CA2248171A1 - Improvements in or relating to detection of salmonella - Google Patents
Improvements in or relating to detection of salmonella Download PDFInfo
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- CA2248171A1 CA2248171A1 CA 2248171 CA2248171A CA2248171A1 CA 2248171 A1 CA2248171 A1 CA 2248171A1 CA 2248171 CA2248171 CA 2248171 CA 2248171 A CA2248171 A CA 2248171A CA 2248171 A1 CA2248171 A1 CA 2248171A1
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Abstract
Disclosed is an immunoglobulin molecule or antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains together with, inter alia, use thereof, and an assay device comprising the antibody.
Description
' CA 02248171 1998-10-22 C 7482 (V) Title: Improvements in or Relating to Detection of Salmonella 5 Field of the Invention This invention relates to a method of preparing antibodies, a method of detecting Salmonella bacteria, monoclonal antibodies substantially specific for Salmonella bacteria, and to cell lines, hybridomas etc. producing the monoclonal 10 antibodies.
Background of the Invention Salmonella bacteria generally are motile in liquid or semi-solid media by means of peritrichous flagella. These 15 organelles are powered by a basal body, which acts as an intracellular motor and transmits torque through the bacterial cell wall to an extracellular structure, the filament. When the organism swims, filaments asociate into twisted bundles whose motion drives the cell through the 20 medium. The filaments are extremely long, helical structures, extending 10 ,um or more from the cell surface, and consist of a single self-polymerising protein, flagellin. Flagellar filaments grow by addition of new flagellin monomers to the distal tip, which they reach 25 after travelling along the hollow central channel of the filament; this channel extends into the cell through so-called hook and rod structures which are co-axial with the filament.
30 Salmonella flagellins carry the H antigen, one of the major antigens that elicit an immune response in an infected animal. In addition to being strongly antigenic, H
antigens are also highly diverse, both in length and in sequence. The amino acid sequence of the antigenic region C 7482 (V) of the flagellin molecule is highly polymorphic: over 50 flagellins can be distinguished by serology. In fact, the diversity of the H antigens, together with that of the somatic lipopolysaccharide (O) antigens, forms the basis of 5 the official (Kauffmann-White) scheme for the classification of Salmonellae. This scheme identifies serovars of Salmonella by a unique antigenic formula of O
and H antigens. Currently there are over 2300 recognised serovars of Salmonella.
Salmonella-mediated food poisoning is a major problem in many countries. Accordingly, there is a need for tests in the food industry, and in the medical and veterinary fields, which are capable of reliably detecting the 15 presence of Salmonellae. Such tests are needed to ensure that foodstuffs and animal feedstuffs are safe for consumption, to aid diagnosis for clinicians and veterinarians, and to assist epidemiologists in tracking outbreaks of disease.
Currently available tests fall into two general categories:
biological or immunological. Biological tests require culture of samples for a prolonged period, detection of Salmonella organisms being based on biochemical and/or 25 growth characteristics of the bacteria. Accordingly, such tests take 3-5 days to produce results and are not rapid enough for many purposes. Immunological tests are based on the serological or antigenic characteristics of the bacteria. Whilst such immunological tests may require some 30 culture of samples, the duration of culture is far shorter than in biological tests, such that immunological tests may generally be performed more rapidly than biological tests.
' CA 02248l7l l998-l0-22 C 7482 (V) Originally, immunological tests relied on the use of polyclonal antisera (reviewed by Thomason, 1980 J. Food Sci. 44, 381), but these were less than ideal in that they were insufficiently specific for Salmonella, giving rise to 5 many false positive results. Subsequently, various monoclonal antibodies were raised against Salmonellae (Mattingly et al., 1985 Food Technology 90-94; Tsang et al., 1987 Infection and Immunity 55, 211-216; Paterson &
Tiffin, 1988 Letts. in Applied Microbiol. 7, 41-43; and 10 Todd et al., 1991 Food Microbiol. 8, 311-324). Monoclonal antibodies have several advantages over polyclonal sera:
they are generally more specific and available at consistent quality.
15 It is desirable that any single antibody should bind to all strains of Salmonella, whilst ideally not binding to any organism which is not a Salmonella species. In laboratory conditions, testing deliberately contaminated samples, the best test devices currently available (using ELISA-based 20 assays) perform very well but in practical conditions, using real food samples, their performance is less satisfactory, often giving rise to false positive results in the presence of one or more of several non-Salmonella organisms. In addition the anti-Salmonella antibodies, 25 upon which conventional ELISAs are based, fail to bind to several Salmonella strains, such that a number of monoclonal antibodies, of different specificities, are required in combination for best results.
30 There is thus an unmet need for an improved immunoglobulin which, ideally, in tests will detect all or virtually all Salmonella organisms whilst giving a lower rate of false positive results than those antibodies currently available.
' ' CA 02248171 1998-10-22 C 7482 (V) One of the objects of the present invention is to provide such an immunoglobulin.
Summary of the Invention 5 In a first aspect, the invention provides an immunoglobulin molecule or an antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 (Seq ID No. 1 in the attached sequence listing) of the flagellin protein of Salmonella typhimurium PIR accession 10 No. A24262, or the equivalent portion from other Salmonella strains. (Flagellins from different strains vary in length, so the amino acid residue numbering is not universal. However sequence comparisons, with the benefit of the present disclosure [e.g. Figure 1], will enable the 15 person skilled in the art readily to identify such equivalent portions).
The immunoglobulin molecule is typically an antibody (most preferably a monoclonal antibody), which may be of any 20 class and sub-type, but is typically IgG. The term "antigen-binding variant" refers to molecules which comprise the antigen-binding portion of an immunoglobulin (which antigen-binding is conferred primarily by the complementarity determining regions, or CDRs), whether 25 obtained by traditional immunisation techniques, or obtained by genetic manipulation of appropriate coding sequences, or by in vi tro screening of (e.g. phage) libraries expressing antibody-like molecules.
30 Examples of molecules falling within the meaning of the term "antigen-binding variant" of an immunoglobulin include, but are not limited to, the following:
' CA 02248l7l l998-l0-22 C 7482 (V) Fv antibody fragments (Skerra & Pluckthun, 1988 Science 240, 1038);
ScFv antibody fragments (Bird et al, 1988 Science 242, 423);
5 bi-specific antibody fragments where one or both binding specificities is in accordance with the invention defined above (Holliger et al, 1993 Proc. Natl. Acad. Sci. USA 90, 6444);
bi-specific antibody fragments where one binding 10 specificity is in accordance with the invention defined above, and the other binding specificity is for a marker molecule, reagent or the like (e.g. an enzyme);
antibody fragments obtainable from, derived from, or encoded by nucleic acid sequences obtainable from Camelidae 15 (WO 94/04678, WO 94/25591); and antibody-type molecules isolated from, or encoded by nucleic acid sequences obtainable from, synthetic biomolecular libraries (e.g. Lerner et al, 1992 Science 258, 1313).
In the following text, references to "immunoglobulin"
should be construed as including reference to antigen-binding variants thereof, where the context so permits.
25 It will be apparent from the foregoing that the immunoglobulin or antigen-binding variant thereof may be a naturally occurring molecule (e.g. antibody) obtained by traditional immunisation techniques, or may be obtained or altered by recombinant DNA techniques so as to comprise a 30 synthetic portion or be wholly synthetic. Such partially or wholly synthetic molecules may be engineered, for example, to alter affinity, or number of binding sites, or number of binding specificities.
C 7482 (V) The immunoglobulin or antigen-binding variant thereof in accordance with the invention may be in isolation from other immunoglobulin molecules (or variants thereof) directed against other epitopes. More especially, the 5 molecules of the invention may preferably be in isolation from immunoglobulin molecules (or variants thereof) directed against portions of Salmonella flagellin other than residues 403-452 or directed against other Salmonella antigens (e.g. "O" antigens of LPS).
The present inventors have found that this portion (i.e.
equivalent to residues 403-452 of S. typhimunium flagellin) of the flagellin protein is well conserved amongst bacteria of the genus Salmonella, whilst being rather different in 15 sequence to the flagellin proteins of other bacterial genera. The inventors have found that antibodies directed against epitopes present in this portion of flagellin protein will tend to bind to all or substantially all strains of Salmonella, whilst binding to very few non-20 Salmonella bacteria, even those closely-related, such as Escherichia, Klebsiella, Citrobacter etc.
Desirably the immunoglobulin molecule (or antigen-binding variant thereof) in accordance with the invention binds to 25 an epitope present within amino acid residues 418 to 452 of the Salmonella typhimurium flagellin protein, which residues tend to be more highly conserved than residues 403-417. More preferably, the immunoglobulin will bind to an epitope present within amino acid residues 427 to 436, 30 or to an epitope present within amino acid residues 444 to 451, of the Salmonella typhimurium flagellin protein, which portions are especially well-conserved among Salmonellae.
' CA 02248171 1998-10-22 C 7482 (V) This sequence of residues 427 to 436 in Salmonella is generally RFDSAITNLG (Seq ID No. 2 in the attached sequence listing), although certain strains have been found to possess a slight variant RFNSAITNLG. Immunoglobulins (or 5 antigen-binding variants thereof) which bind to either one, or both, of these variants may be of use in the present invention. The sequence of residues 444 to 451 is generally SARSRIED (Seq ID No. 3 in the attached sequence listing).
A preferred immunoglobulin in accordance with the present invention is a monoclonal antibody obtainable from a hybridoma designated 5836.1, which antibody binds to an epitope present in residues 427-436 and has been found to 15 detect all motile strains of Salmonella (i.e. those with flagella) so far tested. Another preferred immunoglobulin is a monoclonal antibody obtainable from a hybridoma designated 6035.1, which antibody binds to an epitope present in residues 444 to 451.
The immunoglobulin molecule (or antigen-binding variant) of the invention may be provided as a serum sample, or in the form of a crude culture supernatant, or ascites fluid, or cells producing the immunoglobulin, or may be purified 25 (e.g. by affinity chromatography and the like). Further, the antibody may be frozen, lyophilised, immobilised on a solid support etc. all of which may be performed by methods well-known to those skilled in the art.
The invention also provides a host cell capable of 30 producing an immunoglobulin molecule or antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella ' ' CA 02248171 1998-10-22 C 7482 (V) strains. The host cell may be prokaryotic (e.g.
bacterial), such as a bacterium harbouring a phage encoding the immunoglobulin-like molecule. Typically, however, the host cell is eukaryotic, and preferably mammalian.
5 Desirably the host cell is an immortalised cell line, such as a hybridoma (e.g. hybridoma 5836.1 or 6035.1, mentioned above), and produces a monoclonal antibody molecule.
