purposes.
The slide agglutination test is carried out by suspending a suspected Salmonella growing on a non-selective agar medium in a drop (e.g. 10 ~1) of saline orphosphate buffered saline with or without phenol to give a fairly dense milky suspension and 10 ~1 of suitably diluted monoclonal antibody is added with mixing to the drop of bacterial suspension. Then the slide is rock with gentle rotation for 1 minute and any clear-cut clumpiny of the bacteria is noted as positive agglutination. All positive bacteria should also be tested with either saline or an irrelevant monoclonal antibody in place of the specific anti-Salmonella monoclonal antibody to check for auto-agglutination of the Salmonella bacteria under test.
The monoclonal antibodies can also be used afterattachment to either protein A-Staphylococcal cells (which are available commercially) or to inert latex particles (also available commercially) after suitable purification by DEAE ion exchange chromatography and/or Sepharcyl~ gel filtration chromatography. In either case, this co-agglutination approach would enhance the slide agglutination reaction with positive Salmonella bacteria and at the same time uses less amount of the specific monoclonal antibodies.
* trade mark 2~78~6~

An additional usage of these monoclonal antibodies is to couple them to magnetic and/or immunomagnetic beads for the specific separation and detection of Salmonella.
It should be noted that bacterial that exhibit autoagglutination can not be tested by slide agglutination method using either unconjugated ascitic fluid monoclonal antibodies or monoclonal antibodies conjugated to either protein A-Staphylococcal cells or latex particles.
Furthermore, as a general rule, serotyping of bacteria should not be done on bacteria grown up on selective agar medium because of possible false positive result.
Also, ascitic fluid store under harsh conditions (such as exposed to excessive heat or been through repeated freezing and thawing cycles) may loose their specific reactivities with their corresponding Salmonella. When not in use, the antibodies are best stored at refrigerator temperature of about 4O C or for long term storage at -20O
C.

Nethod for Serotypinq and Detecti~g of Balmonella :
The use of murine hybridoma monoclonal antibodies directed against the outer core region of the Salmonella lipopolysaccharide as genus-specific polyvalent anti-Salmonella sera for the detection of different common serotypes of Salmonella is novel. Other serological reagents that have bean employed for Salmonella detection are either pools of polyclonal reagents directed against the most common O-factors of Salmonella or are polyclonal antiserum against poorly defined cell wall antigens (such as the so-called "Common Structural Antigen, CSA") or monoclonal antibodies against flagellar epitopes common to different Salmonella. Howaver in the case of the latt~r, the monoclonal myeloma proteins are not truly specific for Salmonella since there are cross-reactions with another member in the Family of Enterobacteriaceae, Citrobacter, 12 ~78t 62 and not all Salmonella are detected by such monoclonal myeloma proteins.
For serotyping purposes, slide agglutination test with diluted ascitic fluid induced by the hybridoma cell lines or ascitic fluid antibodies attached to either protein A-Staphylococcal cells or latex particles have been found to be suitable.
The sandwich capture ~LISA can be done with unlabelled monoclonal antibody T6 as capture antibody and enzyme labelled monoclonal antibody T6 as detector antibody for the detection of the most commonly encountered Salmonella serotypes. If enzyme labelled monoclonal antibodies that are directed against the O-antigens of serogroups A, B, C1, C2, D1, E1l and E4 Salmonella are used as detector antibodies, then simultaneous detection and serogrouping can be done in the same test and the results obtained with the enzyme labelled O-factor specific monoclonal antibodies can also be used as a confirmation of the results obtained with monoclonal antibody T6 alone.
The sampl~s to be tested (clinical material, food or environmental samples) are put into a suitable enrichment medium (this may include a pre-enrishment step in a non-selective broth medium followed by incubation in a selective enrichment broth medium for Salm_nella) for incubation to allow even small numbers of Salmonella to multiply to detsctable level by the immunoassay. Commonly available enrichment media ~or the culture of Salmonella include M-Broth (non-selective), Rappaport Vassiliadis medium, Selenite-Cystine and Tetrathionate broths (selective enrichment for Salmonella).
The sandwich capture ELISA is done using Dynatech Immulon 2 microtiter plate coated with the ascitic fluid monoclonal antibody T6. The optimal amount of T6 antibody used for coating is determined by checker-board titration and the diluent for diluting the T6 antibody for coating is * trade mark ~7~ 2 carbonate-bicarbonate buffer, pH 9.6 (1.59 g Na2C03, 2.93 g NaHC03, in one litre of distilled water). Wells are filled with 100 1 of the T6 antibody for coating at 4O C
overnight. Following three washes with phosphate buffered saline (PBS) with 0.05% Tween 20 (PBS-Tween), wells are blocked with 300 1 of 2% bovine serum albumin (BSA) in PBS
at 37O C for 1.5 hour. After three more washes as above, samples are added at a volume of 100 1 per well for incubation at 37~ C for 1 hour. Then wells are washed four more times as above and Salmonella antigens captured are detected by suitable dilution (determined by titration) o~
enzyme (e.g. horseradish peroxidase) conjugated monoclonal antibody T6 using 100 1 per well for incubation at 37O C
for 1 hour. After a final wash of four times as above, wells are developed by addition of 100 1 per well of 0.1 mg/ml of O-phenylenediamine dihydrochloride (OPD) in a substrate buffer made up of 0.05 mol/l Na2HP04, 0.025 mol/l citric acid with 0.003% H202. The colour development is carried out in the dark at 37O C for 30 minutes, and the substrate-enzyme reaction is stopped by addition of 25 per well of 4N H2S04. The colour developed is read with a microtiter plate ELISA reader (e.g. Dynatech MR 710 microtiter plate reader) at the wavelength 492 nm using suitable filter. Controls including standard amounts of a Salmonella Ra-LPS as well as overnight broth culture~ of any common Salmon~lla serovars are included together with buffer without any antigen or Salmonella serving as negative controls. In deciding on an optical density value ~or calling a test sample as positive, mean optical density value from no antigen control experiments plus three times their standard deviation is used. This will allow for more than a 95% confidence limit.
Under normal testing condition, it is recommended that samples be tested in triplicates. Therefore in a 96 wells microtiter plate, 32 samples of unknown, positive and * trade mark ;