The invention further provides a nucleic acid sequence 10 encoding an immunoglobulin molecule or antigen-binding variant thereof, which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella 15 strains. Such a nucleic acid may be obtainable, for example, from hybridoma 5836.1 or 6035.1, by standard nucleic acid isolation methods known to those skilled in the art (e.g. from a phage library).
20 In a further aspect the invention provides a method of producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising immunising a mammal with an immunogen which comprises at least one B-cell epitope present within amino acid residues 403 to 452 of 25 the flagellin protein of Salmonella typhimurium PIR
accession No. A24262, or the equivalent portion of flagellin from other Salmonella strains, said B-cell epitope being in substantial isolation from Salmonella flagellin residues outside the portion 403 to 452.
30 Conveniently the immunogen is a synthetic peptide and preferably several immunisations are performed, typically using adjuvants such as Freund's Complete or Incomplete Adjuvant. Methods of preparing hybridomas, and hence monoclonal antibodies, from suitably immunised animals are ' CA 02248l7l l998-l0-22 C 7482 (V) well known (e.g. Kohler & Milstein, 1975 Nature 256, 495;
Newell et al., 1988 "Making Monoclonals" PHLS, London ppl-94). Preferably the immunogen will comprise the flagellin epitope linked or fused to an amino acid sequence 5 comprising one or more T helper cell epitopes, so as to facilitate induction of an antibody response. Such T
helper cell epitopes may conveniently be provided by fusion of the flagellin epitope to a carrier protein (such as bovine serum albumin, or keyhole limpet haemocyanin), or 10 chemical cross-linking to a protein. Suitable T helper cell epitopes are well known to those skilled in the art, including epitopes present in sperm whale myoglobin.
In an additional aspect, the invention provides a method of 15 producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising the steps of:
obtaining a library of nucleic acid sequences encoding immunoglobulin molecules or antigen-binding variants thereof; screening the library with a screening molecule 20 which comprises at least a portion of amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A 24262, or the equivalent portion of flagellin from other Salmonella strains, said portion being in substantial isolation from Salmonella 25 flagellin residues outside the portion 403 to 452;
selecting those nucleic acid sequences which encode an immunoglobulin or antigen-binding variant which binds to the screening molecule; and causing expression of the selected nucleic acid sequences to obtain the desired 30 immunoglobulin or antigen-binding variant thereof.
Conveniently the screening molecule comprises a synthetic peptide which may optionally be conjugated to, or fused with, a carrier molecule. Appropriate libraries of nucleic . . .... .
' CA 02248l7l l998-l0-22 C 7482 (V) acid sequences in a form suitable for screening are well-known to those skilled in the art (e.g. Lerner et al, 1992 Science 258, 1313; McCafferty et al, 1990 Nature 348, 552).
5 If desired, the selected nucleic acid sequences may be further screened (e.g. with screening molecules which comprise portions of flagellin from non-Salmonella species), to identify immunoglobulin molecules or antigen-binding variants thereof which have optimum specificity for 10 Salmonella flagellin epitopes (i.e. minimum cross-reactivity for non-Salmonella species) but which react with as many strains of Salmonella as possible. (Similar screening will desirably be applied to immunoglobulins obtained by conventional immunisation techniques.) In a further aspect, the invention provides a method for detecting the presence of Salmonella organisms, comprising contacting a sample to be tested with an immunoglobulin molecule or antigen-binding variant thereof which binds to 20 an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR
accession No. A24262, or the equivalent portion of flagellin from other Salmonella strains; and monitoring binding of the immunoglobulin molecule, or antigen-binding 25 portion thereof, to the sample. Desirably the immunoglobulin molecule is an antibody (e.g. monoclonal antibody) having the preferred characteristics defined above.
It will generally be desired for the sample to be subjected 30 to a processing step prior to contact with the immunoglobulin molecule or antigen-binding variant, the purpose of which is to ensure that the epitopes of the flagellin protein of any Salmonella bacteria present in the sample are exposed and available for binding to flagellin-' CA 02248l7l l998-l0-22 C 7482 (V) specific immunoglobulins. Conveniently this is accomplished by boiling of the sample for a short period, or otherwise by processing the sample with denaturing agents such as surfactants and the like.
Those skilled in the art will appreciate that numerous assay formats can be envisaged for performing the method defined above. Preferred embodiments include enzyme-linked immunosorbent assay (ELISA), immunochromatographic assays, 10 or Western blots. Generally, the assay method will normally involve binding of the immunoglobulin molecule (or antigen-binding variant thereof) of the invention to Salmonella flagellin, if present in the sample. The immunoglobulin molecule may be labelled to facilitate 15 detection of such binding. A variety of labels are known to those skilled in the art and include radioactive labels, enzyme labels, coloured latex particles, and biochemical labels (such as biotin and the like). Alternatively, binding of the immunoglobulin molecule to Salmonella 20 flagellin may be detected by use of a second immunoglobulin, which immunoglobulin may be labelled and will preferably also be a monoclonal antibody.
A large number of antibody-based Salmonella-detection 25 methods have been described in the prior art (e.g. Van Poucke, 1990 Appl. Environ. Microbiol. 56, 924-927; Anon., 1987 J. Assoc. Off. Anal. Chem. 70, 396-397; Cerqueira-Campos et al., 1986 Appl. Environ. Microbiol. 52, 124-127;
Ibrahim et al., 1986 J. Food Protect. 49, 92-98) and, in 30 principle, any of these could be adapted for use with an immunoglobulin molecule or variant thereof in accordance with the invention. Further details of specific envisaged assay methods are given in the examples below.
' CA 02248l7l l998-l0-22 C 7482 (V) In a further aspect, the invention provides an assay device for use in detecting the presence of Salmonella organisms, the assay device comprising an immunoglobulin molecule or antigen-binding variant thereof in accordance with the 5 invention. Again, in principle, any of the assay devices known in the prior art (e.g. D'Aoust & Sewell, 1988 J. Food Protect. 51, 538-541; Farber et al., 1985 J. Food Protect.
48, 790-793) could be adapted for use with an immunoglobulin molecule in accordance with the invention.
10 Desirably the device will comprise a solid support (e.g.
microtitre plate, glass slide, latex bead, chromatographic test stick or the like) provided with an immunoglobulin molecule (or antigen-binding variant thereof) in accordance with the invention.
A preferred embodiment of the assay device is an immunochromatographic device, such as the ClearviewTM
device sold by Unipath Limited. Such devices are easy to use and are reliable. In particular, the device may 20 comprise two or more lmmunoglobulin molecules (or antigen-binding variants thereof), each molecule binding a respective epitope present in Salmonella, and in particular, each molecule binding a respective epitope present in the region of 403 to 452 of the Salmonella 25 typhimurium flagellin. Conveniently, one such immunoglobulin molecule will be directed against an epitope present in residues 427-436, and another such immunoglobulin molecule will be directed against an epitope present in residues 444-451. Embodiments of this type 30 facilitate use of a "sandwich" type assay device and method, in which Salmonella organisms are captured on a solid support with a first immunoglobulin, and detected by binding to a second (typically) labelled immunoglobulin.
' CA 02248l7l l998-l0-22 C 7482 (V) The assay method and assay device of the invention are, in preferred embodiments, able to detect the majority (over 85%, preferably over 95%) of pathogenically important Salmonella species (i.e. those which generally cause 5 infections in man), whilst giving a very low (less than 2%) false positive rate. The assay method and device may particularly be used for detecting the presence of Salmonella in foods and/or in food preparation areas.
10 The present inventors consider that the amino acid residues 403 to 452 of the flagellin protein have unexpectedly useful properties (e.g. are useful in preparing monoclonal antibodies in accordance with the invention). Accordingly, in another aspect the invention provides a peptide 15 comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence. The peptide is preferably in 20 antigenic and/or immunogenic form and preferably substantially pure. The peptide preferably comprises at least amino acid residues 427 to 436, and/or at least residues 444 to 451, of the flagellin sequence. The peptide may desirably be fused or chemically coupled to 25 other molecules (such as polypeptides or peptides which comprise T helper cell epitopes).
In addition to raising antibodies, the particular peptide sequence of the flagellin protein identified by the 30 inventors may be useful as a reagent (e.g. included as a component in an assay method and/or an assay kit for detecting the presence of Salmonella organisms). When used as a reagent in an assay, the peptide may desirably be joined to a labelling and/or anchoring moiety (e.g. biotin, C 7482 (V) or avidin) to label the peptide and/or facilitate immobilisation of the peptide to a solid surface.
Accordingly in another aspect the invention provides for a 5 method of detecting the presence of Salmonella organisms, comprising use of a peptide comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in isolation from 10 other portions of the flagellin sequence. The invention also provides for an assay device for detecting the presence of Salmonella organisms, the device comprising the peptide defined above.
15 In one embodiment, the peptide of the invention is used in an assay method and/or device involving displacement. For example, immobilised peptide could be displaced by the introduction of a sample comprising Salmonella organisms.
Preferably the peptide would be provided with a label (e.g.
20 enzyme, or coloured bead) to facilitate detection of the displacement of the peptide. Displacement assays of this type are described, for example, in WO 91/05262 and PCT/EP95/04518.
25 The invention will be further described by way of illustrative example and with reference to the accompanying drawing, Figure 1, which is a comparison of the sequence of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium A24262 with the homologous sequences of 30 various bacteria, including other Salmonella strains, and E. coli and Citrobacter strains.
Exampl~ 1 CA 02248l7l l998-l0-22 C 7482 (V) The inventors and their colleagues first used a Salmonella whole cell extract or a Salmonella flagellin preparation to make monoclonal antibodies against Salmonella "O" antigen (lipopolysaccharide, LPS) or "H" antigen (flagellin) 5 respectively. The binding sites of the various monoclonal antibodies raised against H antigens were roughly mapped by using them to probe Western blots of trypsin-digested flagellin.
10 The inventors observed that antibodies which bound to the central portion of the 490 amino acid flagellin protein tended to be too specific for use in an assay, in that whilst they did not bind to the flagellin protein of non-Salmonella organisms (and so would not give false positive 15 results) they equally would not bind to the flagellin of every Salmonella strain, such that some strains might go undetected in a test. (A similar observation has been reported by van Asten et al., 1995 J. Bacteriol. 177, 1610-1613).
Conversely, antibodies which bound at the N- or C-terminal regions tended to be insufficiently specific, in that whilst they bound to all Salmonella strains, they also bound to large numbers of non-Salmonella organisms.
The inventors hypothesised that antibodies which bound to the regions between the central portion and the N- or C-terminal may combine the desired characteristics of high specificity for Salmonella, whilst still binding to all 30 Salmonella strains. Accordingly the inventors set out to prepare such monoclonal antibodies.
A search of various computer databases was conducted to find reported flagellin sequences. The search included C 7482 (V) protein translations of coding sequences on the GenBank database (National Institutes of Health, MD, USA), the SWISS-PROT database maintained by the University of Geneva and the European Molecular Biology Laboratory (EMBL) data 5 library, and the Protein Information Resource (PIR) Protein sequence database (National Biomedical Research Foundation, Washington D.C.). Flagellin protein sequences recovered from the databases were aligned by the Clustal method using the Megalign facility on DNA-Star software. Sequences 10 available for comparison were limited to a few strains of Salmonella, E. coli, Proteus, Serratia, Yersinia, Pseudomonas and Campylobacter. Of particular interest were the flagellins of Enterobacteriaceae which typically cause false positives in Salmonella immunoassays: Citrobacter, E.
15 coli and Proteus.
From this comparison, a stretch of 50 amino acids, corresponding to residues 403 to 452 of the S. typhimurium sequence PIR accession A24262 was selected as being 20 relatively well conserved among the Salmonellae while exhibiting some differences between Salmonellae and non-Salmonellae. Flagellins from different strains vary in length, so the amino acid numbering is not universal.
25 A few Salmonella flagellin sequences were already known, but the inventors determined several more. Figure 1 shows a comparison of the amino acid sequence in this 50 residue region (403-452) for various strains of Salmonella and some non-Salmonella bacteria. The consensus sequence 30 ("majority") is shown above and generally corresponds to that of S. typhimurium A24262 flagellin. The numbers to the right of the sequence refer to the culture collection numbers of the various strains examined and correlate with those shown in Table 1.
' CA 02248171 1998-10-22 C 7482 (V) Within this fifty amino acid region, the ten amino acid sequence RFNSAITNLG, corresponding to residues 427 to 436 of the S. typhimurium A24262 sequence, was chosen as being 5 especially specific to the Salmonellae. This sequence was therefore selected for synthesis as peptide immunogen.
Since many Salmonella serotypes have the sequence variant RFDSAITNLG it was necessary to select for antibodies which were indifferent to this variation. This was achieved by 10 screening the hybridomas for their ability to bind to flagellins of the two types (represented here by S.
typhimurium and S. tennessee "6388").
The inventors subsequently determined the sequences of the 15 flagellins of a number of additional Salmonellae and non-Salmonellae, most significantly the Citrobacter strains and an unusual strain of E. coli (NCTC 10418). Certain Citrobacter strains and E. coli NCTC 10418 contain the peptide RFNSAITNLG in its entirety and therefore would be 20 expected to bind to any Salmonella-binding MAb raised against that peptide sequence.
Synthesis of Peptide An RFNSAITNLG "Multi-Antigenic Peptide" (MAP) was produced 25 on the NovaSyn GemTM semi-automatic solid phase peptide synthesiser. The solid support was Kieselguhr [NovaSyn KA
100] derivatised with a branching octavalent lysine core with the following structure:
30 Fmocg_Lys4_Lys-2_Lys- -Ala-Solid support The following peptide sequence was built up on the support using standard Fmoc chemistry:
' CA 02248l7l l998-l0-22 C 7482 (V) (Lys-Lys-Arg-Phe-Asn-Ser-Ala-Ile-Thr-Asn-Leu-Gly) 8- [lysine core]- -Ala-Solid support The peptide comprises the residues 427 to 436 of the S.
5 typhimurium sequence, preceded by two Lys residues which were included to improve the water-solubility of the peptide and are not part of the flagellin sequence.
Following synthesis, the peptide was cleaved from the support using trifluoroacetic acid/tri-isopropyl silane 10 solution and concentrated by evaporation. The peptide was extracted three times with diethyl ether and dried under vacuum. The peptide was dissolved in distilled H2O and dialysed overnight against distilled H2O. The peptide was freeze-dried and stored at -20 C. A sample of the MAP was 15 analysed on an Applied Biosystems automated protein sequencer. This confirmed that the sequence had been authentically synthesized as above.
I unisation with Peptide 20 A BALB/C mouse initially received, by the subcutaneous and intraperitoneal routes, a total of 50 g MAP emulsified in Freund's Complete Adjuvant. Two months later it received, by the subcutaneous and intraperitoneal routes, a total of g MAP emulsified in Freund's Incomplete Adjuvant.
25 Immunisation in Freund's Incomplete Adjuvant was repeated after 2.5 weeks. A first test tail bleed followed after 1 week. The mouse was immunised with a further 50 g of MAP
by the intraperitoneal route ten days later. A second test tail bleed followed after two weeks. One month later the 30 mouse was immunised with 50 g of purified flagellin from 5. typhimurium and 50 g of purified flagellin from S.
tennessee. A third test tail bleed was performed nine days later. After seven weeks the mouse received 100 g MAP
.... .. ~ .
' CA 02248171 1998-10-22 C 7482 (V) intravenously. Three days later the animal was sacrificed and its spleen removed for preparation of hybridomas.
Tail Bleed Testing 5 The antigens used for testing by ELISA were MAP, prepared as above, and flagellins purified from plate cultures of S.
typhimurium and S. tennessee by a differential centrifugation protocol modified from that of Kerridge et al. [1962 J. Mol. Biol. 4 pp 227-238]. Flagellin 10 preparations were boiled for 5 minutes prior to dilution in carbonate buffer to improve the exposure of Salmonella-specific epitopes. Greiner high-capacity binding microtitre plates were sensitised overnight at 4 C with 100 l of antigen (MAP diluted to 5 g/ml, or flagellins 15 diluted to 1 g/ml in 0.05 M sodium carbonate buffer, pH
9.6). The wells were washed with PBS and then blocked with 200 l of PBS containing 0.05% Tween-20 and 1% bovine serum albumin ("PBST/BSA") at 37 C for 1 hour. The wells were then washed in PBS. 100 l samples of tail bleed sera, in 20 serial 10-fold or 5-fold dilutions made in PBST, from immunised and non-immunised mice, were added to the wells for 2 hours at 37 C. The wells were again washed with PBS.
100 l of rabbit anti-mouse IgG/IgA/IgM-alkaline phosphatase conjugate [Zymed] diluted 1/1000 with PBST/BSA
25 was added for 1 hour at 37 C. The wells were then thoroughly washed with PBS and 100 l of p-nitrophenyl phosphate [Sigma 104 Phosphatase Substrate tablets, dissolved at lmg/ml in 1 M diethanolamine, pH 9.8] was added for 30-60 minutes at 37 C. Yellow colour development 30 was measured using a spectrophotometer at 410 nm.
Wells coated with MAP gave signals above background levels with the first tail bleed serum from the immunised mouse at a dilution of 1/100,000; whereas wells coated with ' CA 02248171 1998-10-22 C 7482 (V) flagellins reacted at 1/100 dilution of serum. With the second tail bleed, the flagellins reacted with serum at 1/1000 dilution. With the third tail bleed, S. typhimurium flagellin reacted at 1/12500 dilution of serum and S.
5 tennessee flagellin at 1/62500.
Preparation of Hybridomas The spleen removed from the immunised mouse was perfused with RPMI medium to collect spleen cells. Approximately 5 10 x 107 spleen cells were fused with 4 x 107 SP2 mouse myeloma cells in the presence of polyethylene glycol by standard methods (Kohler & Milstein, 1975 Nature 256, 495).
Fused cells were diluted in selective medium (RPMI
containing 10% horse serum, 2.4 mM L-Glutamine, 1.2 mM
15 sodium pyruvate, 60 I.U./ml penicillin, 60 g/ml streptomycin, 10-4 M hypoxanthine and 1 g/ml azaserine) and plated out in twelve 48-well culture plates. The plates were incubated at 37 C in 5% CO2 for one week. The selective medium was then replaced by growth medium (RPMI
20 containing 5% horse serum, 2.4 mM L-Glutamine, 1.2 mM
sodium pyruvate, 60 I.U./ml penicillin, and 60 g/ml streptomycin). Two days later the hybridoma supernatants were screened by ELISA for binding to MAP and flagellins, as detailed below.
Greiner high-capacity binding plates were sensitised with MAP or boiled flagellin from S. typhimurium, washed, and blocked, as described for Tail Bleed Testing above. 100 of individual hybridoma well supernatant was added to the 30 wells for 2 hours at 37 C. Following washing with PBS, the bound antibody was detected with conjugate and colorimetric substrate as described for Tail Bleed Testing. Colour development at 37 C was measured after 1 hour. Of 576 hybridoma wells screened, 38 produced antibodies which .
CA 02248l7l l998-l0-22 C 7482 (V) reacted with both MAP- and flagellin-sensitised wells.
After expansion, these hybridomas were retested in ELISA
for binding of supernatant antibody to 4 different immobilised antigens: MAP; boiled flagellin from S.
5 typhimurium; boiled flagellin from S. tennessee; and bovine serum albumin. The microtitre plate wells were sensitised overnight at 4 C with antigens at 1 g/ml in carbonate buffer, except MAP which was at 5 g/ml. Assay steps were as described above. One hybridoma was found to bind 10 strongly to MAP and the two flagellins, but not to BSA, and was therefore selected for further analysis.
Subcloning The MAP- and flagellin-binding hybridoma was subcloned by 15 the method of limiting dilution. Subclones were re-tested for their ability to bind specifically to MAP and the two flagellin types. The hybridoma was isotyped after subcloning using isotype-specific rabbit anti-mouse-alkaline phosphatase conjugates and found to be of the IgG1 2 0 class.
Specificity testing Overnight cultures of bacterial strains (17 motile strains of Salmonella plus 19 other Enterobacteriaceae) in 25 brain/heart infusion (BHI) broth were boiled for 30 min and centrifuged for 5 minutes at 13,000 rpm in a microcentrifuge. The culture supernatants (which contain shed flagella) were diluted 1:10 with carbonate buffer and 100,ul used to sensitise the wells of Greiner high capacity 30 binding plates overnight at 4 C. Hybridoma supernatants were tested for binding to the wells by ELISA as described above. The subclone showing the most uniform reaction with the Salmonellae and the least reaction with the other Enterobacteriaceae was selected for re-cloning. Following C 7482 (V) re-cloning, the specificity of the hybridoma was re-confirmed using the same bacterial panel. The re-cloned hybridoma cell line was designated 5836.1.
5 Purification of MAb 5836.1 A culture supernatant of cell line 5836.1 in serum-free medium was filtered to remove cells and loaded onto a PROSEPR High Capacity Protein A column [bioProcessing Inc.]
in lM borate buffer pH 8.5 containing 0.15M NaCl.