. : , 2~7~162 negative controls can be accommodated in one run on a single plate.
For simultaneous detection and serogrouping of Salmonella, samples are set up in multiple wells so that some wells will receive enzyme labelled monoclonal antibody T6 while other wells will receive enzyme labelled anti-Salmonella 0-antigen specific monoclonal antibodies.
However, it is worth noting that genuine cross-reactions of the monoclonal antibodies developed against Salmonella lipopolysaccharide antigens with non-Salmonella bacteria may create false positive results. For example it is known that monoclonal antibody T6 will react with bacteria having the disaccharide N-acetyl-D-glucosamine alpha 1,2 linked to D-glucose as in the Escherichia coli having the R2 core type. However such Escherichia coli has been found in only a couple of serotypes that are not commonly encountered.
Also, failure to grow up sufficient numbers of the Salmonella organisms in a suitable enrichment broth media may lead to false negative results.

Multi~le Time Point Immunoa~Yay:
The immunoassay approach that follows the kinetics of the growth of the target infectious microorganism by taking multiple measurements during the culture step of the target microorganism is new and add~ advantage to the existing method by including a self control for each specimen as a reference point to compare results with. This feature serves as a control for non-specific reactions that are prone to occur in immunoassays done on biological as well as food and environmental samples.
By performing the immunoassay or the target microorganism both at 0 hour of incubation and at different times during culturing of the sample for the target organism in a suitable broth medium allows for accurate 2~7~62 documentation of the presence of viable target microorganism. After the base line 0 hour measurement has been noted, the subsequent frequency of taking a sample out from the broth culture to do immunoassay will depend on the specimen type and the anticipated number of target microorganism that may be present in the sample as well as the detection limit or sensitivity that one aims to achieve. In practice, at least two samples should be taken, one at the 0 hour of incubation and one at tha end (preferably after overnight incubation of the broth medium to allow detection of even very low numbers of the target microorganism) of the incubation period for culturing of the target microorganism. If more than two assays can be done on a single sample, then they should include at least the two time points as described above plus samples that have been incubated 4 to 6 hours allowing adequate time for target microorganism multiplication.
This novel approach offers an easy method to discriminate any false positive results due to non-specific stickiness of the immunological reagents in the test from the genuine positive results due to the presence of Salmonella as the latter would increase during incubation of the culture while the former would only remain static.
This approach can also be extended to the deteation of other infectious ayents with enrichment serology method, provided that specific antiserum against individual microorganism is available. Other immunoassay methods such as competitive assay can also be used with this approach.
This applies to the nature of the anti-serum too.
Polyclonal sera instead of monoclonal antibodies can also be used with this method.
However, it has been found that non-physiological pH
and ionic condition of the assay medium may lead to spurious reactions.

~7~2 Embodiments of the invention will now be described, by way of laboratory or commercial tests results, with reference to the accompanying drawings, in which:
Figure 1 shows standard dose-response curves for the detection of Salmonella typhimurium Ra and smooth LPS.

~A) S0rotyping of Salmonella with Nurine Nonoclonal Antibodies:
Table 1 describes the eight murine anti-Salmonella hybridoma monoclonal antibodies, their isotypes and agglutinating titers against homologous serogroup of Salmonella. The slide agglutination reaction is in ~ll cases very strong and occur within on~ minute of mixing the diluted ascitic fluid with the bacterial suspension.
The specificity of the monoclonal antibodies is demonstrated by testing the antibodies with standard strains obtained from thP American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A. as well as clinical isolates of Salmonella obtained from a university teaching hospital in Hong Kong.

17 ~7~2 .i.b.le 1. Isotypes and agglut;nating titers of mouse ascitic fluid monoclonal antibodies against somatic 0-antigens ~,f serogroups ~ to E ~alrnclnella ....______________________________________________________________ Monoclonal SerogroupSlide ac~,~c,lutination ntibod~ specificity Isotype titer agair,st homologGus seroc,roup of Salmonella __ ________________ ____ _____________________ _____ _______~
M0? ~ IgGl1:32 ~Q4-4 S Ic,Gl1:16 Cl-l ~L IgG31:'2 ~10S C2 IgG31:32 MG~ Dl I gGl1: :~52 El-l ElfE4 IqGl. 1:64 *
E -l E2,fr-3 IgGl1:3~
:
Vi Vi antigen IgG2b1:32 of Salmonella and Citrobacter __ ____________ __._______ __ __ ___ ___ ________________ ____ *
strains of serogroups E2 and E3 are phaga modified serogroup El bacteria, an~l they are nol~i classifieci as serogroup E
strains. E2 and E3 are previous designations.

. .

2~7~62 Amongst the 13 non-Salmonella coliform ATCC standard strains, none was found to be agglutinated by the anti-Salmonella monoclonal antibodies. In contrast, 5 standard Salmonella strains obtained from ATCC were all agglutinated by the anti-Salmonella monoclonal antibodies according to their serogroup specificity (Table 2).

19 2~7~2 Table 2. Spe'ci~icity c,f the monoclonal antibodies againsL somatic 0-antigens o~ serogroups ~ to E ,S,,.,~,.l,"m""o,n,e.,,l,,l,"a", teste~ by slide a~91 U Li nation with ~TCC~ ~strains ______________________________________________________________________ , Salmonella Sero- ' ~gglutination with group Serotype ~TCC No M02 M04 C~ 08 M09 E1-1 E~-1 ___ _________________________________________________________________ S paratyphi ~ 9150 ~ - - - - - - ;
C1 S choleraesuis 13312 - - +
C2 S. newport ~962 ~- - - +
D1 S. enteritidis 13076 + - - - . ~ - -S. typhi 19430 - - - - ~ - -.~ . .. . . .. = . ...
Non-Salmonella ~TCC No~ , . -specles Escherichia coli 25922 - - - - ~ - - -Escherich;a coli 35~18 Shigella fle~neri 29903 ' Shigella sonn~i 25931 Klebsiella pneumoniae 13883 Enterobacter 27155 - - - -a~lomerans Enterobacter,cloacae 23355 - - - .- , .-Proteus mirabilis 7002 - - - . .-Providencia.rettgeri 29944 - - - '-' ~ Citrobacter freundii 8090 Serratia marcescens 8100 Edwardsiella tarda 15947 Yersinia enterocolitica 9610 ____ _ _ ___ __ ______________________________ ________ ___________ Strains were purchased from Rmerican Type Culture Collection..