10 Following washing in binding buffer, the MAb was eluted in O.lM citrate buffer, pH 3Ø Purified MAb 5836.1 was tested for binding in ELISA to immobilised crude supernatant antigens (prepared as above) from Salmonella and Enterobacteriaceae strains. The panel comprised:
1. Salmonella serotypes common in food outbreaks (according to UK Public Health Laboratory Service figures):
S. enteritidis, S. typhimurium, and S. virchow.
2. Salmonella serotyopes which include strains known not 20 to bind to some earlier "Salmonella-specific" MAbs: S.
derby, S. tennessee, S. ealing, S. muenchen, and S.
rubislaw.
Background of the Invention Salmonella bacteria generally are motile in liquid or semi-solid media by means of peritrichous flagella. These 15 organelles are powered by a basal body, which acts as an intracellular motor and transmits torque through the bacterial cell wall to an extracellular structure, the filament. When the organism swims, filaments asociate into twisted bundles whose motion drives the cell through the 20 medium. The filaments are extremely long, helical structures, extending 10 ,um or more from the cell surface, and consist of a single self-polymerising protein, flagellin. Flagellar filaments grow by addition of new flagellin monomers to the distal tip, which they reach 25 after travelling along the hollow central channel of the filament; this channel extends into the cell through so-called hook and rod structures which are co-axial with the filament.
30 Salmonella flagellins carry the H antigen, one of the major antigens that elicit an immune response in an infected animal. In addition to being strongly antigenic, H
antigens are also highly diverse, both in length and in sequence. The amino acid sequence of the antigenic region C 7482 (V) of the flagellin molecule is highly polymorphic: over 50 flagellins can be distinguished by serology. In fact, the diversity of the H antigens, together with that of the somatic lipopolysaccharide (O) antigens, forms the basis of 5 the official (Kauffmann-White) scheme for the classification of Salmonellae. This scheme identifies serovars of Salmonella by a unique antigenic formula of O
and H antigens. Currently there are over 2300 recognised serovars of Salmonella.
Salmonella-mediated food poisoning is a major problem in many countries. Accordingly, there is a need for tests in the food industry, and in the medical and veterinary fields, which are capable of reliably detecting the 15 presence of Salmonellae. Such tests are needed to ensure that foodstuffs and animal feedstuffs are safe for consumption, to aid diagnosis for clinicians and veterinarians, and to assist epidemiologists in tracking outbreaks of disease.
Currently available tests fall into two general categories:
biological or immunological. Biological tests require culture of samples for a prolonged period, detection of Salmonella organisms being based on biochemical and/or 25 growth characteristics of the bacteria. Accordingly, such tests take 3-5 days to produce results and are not rapid enough for many purposes. Immunological tests are based on the serological or antigenic characteristics of the bacteria. Whilst such immunological tests may require some 30 culture of samples, the duration of culture is far shorter than in biological tests, such that immunological tests may generally be performed more rapidly than biological tests.
' CA 02248l7l l998-l0-22 C 7482 (V) Originally, immunological tests relied on the use of polyclonal antisera (reviewed by Thomason, 1980 J. Food Sci. 44, 381), but these were less than ideal in that they were insufficiently specific for Salmonella, giving rise to 5 many false positive results. Subsequently, various monoclonal antibodies were raised against Salmonellae (Mattingly et al., 1985 Food Technology 90-94; Tsang et al., 1987 Infection and Immunity 55, 211-216; Paterson &
Tiffin, 1988 Letts. in Applied Microbiol. 7, 41-43; and 10 Todd et al., 1991 Food Microbiol. 8, 311-324). Monoclonal antibodies have several advantages over polyclonal sera:
they are generally more specific and available at consistent quality.
15 It is desirable that any single antibody should bind to all strains of Salmonella, whilst ideally not binding to any organism which is not a Salmonella species. In laboratory conditions, testing deliberately contaminated samples, the best test devices currently available (using ELISA-based 20 assays) perform very well but in practical conditions, using real food samples, their performance is less satisfactory, often giving rise to false positive results in the presence of one or more of several non-Salmonella organisms. In addition the anti-Salmonella antibodies, 25 upon which conventional ELISAs are based, fail to bind to several Salmonella strains, such that a number of monoclonal antibodies, of different specificities, are required in combination for best results.
30 There is thus an unmet need for an improved immunoglobulin which, ideally, in tests will detect all or virtually all Salmonella organisms whilst giving a lower rate of false positive results than those antibodies currently available.
' ' CA 02248171 1998-10-22 C 7482 (V) One of the objects of the present invention is to provide such an immunoglobulin.
Summary of the Invention 5 In a first aspect, the invention provides an immunoglobulin molecule or an antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 (Seq ID No. 1 in the attached sequence listing) of the flagellin protein of Salmonella typhimurium PIR accession 10 No. A24262, or the equivalent portion from other Salmonella strains. (Flagellins from different strains vary in length, so the amino acid residue numbering is not universal. However sequence comparisons, with the benefit of the present disclosure [e.g. Figure 1], will enable the 15 person skilled in the art readily to identify such equivalent portions).
The immunoglobulin molecule is typically an antibody (most preferably a monoclonal antibody), which may be of any 20 class and sub-type, but is typically IgG. The term "antigen-binding variant" refers to molecules which comprise the antigen-binding portion of an immunoglobulin (which antigen-binding is conferred primarily by the complementarity determining regions, or CDRs), whether 25 obtained by traditional immunisation techniques, or obtained by genetic manipulation of appropriate coding sequences, or by in vi tro screening of (e.g. phage) libraries expressing antibody-like molecules.
30 Examples of molecules falling within the meaning of the term "antigen-binding variant" of an immunoglobulin include, but are not limited to, the following:
' CA 02248l7l l998-l0-22 C 7482 (V) Fv antibody fragments (Skerra & Pluckthun, 1988 Science 240, 1038);
ScFv antibody fragments (Bird et al, 1988 Science 242, 423);
5 bi-specific antibody fragments where one or both binding specificities is in accordance with the invention defined above (Holliger et al, 1993 Proc. Natl. Acad. Sci. USA 90, 6444);
bi-specific antibody fragments where one binding 10 specificity is in accordance with the invention defined above, and the other binding specificity is for a marker molecule, reagent or the like (e.g. an enzyme);
antibody fragments obtainable from, derived from, or encoded by nucleic acid sequences obtainable from Camelidae 15 (WO 94/04678, WO 94/25591); and antibody-type molecules isolated from, or encoded by nucleic acid sequences obtainable from, synthetic biomolecular libraries (e.g. Lerner et al, 1992 Science 258, 1313).
In the following text, references to "immunoglobulin"
should be construed as including reference to antigen-binding variants thereof, where the context so permits.
25 It will be apparent from the foregoing that the immunoglobulin or antigen-binding variant thereof may be a naturally occurring molecule (e.g. antibody) obtained by traditional immunisation techniques, or may be obtained or altered by recombinant DNA techniques so as to comprise a 30 synthetic portion or be wholly synthetic. Such partially or wholly synthetic molecules may be engineered, for example, to alter affinity, or number of binding sites, or number of binding specificities.
C 7482 (V) The immunoglobulin or antigen-binding variant thereof in accordance with the invention may be in isolation from other immunoglobulin molecules (or variants thereof) directed against other epitopes. More especially, the 5 molecules of the invention may preferably be in isolation from immunoglobulin molecules (or variants thereof) directed against portions of Salmonella flagellin other than residues 403-452 or directed against other Salmonella antigens (e.g. "O" antigens of LPS).
The present inventors have found that this portion (i.e.
equivalent to residues 403-452 of S. typhimunium flagellin) of the flagellin protein is well conserved amongst bacteria of the genus Salmonella, whilst being rather different in 15 sequence to the flagellin proteins of other bacterial genera. The inventors have found that antibodies directed against epitopes present in this portion of flagellin protein will tend to bind to all or substantially all strains of Salmonella, whilst binding to very few non-20 Salmonella bacteria, even those closely-related, such as Escherichia, Klebsiella, Citrobacter etc.
Desirably the immunoglobulin molecule (or antigen-binding variant thereof) in accordance with the invention binds to 25 an epitope present within amino acid residues 418 to 452 of the Salmonella typhimurium flagellin protein, which residues tend to be more highly conserved than residues 403-417. More preferably, the immunoglobulin will bind to an epitope present within amino acid residues 427 to 436, 30 or to an epitope present within amino acid residues 444 to 451, of the Salmonella typhimurium flagellin protein, which portions are especially well-conserved among Salmonellae.
' CA 02248171 1998-10-22 C 7482 (V) This sequence of residues 427 to 436 in Salmonella is generally RFDSAITNLG (Seq ID No. 2 in the attached sequence listing), although certain strains have been found to possess a slight variant RFNSAITNLG. Immunoglobulins (or 5 antigen-binding variants thereof) which bind to either one, or both, of these variants may be of use in the present invention. The sequence of residues 444 to 451 is generally SARSRIED (Seq ID No. 3 in the attached sequence listing).
A preferred immunoglobulin in accordance with the present invention is a monoclonal antibody obtainable from a hybridoma designated 5836.1, which antibody binds to an epitope present in residues 427-436 and has been found to 15 detect all motile strains of Salmonella (i.e. those with flagella) so far tested. Another preferred immunoglobulin is a monoclonal antibody obtainable from a hybridoma designated 6035.1, which antibody binds to an epitope present in residues 444 to 451.
The immunoglobulin molecule (or antigen-binding variant) of the invention may be provided as a serum sample, or in the form of a crude culture supernatant, or ascites fluid, or cells producing the immunoglobulin, or may be purified 25 (e.g. by affinity chromatography and the like). Further, the antibody may be frozen, lyophilised, immobilised on a solid support etc. all of which may be performed by methods well-known to those skilled in the art.
The invention also provides a host cell capable of 30 producing an immunoglobulin molecule or antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella ' ' CA 02248171 1998-10-22 C 7482 (V) strains. The host cell may be prokaryotic (e.g.
bacterial), such as a bacterium harbouring a phage encoding the immunoglobulin-like molecule. Typically, however, the host cell is eukaryotic, and preferably mammalian.
5 Desirably the host cell is an immortalised cell line, such as a hybridoma (e.g. hybridoma 5836.1 or 6035.1, mentioned above), and produces a monoclonal antibody molecule.
The invention further provides a nucleic acid sequence 10 encoding an immunoglobulin molecule or antigen-binding variant thereof, which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella 15 strains. Such a nucleic acid may be obtainable, for example, from hybridoma 5836.1 or 6035.1, by standard nucleic acid isolation methods known to those skilled in the art (e.g. from a phage library).
20 In a further aspect the invention provides a method of producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising immunising a mammal with an immunogen which comprises at least one B-cell epitope present within amino acid residues 403 to 452 of 25 the flagellin protein of Salmonella typhimurium PIR
accession No. A24262, or the equivalent portion of flagellin from other Salmonella strains, said B-cell epitope being in substantial isolation from Salmonella flagellin residues outside the portion 403 to 452.
30 Conveniently the immunogen is a synthetic peptide and preferably several immunisations are performed, typically using adjuvants such as Freund's Complete or Incomplete Adjuvant. Methods of preparing hybridomas, and hence monoclonal antibodies, from suitably immunised animals are ' CA 02248l7l l998-l0-22 C 7482 (V) well known (e.g. Kohler & Milstein, 1975 Nature 256, 495;
Newell et al., 1988 "Making Monoclonals" PHLS, London ppl-94). Preferably the immunogen will comprise the flagellin epitope linked or fused to an amino acid sequence 5 comprising one or more T helper cell epitopes, so as to facilitate induction of an antibody response. Such T
helper cell epitopes may conveniently be provided by fusion of the flagellin epitope to a carrier protein (such as bovine serum albumin, or keyhole limpet haemocyanin), or 10 chemical cross-linking to a protein. Suitable T helper cell epitopes are well known to those skilled in the art, including epitopes present in sperm whale myoglobin.
In an additional aspect, the invention provides a method of 15 producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising the steps of:
obtaining a library of nucleic acid sequences encoding immunoglobulin molecules or antigen-binding variants thereof; screening the library with a screening molecule 20 which comprises at least a portion of amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A 24262, or the equivalent portion of flagellin from other Salmonella strains, said portion being in substantial isolation from Salmonella 25 flagellin residues outside the portion 403 to 452;
selecting those nucleic acid sequences which encode an immunoglobulin or antigen-binding variant which binds to the screening molecule; and causing expression of the selected nucleic acid sequences to obtain the desired 30 immunoglobulin or antigen-binding variant thereof.
Conveniently the screening molecule comprises a synthetic peptide which may optionally be conjugated to, or fused with, a carrier molecule. Appropriate libraries of nucleic . . .... .
' CA 02248l7l l998-l0-22 C 7482 (V) acid sequences in a form suitable for screening are well-known to those skilled in the art (e.g. Lerner et al, 1992 Science 258, 1313; McCafferty et al, 1990 Nature 348, 552).
5 If desired, the selected nucleic acid sequences may be further screened (e.g. with screening molecules which comprise portions of flagellin from non-Salmonella species), to identify immunoglobulin molecules or antigen-binding variants thereof which have optimum specificity for 10 Salmonella flagellin epitopes (i.e. minimum cross-reactivity for non-Salmonella species) but which react with as many strains of Salmonella as possible. (Similar screening will desirably be applied to immunoglobulins obtained by conventional immunisation techniques.) In a further aspect, the invention provides a method for detecting the presence of Salmonella organisms, comprising contacting a sample to be tested with an immunoglobulin molecule or antigen-binding variant thereof which binds to 20 an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR
accession No. A24262, or the equivalent portion of flagellin from other Salmonella strains; and monitoring binding of the immunoglobulin molecule, or antigen-binding 25 portion thereof, to the sample. Desirably the immunoglobulin molecule is an antibody (e.g. monoclonal antibody) having the preferred characteristics defined above.
It will generally be desired for the sample to be subjected 30 to a processing step prior to contact with the immunoglobulin molecule or antigen-binding variant, the purpose of which is to ensure that the epitopes of the flagellin protein of any Salmonella bacteria present in the sample are exposed and available for binding to flagellin-' CA 02248l7l l998-l0-22 C 7482 (V) specific immunoglobulins. Conveniently this is accomplished by boiling of the sample for a short period, or otherwise by processing the sample with denaturing agents such as surfactants and the like.
Those skilled in the art will appreciate that numerous assay formats can be envisaged for performing the method defined above. Preferred embodiments include enzyme-linked immunosorbent assay (ELISA), immunochromatographic assays, 10 or Western blots. Generally, the assay method will normally involve binding of the immunoglobulin molecule (or antigen-binding variant thereof) of the invention to Salmonella flagellin, if present in the sample. The immunoglobulin molecule may be labelled to facilitate 15 detection of such binding. A variety of labels are known to those skilled in the art and include radioactive labels, enzyme labels, coloured latex particles, and biochemical labels (such as biotin and the like). Alternatively, binding of the immunoglobulin molecule to Salmonella 20 flagellin may be detected by use of a second immunoglobulin, which immunoglobulin may be labelled and will preferably also be a monoclonal antibody.
A large number of antibody-based Salmonella-detection 25 methods have been described in the prior art (e.g. Van Poucke, 1990 Appl. Environ. Microbiol. 56, 924-927; Anon., 1987 J. Assoc. Off. Anal. Chem. 70, 396-397; Cerqueira-Campos et al., 1986 Appl. Environ. Microbiol. 52, 124-127;
Ibrahim et al., 1986 J. Food Protect. 49, 92-98) and, in 30 principle, any of these could be adapted for use with an immunoglobulin molecule or variant thereof in accordance with the invention. Further details of specific envisaged assay methods are given in the examples below.
' CA 02248l7l l998-l0-22 C 7482 (V) In a further aspect, the invention provides an assay device for use in detecting the presence of Salmonella organisms, the assay device comprising an immunoglobulin molecule or antigen-binding variant thereof in accordance with the 5 invention. Again, in principle, any of the assay devices known in the prior art (e.g. D'Aoust & Sewell, 1988 J. Food Protect. 51, 538-541; Farber et al., 1985 J. Food Protect.
48, 790-793) could be adapted for use with an immunoglobulin molecule in accordance with the invention.
10 Desirably the device will comprise a solid support (e.g.
microtitre plate, glass slide, latex bead, chromatographic test stick or the like) provided with an immunoglobulin molecule (or antigen-binding variant thereof) in accordance with the invention.
A preferred embodiment of the assay device is an immunochromatographic device, such as the ClearviewTM
device sold by Unipath Limited. Such devices are easy to use and are reliable. In particular, the device may 20 comprise two or more lmmunoglobulin molecules (or antigen-binding variants thereof), each molecule binding a respective epitope present in Salmonella, and in particular, each molecule binding a respective epitope present in the region of 403 to 452 of the Salmonella 25 typhimurium flagellin. Conveniently, one such immunoglobulin molecule will be directed against an epitope present in residues 427-436, and another such immunoglobulin molecule will be directed against an epitope present in residues 444-451. Embodiments of this type 30 facilitate use of a "sandwich" type assay device and method, in which Salmonella organisms are captured on a solid support with a first immunoglobulin, and detected by binding to a second (typically) labelled immunoglobulin.
' CA 02248l7l l998-l0-22 C 7482 (V) The assay method and assay device of the invention are, in preferred embodiments, able to detect the majority (over 85%, preferably over 95%) of pathogenically important Salmonella species (i.e. those which generally cause 5 infections in man), whilst giving a very low (less than 2%) false positive rate. The assay method and device may particularly be used for detecting the presence of Salmonella in foods and/or in food preparation areas.
10 The present inventors consider that the amino acid residues 403 to 452 of the flagellin protein have unexpectedly useful properties (e.g. are useful in preparing monoclonal antibodies in accordance with the invention). Accordingly, in another aspect the invention provides a peptide 15 comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence. The peptide is preferably in 20 antigenic and/or immunogenic form and preferably substantially pure. The peptide preferably comprises at least amino acid residues 427 to 436, and/or at least residues 444 to 451, of the flagellin sequence. The peptide may desirably be fused or chemically coupled to 25 other molecules (such as polypeptides or peptides which comprise T helper cell epitopes).
In addition to raising antibodies, the particular peptide sequence of the flagellin protein identified by the 30 inventors may be useful as a reagent (e.g. included as a component in an assay method and/or an assay kit for detecting the presence of Salmonella organisms). When used as a reagent in an assay, the peptide may desirably be joined to a labelling and/or anchoring moiety (e.g. biotin, C 7482 (V) or avidin) to label the peptide and/or facilitate immobilisation of the peptide to a solid surface.
Accordingly in another aspect the invention provides for a 5 method of detecting the presence of Salmonella organisms, comprising use of a peptide comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in isolation from 10 other portions of the flagellin sequence. The invention also provides for an assay device for detecting the presence of Salmonella organisms, the device comprising the peptide defined above.
15 In one embodiment, the peptide of the invention is used in an assay method and/or device involving displacement. For example, immobilised peptide could be displaced by the introduction of a sample comprising Salmonella organisms.
Preferably the peptide would be provided with a label (e.g.
20 enzyme, or coloured bead) to facilitate detection of the displacement of the peptide. Displacement assays of this type are described, for example, in WO 91/05262 and PCT/EP95/04518.
25 The invention will be further described by way of illustrative example and with reference to the accompanying drawing, Figure 1, which is a comparison of the sequence of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium A24262 with the homologous sequences of 30 various bacteria, including other Salmonella strains, and E. coli and Citrobacter strains.
Exampl~ 1 CA 02248l7l l998-l0-22 C 7482 (V) The inventors and their colleagues first used a Salmonella whole cell extract or a Salmonella flagellin preparation to make monoclonal antibodies against Salmonella "O" antigen (lipopolysaccharide, LPS) or "H" antigen (flagellin) 5 respectively. The binding sites of the various monoclonal antibodies raised against H antigens were roughly mapped by using them to probe Western blots of trypsin-digested flagellin.
10 The inventors observed that antibodies which bound to the central portion of the 490 amino acid flagellin protein tended to be too specific for use in an assay, in that whilst they did not bind to the flagellin protein of non-Salmonella organisms (and so would not give false positive 15 results) they equally would not bind to the flagellin of every Salmonella strain, such that some strains might go undetected in a test. (A similar observation has been reported by van Asten et al., 1995 J. Bacteriol. 177, 1610-1613).
Conversely, antibodies which bound at the N- or C-terminal regions tended to be insufficiently specific, in that whilst they bound to all Salmonella strains, they also bound to large numbers of non-Salmonella organisms.
The inventors hypothesised that antibodies which bound to the regions between the central portion and the N- or C-terminal may combine the desired characteristics of high specificity for Salmonella, whilst still binding to all 30 Salmonella strains. Accordingly the inventors set out to prepare such monoclonal antibodies.
A search of various computer databases was conducted to find reported flagellin sequences. The search included C 7482 (V) protein translations of coding sequences on the GenBank database (National Institutes of Health, MD, USA), the SWISS-PROT database maintained by the University of Geneva and the European Molecular Biology Laboratory (EMBL) data 5 library, and the Protein Information Resource (PIR) Protein sequence database (National Biomedical Research Foundation, Washington D.C.). Flagellin protein sequences recovered from the databases were aligned by the Clustal method using the Megalign facility on DNA-Star software. Sequences 10 available for comparison were limited to a few strains of Salmonella, E. coli, Proteus, Serratia, Yersinia, Pseudomonas and Campylobacter. Of particular interest were the flagellins of Enterobacteriaceae which typically cause false positives in Salmonella immunoassays: Citrobacter, E.