M02 and M04 were used at a dilution of 1:8~ while C1-1, M08~ M09, E1-1 and E2-1 were used at a dilution of 1:16 - . , 2~J7~1~2 Further studies with clinical wild isolates from patients suffering from salmonellosis substantiated their specificity (Table 3).

2~7~

Table ~ Sero~yping of 10~ clinlcal isolates of ,',~,c~ln!on~,l,l,,a, by slid~-agglutination .est uslng a panel or anti--,S"al"monel,,l,,a mono clonal antibodies ______________________________ _____________________________________ :K `t ~ero- Serotype No of ~gglutinatlon with r~bs :
group stl-alns MO M04 Cl-l riO~ MO~ El-l E2-1.
tested ..____________________________________________________________________ S paratyphi ~ S ~- ~
- C S typhimurium 17 - +
S d~rby 13 - +
S~ sa;ntpaul 4 - +
S agona ~ - +
' ~. paratyplli' S 1 . - +
01 S infantis 8 - -S. braenderup, ,, 1, , - , -S thompson 1- - -C2 S manhattan 14 - -'' S litchfield 4 , - -S chailey ~ - -S bloc~ley 2 - -S newport 2 - -Dl S. enteritidis 6 + -S panama 2 ~
S~ durban , 1 - -S typhi~ 2" +~- -.. ,: . .. .. . . . .. .
Ei S, anatum S - - - - - +
'S~ weltevreden 3 - - - - - +
S meleagridis 1 - - - ~ ~ + ~
E4 S krefeld ~ - - - - - +
E2 S portsmouth 4 - -' - - - -____________________________________________________________________ Total No = 107 *

Serogroup and serotypes of S"al,"moc,el,l,a, were identified by slide agglutination test using commerical ,S,,,,a,l,m"o,n,e,ll,,,a agglutlnatln~
, antisera.
+
M~bs M02 and M04 were used at a dilution of 1:8, and M~bs Cl-l~ ~08, MO97 El-l and E2-1 were used at a dllution of'l:l6 in the slide agglutination test.
One strain of S~, typhi was agglutinated by both M02 and M09 while the other strain of S.,_,t,y~h,i was agglutinated by ~09 only .... ,.. - - - :

22 2~7~2 The only result that did not show concordance with the conventional polyclonal antisera was found with the mono~lonal antibody M02 which is specific or the dideoxyhexose paratose linked to the backbone somatic 0-antigen of serogroup A almonella. In the study of theSalmonella clinical isolates, it was noted that monoclonal antibody M02 which besides ayglutinating the serogroup A
Salmonella was also found to agglutinate some serogroup D
strains. In closer examination of the serogroup D1 Salmonella, it was found that these strains show close resemblance to those belonging to serogroup A. The major difference lies in their specific 0-antigen which is specified by the dideoxyhexose, paratose in serogroup A and tyvelose in serogroup D. Furthermore the biosynthetic pathway of these two dideoxyhexoses is also inter-related in that the two sugars can be interconverted by an isomerasa enzyme which is present in the serogroup D
Salmonella. Hence it is not surprising that stains of serogroup D Salmonella may express the dideoxyhexose paratose in its cell surface which may then be detected by the monoclonal antibody M02. Nevertheless this unusual cross-reactions between the serogroup D Salmonella with the M02 monoclonal antibody does not seem to cause confusion in the differentiation of these two serogroups of organisms since the cross-reactions seems to occur in one direction only and there is no similar cross-reactions of serogroup A Salmonella strains with the M09 monoclonal antibody.
Therefore strains of Salmonella reacting with M02 alone belongs to serogroup A while strains reacting to either M09 alone or both M09 and M02 belong to serogroup D.
Further evaluation of the anti-Salmonella monoclonal antibodies as serotyping reagents for Salmonella have been carried out at the German National Salmonella Centre at Hamburg, Germany and at the National Laboratory for Enteric Pathogens of the Laboratory Centre for Disease Control, ~7~.62 Health and Welfare Canada, Ottawa, Canada. Part of the evaluation done in Germany is summarized in Table 4.