15 coli and Proteus.
From this comparison, a stretch of 50 amino acids, corresponding to residues 403 to 452 of the S. typhimurium sequence PIR accession A24262 was selected as being 20 relatively well conserved among the Salmonellae while exhibiting some differences between Salmonellae and non-Salmonellae. Flagellins from different strains vary in length, so the amino acid numbering is not universal.
25 A few Salmonella flagellin sequences were already known, but the inventors determined several more. Figure 1 shows a comparison of the amino acid sequence in this 50 residue region (403-452) for various strains of Salmonella and some non-Salmonella bacteria. The consensus sequence 30 ("majority") is shown above and generally corresponds to that of S. typhimurium A24262 flagellin. The numbers to the right of the sequence refer to the culture collection numbers of the various strains examined and correlate with those shown in Table 1.
' CA 02248171 1998-10-22 C 7482 (V) Within this fifty amino acid region, the ten amino acid sequence RFNSAITNLG, corresponding to residues 427 to 436 of the S. typhimurium A24262 sequence, was chosen as being 5 especially specific to the Salmonellae. This sequence was therefore selected for synthesis as peptide immunogen.
Since many Salmonella serotypes have the sequence variant RFDSAITNLG it was necessary to select for antibodies which were indifferent to this variation. This was achieved by 10 screening the hybridomas for their ability to bind to flagellins of the two types (represented here by S.
typhimurium and S. tennessee "6388").
The inventors subsequently determined the sequences of the 15 flagellins of a number of additional Salmonellae and non-Salmonellae, most significantly the Citrobacter strains and an unusual strain of E. coli (NCTC 10418). Certain Citrobacter strains and E. coli NCTC 10418 contain the peptide RFNSAITNLG in its entirety and therefore would be 20 expected to bind to any Salmonella-binding MAb raised against that peptide sequence.
Synthesis of Peptide An RFNSAITNLG "Multi-Antigenic Peptide" (MAP) was produced 25 on the NovaSyn GemTM semi-automatic solid phase peptide synthesiser. The solid support was Kieselguhr [NovaSyn KA
100] derivatised with a branching octavalent lysine core with the following structure:
30 Fmocg_Lys4_Lys-2_Lys- -Ala-Solid support The following peptide sequence was built up on the support using standard Fmoc chemistry:
' CA 02248l7l l998-l0-22 C 7482 (V) (Lys-Lys-Arg-Phe-Asn-Ser-Ala-Ile-Thr-Asn-Leu-Gly) 8- [lysine core]- -Ala-Solid support The peptide comprises the residues 427 to 436 of the S.
5 typhimurium sequence, preceded by two Lys residues which were included to improve the water-solubility of the peptide and are not part of the flagellin sequence.
Following synthesis, the peptide was cleaved from the support using trifluoroacetic acid/tri-isopropyl silane 10 solution and concentrated by evaporation. The peptide was extracted three times with diethyl ether and dried under vacuum. The peptide was dissolved in distilled H2O and dialysed overnight against distilled H2O. The peptide was freeze-dried and stored at -20 C. A sample of the MAP was 15 analysed on an Applied Biosystems automated protein sequencer. This confirmed that the sequence had been authentically synthesized as above.
I unisation with Peptide 20 A BALB/C mouse initially received, by the subcutaneous and intraperitoneal routes, a total of 50 g MAP emulsified in Freund's Complete Adjuvant. Two months later it received, by the subcutaneous and intraperitoneal routes, a total of g MAP emulsified in Freund's Incomplete Adjuvant.
25 Immunisation in Freund's Incomplete Adjuvant was repeated after 2.5 weeks. A first test tail bleed followed after 1 week. The mouse was immunised with a further 50 g of MAP
by the intraperitoneal route ten days later. A second test tail bleed followed after two weeks. One month later the 30 mouse was immunised with 50 g of purified flagellin from 5. typhimurium and 50 g of purified flagellin from S.
tennessee. A third test tail bleed was performed nine days later. After seven weeks the mouse received 100 g MAP
.... .. ~ .
' CA 02248171 1998-10-22 C 7482 (V) intravenously. Three days later the animal was sacrificed and its spleen removed for preparation of hybridomas.
Tail Bleed Testing 5 The antigens used for testing by ELISA were MAP, prepared as above, and flagellins purified from plate cultures of S.
typhimurium and S. tennessee by a differential centrifugation protocol modified from that of Kerridge et al. [1962 J. Mol. Biol. 4 pp 227-238]. Flagellin 10 preparations were boiled for 5 minutes prior to dilution in carbonate buffer to improve the exposure of Salmonella-specific epitopes. Greiner high-capacity binding microtitre plates were sensitised overnight at 4 C with 100 l of antigen (MAP diluted to 5 g/ml, or flagellins 15 diluted to 1 g/ml in 0.05 M sodium carbonate buffer, pH
9.6). The wells were washed with PBS and then blocked with 200 l of PBS containing 0.05% Tween-20 and 1% bovine serum albumin ("PBST/BSA") at 37 C for 1 hour. The wells were then washed in PBS. 100 l samples of tail bleed sera, in 20 serial 10-fold or 5-fold dilutions made in PBST, from immunised and non-immunised mice, were added to the wells for 2 hours at 37 C. The wells were again washed with PBS.
100 l of rabbit anti-mouse IgG/IgA/IgM-alkaline phosphatase conjugate [Zymed] diluted 1/1000 with PBST/BSA
25 was added for 1 hour at 37 C. The wells were then thoroughly washed with PBS and 100 l of p-nitrophenyl phosphate [Sigma 104 Phosphatase Substrate tablets, dissolved at lmg/ml in 1 M diethanolamine, pH 9.8] was added for 30-60 minutes at 37 C. Yellow colour development 30 was measured using a spectrophotometer at 410 nm.
Wells coated with MAP gave signals above background levels with the first tail bleed serum from the immunised mouse at a dilution of 1/100,000; whereas wells coated with ' CA 02248171 1998-10-22 C 7482 (V) flagellins reacted at 1/100 dilution of serum. With the second tail bleed, the flagellins reacted with serum at 1/1000 dilution. With the third tail bleed, S. typhimurium flagellin reacted at 1/12500 dilution of serum and S.
5 tennessee flagellin at 1/62500.
Preparation of Hybridomas The spleen removed from the immunised mouse was perfused with RPMI medium to collect spleen cells. Approximately 5 10 x 107 spleen cells were fused with 4 x 107 SP2 mouse myeloma cells in the presence of polyethylene glycol by standard methods (Kohler & Milstein, 1975 Nature 256, 495).
Fused cells were diluted in selective medium (RPMI
containing 10% horse serum, 2.4 mM L-Glutamine, 1.2 mM
15 sodium pyruvate, 60 I.U./ml penicillin, 60 g/ml streptomycin, 10-4 M hypoxanthine and 1 g/ml azaserine) and plated out in twelve 48-well culture plates. The plates were incubated at 37 C in 5% CO2 for one week. The selective medium was then replaced by growth medium (RPMI
20 containing 5% horse serum, 2.4 mM L-Glutamine, 1.2 mM
sodium pyruvate, 60 I.U./ml penicillin, and 60 g/ml streptomycin). Two days later the hybridoma supernatants were screened by ELISA for binding to MAP and flagellins, as detailed below.
Greiner high-capacity binding plates were sensitised with MAP or boiled flagellin from S. typhimurium, washed, and blocked, as described for Tail Bleed Testing above. 100 of individual hybridoma well supernatant was added to the 30 wells for 2 hours at 37 C. Following washing with PBS, the bound antibody was detected with conjugate and colorimetric substrate as described for Tail Bleed Testing. Colour development at 37 C was measured after 1 hour. Of 576 hybridoma wells screened, 38 produced antibodies which .
CA 02248l7l l998-l0-22 C 7482 (V) reacted with both MAP- and flagellin-sensitised wells.
After expansion, these hybridomas were retested in ELISA
for binding of supernatant antibody to 4 different immobilised antigens: MAP; boiled flagellin from S.
5 typhimurium; boiled flagellin from S. tennessee; and bovine serum albumin. The microtitre plate wells were sensitised overnight at 4 C with antigens at 1 g/ml in carbonate buffer, except MAP which was at 5 g/ml. Assay steps were as described above. One hybridoma was found to bind 10 strongly to MAP and the two flagellins, but not to BSA, and was therefore selected for further analysis.
Subcloning The MAP- and flagellin-binding hybridoma was subcloned by 15 the method of limiting dilution. Subclones were re-tested for their ability to bind specifically to MAP and the two flagellin types. The hybridoma was isotyped after subcloning using isotype-specific rabbit anti-mouse-alkaline phosphatase conjugates and found to be of the IgG1 2 0 class.
Specificity testing Overnight cultures of bacterial strains (17 motile strains of Salmonella plus 19 other Enterobacteriaceae) in 25 brain/heart infusion (BHI) broth were boiled for 30 min and centrifuged for 5 minutes at 13,000 rpm in a microcentrifuge. The culture supernatants (which contain shed flagella) were diluted 1:10 with carbonate buffer and 100,ul used to sensitise the wells of Greiner high capacity 30 binding plates overnight at 4 C. Hybridoma supernatants were tested for binding to the wells by ELISA as described above. The subclone showing the most uniform reaction with the Salmonellae and the least reaction with the other Enterobacteriaceae was selected for re-cloning. Following C 7482 (V) re-cloning, the specificity of the hybridoma was re-confirmed using the same bacterial panel. The re-cloned hybridoma cell line was designated 5836.1.
5 Purification of MAb 5836.1 A culture supernatant of cell line 5836.1 in serum-free medium was filtered to remove cells and loaded onto a PROSEPR High Capacity Protein A column [bioProcessing Inc.]
in lM borate buffer pH 8.5 containing 0.15M NaCl.
10 Following washing in binding buffer, the MAb was eluted in O.lM citrate buffer, pH 3Ø Purified MAb 5836.1 was tested for binding in ELISA to immobilised crude supernatant antigens (prepared as above) from Salmonella and Enterobacteriaceae strains. The panel comprised:
1. Salmonella serotypes common in food outbreaks (according to UK Public Health Laboratory Service figures):
S. enteritidis, S. typhimurium, and S. virchow.
2. Salmonella serotyopes which include strains known not 20 to bind to some earlier "Salmonella-specific" MAbs: S.
derby, S. tennessee, S. ealing, S. muenchen, and S.
rubislaw.
3. Type strains of Enterobacteriaceae (potential cross-reactors).
25 4. An unusual strain of E. coli (NCTC 10418) which the inventors' studies have shown to contain sequence identities with Salmonella in the region of the flagellin MAP peptide.
30 The results are shown in Table 1. Monoclonal antibody from the 5836.1 hybridoma is able to detect all motile strains of Salmonella so far tested. It cross-reacts with E. coli NCTC 10418 and, to a certain extent, with certain . CA 02248171 1998-10-22 C 7482 (V) Citrobacter strains which are now known to have flagella possessing the Salmonella sequence RFNSAITNLG.
CA 02248l7l l998-l0-22 C 7482 (V) Table 1 Strain Detected by 5836.1 Co ents S. Typhimurium NCTC 12416 YES Type Strain S. Enteritidis NCTC 12694 YES Type Strain S. Virchow NCTC 5742 YES
S. Tennessee NCTC 6388 YES
S. Derby NCTC 5721 YES
S. Ealing NCTC 11948 YES
S. Muenchen ATCC 8388 YES
S. Rubislaw ATCC 10717 YES
Escherichia coli NCTC 10418 YES Flagellin has "Salmonella"
epitopes E. coli NCTC 9001 YES [Weak]
E. coli NCTC 11954 NO
Citrobacter diversus NO
C. feundii NCTC 6267 YES [Very weak]
C. freundii NCTC 9750 NO Type Strain C. koseri NcTC 10786 NO Type Strain Proteus mirabilis NCTC 11938 NO Type Straln Klebsiella sp. NO
Enterobacter NCTC 10005 NO Type Strain Edwardsiella tarda NCTC NO Type Straln ~ CA 02248171 1998-10-22 C 7482 (V) Example 2 A second peptide was prepared, using conventional peptide synthesising chemistry, which comprised the sequence SARSRIED, corresponding to amino acid residues 444-451 of 5 the flagellin protein of S. typhimurium strain A24262, fused at its N terminal to a T cell epitope from sperm whale myoglobin (which is known to be well-recognised by the MHC Class II molecules of BALB/c mice - Prieto et al, 1995 Eur. J. Immunol. 25, 877-885). The sequence of the 10 resulting peptide, known as T2-SARS, is shown below:
(Seq ID No. 4 in the attached sequence listing) FISEAIIHVLHSRSSARSRIED
15 Preparation of anti-T2-SARS hybridomas A set of four BALB/c mice were immunised with the T2-SARS
peptide essentially as described in Example 1 above.
The three responding mice from the set were sacrificed and 20 their spleen cells used to create hybridomas. The initial screenings (using as solid phase antigens S. typhimurium flagellin, or T2-SARS peptide and BSA) indicated that of 1440 clones tested, five reacted with both flagellin and T2-SARS peptide, while six reacted with T2-SARS peptide 25 alone. In addition, and rather surprisingly, many clones were found which reacted with flagellin alone; most of these originated from the mouse which had shown the strongest anti-flagellin serum titres.
30 The most promising hybridomas were subcloned and tested in ELISA for binding to crude supernatant flagellin antigens of Salmonella and cross-reactor strains. Only one hybridoma, 6035.1, showed a satisfactory degree of specificity for Salmonella, with cross-reactivity to E.
CA 02248l7l l998-l0-22 C 7482 (V) coli NCTC 10418 and Citrobacter strain CMCC 3389. There was very little reaction of 6035.1 with Citrobacter strains NCTC 6267 and CMCC 3022, (i. e. those containing the epitope EARSRIED rather than SARSRIED). Thus the object of the 5 immunisations was accomplished and a monoclonal antibody identified which could distinguish Citrobacter strains on the basis of a single amino acid alteration.
The results of the ELISA tests (conducted as described in 10 Example 1) are shown in Table 2, which details the results obtained with the monoclonal antibodies from hybridoma 6035.1 (described above), 5836.1 (described in Example 1), and a further hybridoma obtained by the inventors (5972.1), which are compared with two monoclonal antibodies produced by prior art hybridomas (3034.13 and 3020.13).
CA 02248l7l l998-l0-22 C 7482 (V) Table 2 Strain ELISA
Result 3034.13 3020.13 5836.1 5972.1 6035.1 S. Typhimurium NCTC 12416 [T] + + + + +
S. Enteritidis NCTC 12694 [T] + + + + +
S. Virchow NCTC 5742 + + + + +
S. Tennessee NCTC 6388 + - + + +
S. Derby NCTC 5721 + - + + +
S. Ealing CMCC 2071 + - + + +
S. Ealing NCTC 11948 + + + + +
S. Muenchen ATCC 8388 + - + + +
S. Rubislaw ATCC 10717 + + + + +
E. coli NCTC 10418 + - + + +
E. coli NCTC 9001 - -- +
Citrobacter diversus CMCC 3022 + + - +
Citrobacter koseri +
NCTC10786[T~
Citrobacter freundii NCTC 6267 + + + +
Citrobafter freundii CMCC3389 + - + + +
Citrobacter freundii + - - +
NCTC9750[T]
Proteus mirabilis NCTC +
11938~T~
Klebsiella sp. CMCC 2703 Enterobacter NCTC 10005 [Tl +
Edwardsiella tarda NCTC 10396 +
+ = positive ELISA result - = negative ELISA result (Strains with the suffix CMCC are proprietary to the applicant) .. . . ... .. . ~ ~
C 7482 (V) Example 3 Immunoglobulins in accordance with the invention, as exemplified by the monoclonal antibody obtainable from hybridoma 5836.1 or 6035.1, could be useful in any of a 5 large number of different assay formats for detection of Salmonella. Such assays include ELISA, immunochromatographic and Western blot-type assays.
Details of such assays are proposed below.
10 In one embodiment, flagella antigen present in a sample to be tested could be deposited on a solid phase (e.g.
microtitre well surface) and used to capture Salmonella flagellin-specific immunoglobulin from solution. Bound immunoglobulin could be detected using, for example, an 15 anti-mouse antibody/enzyme conjugate. Alternatively, the immunoglobulin itself could be used as a conjugate with the enzyme. Methods for preparing such conjugates are well-known to those skilled in the art.
20 In an alternative embodiment, flagellin-specific immunoglobulin (e.g. a monoclonal or MAb) could be attached to a solid phase and used to capture flagella antigen from bacterial culture. Antigen would be detected by a labelled second antibody (e.g. conjugated to enzyme). The second 25 antibody would be capable of binding all the Salmonella strains bound by the first antibody/MAb and might additionally bind some Enterobacterial strains not bound by the first antibody. The overall specificity, however, would remain that of the MAb. Alternatively, the second 30 antibody could be bound to the solid phase and used to capture antigen and the MAb used to detect the antigen.
In another format Multi-Antigenic Peptide (MAP) or similar antigen could be attached to a solid phase, and flagella ' CA 02248l7l l998-l0-22 C 7482 (V) antigen detected through its capacity to inhibit binding of immunoglobulin to MAP, by competition of MAP and flagella antigen for binding sites of the immunoglobulin.
5 Alternatively Multi-Antigenic Peptide (MAP) or similar antigen could be attached to a solid phase and two antibodies would be set in competition for binding to the MAP: a labelled anti-MAP antibody which does not bind to flagellin (the inventors have produced a monoclonal 10 antibody with such a specificity as a by-product of preparing the 5836.1 hybridoma)i and a MAb, which binds both MAP and flagellin. In the presence of flagellin antigen, the binding site of the MAb would be occupied and unable to compete with the MAP-specific antibody for 15 binding to the solid phase. The consequence would be a rise in the ELISA signal in the presence of flagellin antigen.
The foregoing formats may generally be considered as ELISA-20 type detection methods. A different format would be the use of an immunochromatographic device, using similar apparatus to the Unipath CLEARVIEWTM device.
For example, a MAb could be used as either the capture 25 antibody (nitrocellulose-linked) or the detection antibody (latex-linked) in the "CLEARVIEW"TM format for detection of flagellin antigen. A second antibody would be required to fulfil the other role. The second antibody would preferably be capable of binding all the Salmonella strains 30 bound by the MAb and might additionally bind some Enterobacterial strains not bound by the MAb. The overall specificity, however, would remain that of the MAb.
C 7482 (V) Finally, the MAb could be used to detect Salmonella flagellin antigen in Western blots of bacterial proteins by standard protocols.
5 Example 4 Detection of Food-Borne 5~7~on~7 7~ Using MAb from hybridoma 5836.1 A food sample would be mixed with a pre-enrichment medium and incubated for a period, typically overnight, at a 10 temperature conducive to the recovery and growth of Salmonella cells (e.g. 35-37 C). A portion of the pre-enrichment broth would then be added to selective medium and incubated for a period, typically overnight, at a temperature favouring growth of Salmonella over competitor 15 organisms (e.g. 35-37 C, or 43 C). A portion of the selection broth would then be added to a post-enrichment broth and incubated for a period, typically 6 hours, at a temperature conducive to the expression of flagellin antigen (e.g. 37 C).
Conceivably, the post-enrichment broth could be omitted and/or the pre-enrichment and selective media combined to streamline the culturing process.
Following such traditional or streamlined culture of the 25 organisms, a sample of the culture would be treated, typically by heating at 100 C for 10 minutes, but conceivably otherwise by acid, alkali or other chemical treatment, in order to expose the epitopes recognised by the antibodies in the test.
The presence of Salmonella flagella antigen in the sample would be revealed using an assay format such as that described in Example 3 (ideally by ELISA or use of an immunochromatograph device) employing a MAb such as that C 7482 (V) obtainable from hybridoma 5836.1. A positive reaction in the assay would indicate the presence of Salmonella organisms in the original food sample. A negative result would indicate the absence of Salmonella, or their presence 5 below a certain threshold value.~
~ . .. . --CA 02248l7l l999-0l-29 ., SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Unilever PLC
(B) STREET: Unilever House, Blackfriars (C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): EC4P 4BQ
(G) TELEPHONE: (01234) 781781 (H) TELEFAX: (01234) 222633 (ii) TITLE OF INVENTION: Improvements in or Relating to Detection of Salmonella (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BERESKIN ~ PARR
(B) STREET: 40 King Street West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: M5H 3Y2 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,248,171 (B) FILING DATE: 22-OCT-1998 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Langton, Daivd W.R.
(B) REGISTRATION NUMBER: 27,747 (C) REFERENCE/DOCKET NUMBER: 748-2427 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311 (B) TELEFAX: (416) 361-1398 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Salmonella typhimurium (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Pro Leu Ala Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Ala Leu Arg CA 02248l7l l999-0l-29 ..
Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Ser Ser Ala Arg Ser Arg Ile Glu Asp Ala (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Arg Phe Asp Ser Ala Ile Thr Asn Leu Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Ala Arg Ser Arg Ile Glu Asp (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Phe Ile Ser Glu Ala Ile Ile His Val Leu His Ser Arg Ser Ser Ala Arg Ser Arg Ile Glu Asp CA 02248l7l l998-l0-22 C 7482 (V) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Ala Arg Ser Arg Ile Glu Asp (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Phe Ile Ser Glu Ala Ile Ile His Val Leu His Ser Arg Ser Ser Ala Arg Ser Arg Ile Glu Asp _ _ ... .