TABLE 4 Serotyping of Groups Cl and C2 and Other Related O Serogroups of Salmonella by Monoclonal Antibodies Cl-lICI-2 (Specific for Group C~) and M08 (Specific for Group C2) in the National Reference Center for Salmonella, Ger~nany Agglutinating Results with Different Dilutions o~
MAbs' Cl-1 C1-2 M08 Test Strains 1:101:20 1:301:10 1:201.301:10 S. choleraesuis 6~,7:-:1,5 + + + + + + + + + + + +
62,7:-:1,5 + + + + + + + + + (+
S. Iomita 62,7:e,h:1,5 + + + + + + + + + (+) S. thompson 6l,2,7:k:1,5 + + + + + + + + + +
S. mantevideo 6t,2,7:g,m,s:- + + + + + + + + + + +
S. oranienburg 6~,2,7:m,t:- + +. + + + + + + + + + .
S. bareilly : .
6l.2,7:y:1,5 + +. + + + + + + + + + +
S. infantis 6,12,7:r:1,5 '+ + + + + + + + + +
S. virchow 6~,2,7:r:1,2 ' + +,+ + `'+ + + + + + +
S. oslo 6, 2,7:a:e,n,x + + + + + + + + + + + +
S. paratyphi C
6l,2,7,Vi:c:1,5 + + + + + ~ + + + + +
S. georgia 6J,2,7:b:e,n,z~s + + + + + + + + + +
S. potsdam 6~ 2,7:1,v:26 + + + +
S. braenderup 6~,2,7:e,h:e,n,z,5 + + + + + + + + + + +
5. II 6~.2,7:b:z39 + + + + ++ + + + (+
S. II 6~,2,7:g,m,s,t:- + + + + + ++ .~ + + +
.
Gtoup C, 0:14 +
S. ohio 6,7,14:b:1,w + .~ + _ + + (+
S. eimsbuttel 6,7,14:d:1,w + + + + _ + + + _ _ 24 2~7~62 TA~LE 4 C~ntinued - i Agglutinating Results with Different Dilutions of MAbs~
~ 1 C1-2 MO8 Test Strains 1:10 1:201:30 1:10 1:20 1:30 1:10 S. livingstone 6,7,14:d:1,w ++ + _ ++ +
S. nieukerk 6,7,14:d:7~ + + + _ + + (+
S. rnontevideo 6,7,14:g,m,s:- + + + - + + +
S. oranienburg 6,7,14:m,t:- + + + _ + + +
S. thompson .
6,7,14:k:1,5 + + + - + + +
S. infantis 6,7,14:r:1,5 + + + - + + +
S. Iille .
6,7,14:z38:- + + + ; ~ + + (~) ~ ~
5. II " , 6,7,14:g,m,s,t:- + + + + ....... - + + +
Arizona 0:6,7 5. IIlb 6,7:1,v:z53 + + + _ + + +
5. IV
6,7:z~,z23:- + + + (+) + + +
S. Vl 6,7: ~:1,7 + + + (+) + + +
Group 0:18 S. rawash 6,14,18:c:e,n,x + + + (+) + + (+) S. usumburu 6,14,18:d:1,7 + + + (+) + + (+) S. cerro 6,I4,18:z~,Z23:- + + + (+) + + (+) . .;
-- Cl-1 a-2 MO8 1:10 1:10 1:10 1:20 1:30 Group O:H
5. carrau 6,14,24:y:1,7 - - Not done S. bahrenkld 6,14,24:e,h,:1,5 - - Not done S. albuguerque 1,6,14,24:d:z6 - - Not done S. onderstepoort 1,6,14,24:e,h:1,5 - - Not done S. boecker 1,6,14,25:1,v:1,7 -- -- Not done S. fischerkietz 1,6,14,25:y:e,n,x - - Not done S. uzaramo 1,6,14,25:7l,z2~:- -- -- Not done S. lIlb 6,14:b:e,n,x ~ ~ Not done TABLE 4 Continued . 2 7 16 - -- V o Agglutinating Results with ~ifferent Dilutions of MAbs~
Cl-1 C1-2 M08 1-10 1:101:101:20 1:30 S. IV
6,14:z~,zu:- - - Not done 5. Il 1,6,14:Zlo:1,5 ~ -- - Not done Group 0:8 (=C2) S. muenchen 6,8:d:1,2 - -- + + + + (+
5. manhathn 6,8:d:1,5 _ _ + + +
5. neu~ort 6,8:e,h:1,2 _ _ + + +
6,8:m,t:1,5 - - + + +
5. blockley 6,8:k:1,5 -- -- + + +
5. Iitckfield 6,8:1,v:1,2 . -- -- + + +
5. rnanchester 6,al,vl,7 , - - ++ +
5. bovtsmor~ificans . + + (+) 5. goldcoast 6,8:r:1,w _ .
6,8 Z~C:e,n,x -- -- + +
5. hiduddih 6,8:1,z~,z~3:1,5 -- -- + + + (+) 5. kottbus 6,8:e,h:1,5 - - + + +
6,8 z~,Zu:e,n,Zl~ - -- + + + ( ) '~
5. herston 6,8:d:e,n,z~5 - - + + + (+) 6,8:zz9:e,njx - - + + + ( + j 8 k 1 5 - - - Not done S. kentucky Not done -S. amherstiana _ _ Not done 8,20.g,m,s:- -- -- Not done 5. enteritidts Not done 5. senftenberg Not done 5. aberde~t Not done 3,10-.1,v:1,6 -- -- Not done 1,13,22:z:1,6 - - Not done .:

26 2~7~162 TABLE 4 Continued I
Agglutinating Results with Different Dilutions of MAbs' Cl-1 Cl-2 MO8 1:101:10 1:10 1:20 1:3Q
5. waycross 41~ Not done S. uccle 54:g,s,t:---- -- -- Not done ~ , quick and strong positive reaction; +, positive agglu~nation; (+)~ late positive agglutin-ation: and -, negative agglutination.
. . .

~;

-- . .

27 2~78:~fi2 (B) Detection of different common serotype~ of Salmonell~
by ~andwich capture ELIS~ technique using murine monoclonal antibody directed against the genu8-speaific apitope of the Salmonella lipopolysaacharide outer core structure:
The sandwich capture ELISA technique using monoclonal antibody T6, which is directed against the genus specific epitope found in the outer core region of the Salmonella lipopolysaccharide molecule, has been found to be able to detect both smooth and rough lipopolysaccharide antigens of Salmonella and that the method is reproducible as shown in Figure 1. The antibody (T6) and the method ~sandwich capture ELISA) using monoclonal antibody T6 are both specific for the detection of Salmonella organism since 21 non-Salmonella standard strains obtained from American Type Culture Collection tested did not give positive result even when tested at a number of >109 per ml (e.g. overnight broth culture in Brain Heart Infusion medium).
In contrast to the overnight broth cultures of non-Salmonella bacteria which uniformly gave negative result in the sandwich capture ELISA test with monoclonal antibody T6, four hour broth cultures of seven standard Salmonella strains and 24 clinically isolated wild Salmonella strains from the common O serogroups of A to E were all detected by this method (Table 5).

2~17~ 62 . ~ ~ ,~ _ `'C--1 . _ + 't + + + ~ + t- + + + + + + T + + + + + + + T +
tr)~1:~ _ _ ~
e ~- ~ C~ ~ ~ r' ~ r- ~ o g ~o o -r o = ~ r~
u :~ o ~ _ O O O _ O O O ~ O O O O c~ O O O _ O _ _ _ O O
c . .

a a ~ , 3 ~ 3 ~ ~ ~ a ~ ~ ~ 3 E ~ a ~ E
6 ~
c O
u cc ~ 0~ tq ~ ~ a ~

c _ _ . ~. ~ + + ^ + + + + I î I î I I I I I I I t r ~ O ' o ~ O ~ ~ ~ ~ ~ O O ~ O O o O ~ ~ o ~

o~ ~ C~ O ~ ~ O g 00 0 ~ U~ ~ ~ ~ ~ _ v~ o ~ o Ch U~ ~_ e ~ ~ ~ k ~ ~ ..

¦ ~

~ ~ 7 ~

Using a combination of anti-Salmonella o and LPS core specific monoclonal antibodies in the sandwich capture ELISA, it has been demonstrated that not only can Salmonella strains be detected, their serogroup identity can also be found out at the same time (simultaneous serogrouping). Table 6 illustrates the results obtained using horseradish peroxidase enzyme conjugated monoclonal antibodies M02, Cl-1, M08, MO9 and E1-1 as detector antibodies and monoclonal antibody T6 as capture antibody in the sandwich capture ELISA test for the detection of Salmonella.

~7~2 Table 6. Simultaneous detection and serogrouping of Salmonella using a combination of anti-O and anti-core specific monoclonal antibodies in sandwich capture ELISA.

______ ______________________________ _______ _______________________ Mean ELISA O.D. and (Reactivity) for:
Salmonella strains HRPO conjugated monoclonal antibodies (serogroup) MO2 C1-1 MO8 MO9 El-l _________.__________________________________________________________ _ S. paratyphi A 0.~70 0.005 0.004 0.056 0.006 (serogroup A) (+) (-) (~) (~) (~) S. typhimurium 0.016 0.028 0.029 0.014 0.025 (serogroup B) (-) (-) (-) (-) (-) S. choleraesuis 0.041 0.995 0.038 0.013 0.034 (serogroup Cl) (-) (+) (~) (~) (~) S. newport 0.024 0.029 1.011 0.014 0.021 (serogroup C2 ) (-) (-) (+) (-) (-) S. enteritidis 0.038 0.009 0.016 1.057 0.009 (serogroup Dl) (-) (-) (~) (+) (~) S. anatum 0.005 0.012 - 0.013 0.013 0.933 (serogroup El) . (-) (-) (-) (-) (+) _____ _ _ _ ______________________ __ _________ __________________ _ Escherichia coli 0.035 0.057 0.049 0.006 0.034 Brain Heart Infusion 0.004 0.000 0.010 0.019 0.012 medium ___________________________________________________ ________________ *

mean of triplicate determinations 31 2~7~

~C~ Deteation of almo~ella in different specimen3 using multiplo time point immunoassay:
~i) Detection of Salmonella enteritidis phage type PT 8 in egg specimens:
Homogenized grade A eggs were first tested for the absence of Salmonella using standard bacteriological culture techniques. After assuring that no Salmonella was present in the egg specimens, different numbers of S.
entexitidis PT8 was used to spike the homogenized egg.
ml of spiked egg specimen was inoculated onto 9 ml of Selenite Cystine broth for incubation at 37~ C. Samples were removed at different time periods (0, 4, and 18 hours), boiled for 30 minutes and assayed in the sandwich capture ELISA.
The results of Table 7 indicates that the sandwich capture ELISA using monoclonal antibody T6 can detect less than 10 S. enteritidis present in egg specimen after suitable enrichment culture in Selenite Cystine broth medium. Similar results were obtained with another isolate of S. enteritidis PT4 (data not shown).

' 32 2~7~ fi2 Table 7. ~etection or S, ,en,teritidis phage type ~ in eggs using sandwich cap~ure ELIS~ with murine monoclonal antibod~" T6 using a multiple time point assa~

Inoculumn size ~ean ELIS~ O.D. obtained at: 1 used to spike 0 hour 4 hour 1~ hour eggs f~c~u)*
~._______ ____ ______________________________________________ Norle 0~09~ 0~076 0 100 1.25 x lO6 0.104 1 47~ 1.570 1.25 x 105 0.0~1 0~322 1 ~0 1.25 ~ 104 0 0~ 0.072 l.45.`s I.25 x 103 , 0 039 0 059 1 553 L.25 x 102 O.lOl 0.065 1.462 1 25 x 10' 0~0~3 0 061 1.447 . 25 0.0~7 0.074 1.26~
0 125 O lY~2 0.0~9 0 10~ ', I

______,_ ___ ____ __________________ _ - * col ony formi ng uni ts ..

~7~1 ~2 (ii) Detection of different serotypes of Salmonella in faecal specimens obtained from patients suffering from gastroenteritis:
specimens were tested immediately (O hour incubation) by sandwich capture ELISA after inoculation onto Selenite Cystine Broths as well as after overnight incubation at 370 (Table 8).

Table 8 ~etection of S,a,l,m,one,l"l..a, from human faecal specimens by sand~ich capture ELIS~ ~Jith monoclonal antibody T6 using a multiple time point assa~f Specimen ~ean ELIS~ O D. reading after Culture for ~umber 0 hour overnight incubation Sal.rnonella _______________________________________ __ _______~_____ , .
~43C,~ o oC~o O.f99 positive 64839 0 0670~387 positi~fe 3 0 2521~15~ positivP
61474 0 6100 911 negative ~'~2914 b 223 0.176 - ' negative . .. ..
- ~3620 0 9~0 945 negativ~
~3871 '0 5360 575 negative 61057 0 0390 044 negativci ~1664 0.034 0.052 negative 64619 0.0420~055 negative .
__ ___________________________________________________________ - . .... .. . .

2~7~ ~2 The results of Table 8 clearly shown that for some specimens, if only a single assay was done, say after overnight incubation, the high reading obtained would have suggested the presence of Salmonella. However high reading was already found in the specimen without any incubation, and standard bacteriological culture done on the specimen both before and after the incubation in Selenite Cystine Broth had not provided any positive isolation of Salmonella. Therefore the high ELISA reading obtained at the O hour of incubation of the enrichment broth as well as after overnight incubation most likely was due to some non-specific reaction unrelated to the presence of Salmonella.
Specimens with positive isolation of Salmonella all gave significantly increased (at least two folds) ELISA O.D.
between the two time-point measurements.

Claims (10)

1. A reagent for use in the serotyping of Salmonella, comprising a panel of murine hybridoma anti-Salmonella monoclonal antibodies against the Vi and the somatic O-antigens of common serogroups of Salmonella which can be used without prior absorption as factor specific sera, said panel comprising one or more monoclonal antibodies selected from the group consisting of MO2, MO4, Cl-1, MO8, MO9, E1-1, E2-1 and Vi; and said common serogroups including A, B, C1, C2, D1, E1, E4 and phage modified E1 Salmonella.

2. A reagent for specific detection of different common serotypes of Salmonella which comprises a murine hybridoma monoclonal antibody directed against the genus-specific epitope of the Salmonella lipopolysaccharide outer core region, said monoclonal antibody consists of T6 murine hybridoma monoclonal antibody.

3. A reagent according to claim 1 or 2, wherein said reagent is conjugated to protein A-Staphylococcal cells or inert latex particles for coagglutination.

4. A reagent according to claim 1 or 2, wherein said reagent is coupled to magnetic and/or immunomagnetic beads for the specific separation and detection of Salmonella.

5. A method for specific detection of different common serotypes of Salmonella, which comprises, (a) treating a test sample in an enrichment culture medium for incubation; and (b) applying the resulting enriched test sample to a sandwich capture ELISA assay with murine hybridoma monoclonal antibodies directed against the genus-specific epitope of the Salmonella lipopolysaccharide outer core region, said monoclonal antibodies consisting of T6 murine hybridoma monoclonal antibodies.

6. A method for simultaneously detecting and serogrouping Salmonella serotypes, which comprises applying sandwich capture ELISA assay to an enriched test sample using unlabelled monoclonal antibody T6 as capture antibody, and enzyme labelled monoclonal antibodies that are directed against the Vi or O-antigens of serogroups A, B, C1, C2, D1, E1, E4 and phage modified E1 Salmonella as detector antibodies.

7. A quantitative immunoassay method for the detection of Salmonella, which comprises:
(a) treating a test sample in an enrichment culture medium for incubation;
(b) applying the resulting enriched test sample to a sandwich capture ELISA assay with a panel of murine hybridoma anti-Salmonella lipopolysaccharide monoclonal antibodies, either against the Vi antigen or the somatic O-antigens of common serogroups of Salmonella and/or the genus-specific epitope present in the Salmonella lipopolysaccharide outer core region, as capture and detector antibodies, said panel comprising one or more monoclonal antibodies selected from the group consisting of Vi, MO2, MO4, Cl-1, MO8, MO9, E1-1, E2-1 and T6;
said common serogroups including A, B, C1, C2, D1, E1, E4 and phage modified E1 Salmonella; and (c) taking multiple time point ELISA
measurements on the enrichment culture at 0 hour of incubation and at subsequent time during the Salmonella culture step.

8. A quantitative immunoassay method for the detection of infectious agents, which comprises taking multiple time point ELISA measurements of a test sample undergoing enrichment serology testing in the presence of a specific antiserum against the individual infectious microorganism at 0 hour of incubation and at subsequent time during a culture step.

9. A quantitative immunoassay method according to claim 8, wherein the infectious agent is present in eggs.

10. A quantitative immunoassay method according to claim 8, wherein the infectious agent is present in faecal specimens.