25 4. An unusual strain of E. coli (NCTC 10418) which the inventors' studies have shown to contain sequence identities with Salmonella in the region of the flagellin MAP peptide.
30 The results are shown in Table 1. Monoclonal antibody from the 5836.1 hybridoma is able to detect all motile strains of Salmonella so far tested. It cross-reacts with E. coli NCTC 10418 and, to a certain extent, with certain . CA 02248171 1998-10-22 C 7482 (V) Citrobacter strains which are now known to have flagella possessing the Salmonella sequence RFNSAITNLG.
CA 02248l7l l998-l0-22 C 7482 (V) Table 1 Strain Detected by 5836.1 Co ents S. Typhimurium NCTC 12416 YES Type Strain S. Enteritidis NCTC 12694 YES Type Strain S. Virchow NCTC 5742 YES
S. Tennessee NCTC 6388 YES
S. Derby NCTC 5721 YES
S. Ealing NCTC 11948 YES
S. Muenchen ATCC 8388 YES
S. Rubislaw ATCC 10717 YES
Escherichia coli NCTC 10418 YES Flagellin has "Salmonella"
epitopes E. coli NCTC 9001 YES [Weak]
E. coli NCTC 11954 NO
Citrobacter diversus NO
C. feundii NCTC 6267 YES [Very weak]
C. freundii NCTC 9750 NO Type Strain C. koseri NcTC 10786 NO Type Strain Proteus mirabilis NCTC 11938 NO Type Straln Klebsiella sp. NO
Enterobacter NCTC 10005 NO Type Strain Edwardsiella tarda NCTC NO Type Straln ~ CA 02248171 1998-10-22 C 7482 (V) Example 2 A second peptide was prepared, using conventional peptide synthesising chemistry, which comprised the sequence SARSRIED, corresponding to amino acid residues 444-451 of 5 the flagellin protein of S. typhimurium strain A24262, fused at its N terminal to a T cell epitope from sperm whale myoglobin (which is known to be well-recognised by the MHC Class II molecules of BALB/c mice - Prieto et al, 1995 Eur. J. Immunol. 25, 877-885). The sequence of the 10 resulting peptide, known as T2-SARS, is shown below:
(Seq ID No. 4 in the attached sequence listing) FISEAIIHVLHSRSSARSRIED
15 Preparation of anti-T2-SARS hybridomas A set of four BALB/c mice were immunised with the T2-SARS
peptide essentially as described in Example 1 above.
The three responding mice from the set were sacrificed and 20 their spleen cells used to create hybridomas. The initial screenings (using as solid phase antigens S. typhimurium flagellin, or T2-SARS peptide and BSA) indicated that of 1440 clones tested, five reacted with both flagellin and T2-SARS peptide, while six reacted with T2-SARS peptide 25 alone. In addition, and rather surprisingly, many clones were found which reacted with flagellin alone; most of these originated from the mouse which had shown the strongest anti-flagellin serum titres.
30 The most promising hybridomas were subcloned and tested in ELISA for binding to crude supernatant flagellin antigens of Salmonella and cross-reactor strains. Only one hybridoma, 6035.1, showed a satisfactory degree of specificity for Salmonella, with cross-reactivity to E.
CA 02248l7l l998-l0-22 C 7482 (V) coli NCTC 10418 and Citrobacter strain CMCC 3389. There was very little reaction of 6035.1 with Citrobacter strains NCTC 6267 and CMCC 3022, (i. e. those containing the epitope EARSRIED rather than SARSRIED). Thus the object of the 5 immunisations was accomplished and a monoclonal antibody identified which could distinguish Citrobacter strains on the basis of a single amino acid alteration.
The results of the ELISA tests (conducted as described in 10 Example 1) are shown in Table 2, which details the results obtained with the monoclonal antibodies from hybridoma 6035.1 (described above), 5836.1 (described in Example 1), and a further hybridoma obtained by the inventors (5972.1), which are compared with two monoclonal antibodies produced by prior art hybridomas (3034.13 and 3020.13).
CA 02248l7l l998-l0-22 C 7482 (V) Table 2 Strain ELISA
Result 3034.13 3020.13 5836.1 5972.1 6035.1 S. Typhimurium NCTC 12416 [T] + + + + +
S. Enteritidis NCTC 12694 [T] + + + + +
S. Virchow NCTC 5742 + + + + +
S. Tennessee NCTC 6388 + - + + +
S. Derby NCTC 5721 + - + + +
S. Ealing CMCC 2071 + - + + +
S. Ealing NCTC 11948 + + + + +
S. Muenchen ATCC 8388 + - + + +
S. Rubislaw ATCC 10717 + + + + +
E. coli NCTC 10418 + - + + +
E. coli NCTC 9001 - -- +
Citrobacter diversus CMCC 3022 + + - +
Citrobacter koseri +
NCTC10786[T~
Citrobacter freundii NCTC 6267 + + + +
Citrobafter freundii CMCC3389 + - + + +
Citrobacter freundii + - - +
NCTC9750[T]
Proteus mirabilis NCTC +
11938~T~
Klebsiella sp. CMCC 2703 Enterobacter NCTC 10005 [Tl +
Edwardsiella tarda NCTC 10396 +
+ = positive ELISA result - = negative ELISA result (Strains with the suffix CMCC are proprietary to the applicant) .. . . ... .. . ~ ~
C 7482 (V) Example 3 Immunoglobulins in accordance with the invention, as exemplified by the monoclonal antibody obtainable from hybridoma 5836.1 or 6035.1, could be useful in any of a 5 large number of different assay formats for detection of Salmonella. Such assays include ELISA, immunochromatographic and Western blot-type assays.
Details of such assays are proposed below.
10 In one embodiment, flagella antigen present in a sample to be tested could be deposited on a solid phase (e.g.
microtitre well surface) and used to capture Salmonella flagellin-specific immunoglobulin from solution. Bound immunoglobulin could be detected using, for example, an 15 anti-mouse antibody/enzyme conjugate. Alternatively, the immunoglobulin itself could be used as a conjugate with the enzyme. Methods for preparing such conjugates are well-known to those skilled in the art.
20 In an alternative embodiment, flagellin-specific immunoglobulin (e.g. a monoclonal or MAb) could be attached to a solid phase and used to capture flagella antigen from bacterial culture. Antigen would be detected by a labelled second antibody (e.g. conjugated to enzyme). The second 25 antibody would be capable of binding all the Salmonella strains bound by the first antibody/MAb and might additionally bind some Enterobacterial strains not bound by the first antibody. The overall specificity, however, would remain that of the MAb. Alternatively, the second 30 antibody could be bound to the solid phase and used to capture antigen and the MAb used to detect the antigen.
In another format Multi-Antigenic Peptide (MAP) or similar antigen could be attached to a solid phase, and flagella ' CA 02248l7l l998-l0-22 C 7482 (V) antigen detected through its capacity to inhibit binding of immunoglobulin to MAP, by competition of MAP and flagella antigen for binding sites of the immunoglobulin.
5 Alternatively Multi-Antigenic Peptide (MAP) or similar antigen could be attached to a solid phase and two antibodies would be set in competition for binding to the MAP: a labelled anti-MAP antibody which does not bind to flagellin (the inventors have produced a monoclonal 10 antibody with such a specificity as a by-product of preparing the 5836.1 hybridoma)i and a MAb, which binds both MAP and flagellin. In the presence of flagellin antigen, the binding site of the MAb would be occupied and unable to compete with the MAP-specific antibody for 15 binding to the solid phase. The consequence would be a rise in the ELISA signal in the presence of flagellin antigen.
The foregoing formats may generally be considered as ELISA-20 type detection methods. A different format would be the use of an immunochromatographic device, using similar apparatus to the Unipath CLEARVIEWTM device.
For example, a MAb could be used as either the capture 25 antibody (nitrocellulose-linked) or the detection antibody (latex-linked) in the "CLEARVIEW"TM format for detection of flagellin antigen. A second antibody would be required to fulfil the other role. The second antibody would preferably be capable of binding all the Salmonella strains 30 bound by the MAb and might additionally bind some Enterobacterial strains not bound by the MAb. The overall specificity, however, would remain that of the MAb.
C 7482 (V) Finally, the MAb could be used to detect Salmonella flagellin antigen in Western blots of bacterial proteins by standard protocols.
5 Example 4 Detection of Food-Borne 5~7~on~7 7~ Using MAb from hybridoma 5836.1 A food sample would be mixed with a pre-enrichment medium and incubated for a period, typically overnight, at a 10 temperature conducive to the recovery and growth of Salmonella cells (e.g. 35-37 C). A portion of the pre-enrichment broth would then be added to selective medium and incubated for a period, typically overnight, at a temperature favouring growth of Salmonella over competitor 15 organisms (e.g. 35-37 C, or 43 C). A portion of the selection broth would then be added to a post-enrichment broth and incubated for a period, typically 6 hours, at a temperature conducive to the expression of flagellin antigen (e.g. 37 C).
Conceivably, the post-enrichment broth could be omitted and/or the pre-enrichment and selective media combined to streamline the culturing process.
Following such traditional or streamlined culture of the 25 organisms, a sample of the culture would be treated, typically by heating at 100 C for 10 minutes, but conceivably otherwise by acid, alkali or other chemical treatment, in order to expose the epitopes recognised by the antibodies in the test.
The presence of Salmonella flagella antigen in the sample would be revealed using an assay format such as that described in Example 3 (ideally by ELISA or use of an immunochromatograph device) employing a MAb such as that C 7482 (V) obtainable from hybridoma 5836.1. A positive reaction in the assay would indicate the presence of Salmonella organisms in the original food sample. A negative result would indicate the absence of Salmonella, or their presence 5 below a certain threshold value.~
~ . .. . --CA 02248l7l l999-0l-29 ., SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Unilever PLC
(B) STREET: Unilever House, Blackfriars (C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): EC4P 4BQ
(G) TELEPHONE: (01234) 781781 (H) TELEFAX: (01234) 222633 (ii) TITLE OF INVENTION: Improvements in or Relating to Detection of Salmonella (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BERESKIN ~ PARR
(B) STREET: 40 King Street West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: M5H 3Y2 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,248,171 (B) FILING DATE: 22-OCT-1998 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Langton, Daivd W.R.
(B) REGISTRATION NUMBER: 27,747 (C) REFERENCE/DOCKET NUMBER: 748-2427 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311 (B) TELEFAX: (416) 361-1398 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Salmonella typhimurium (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Pro Leu Ala Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Ala Leu Arg CA 02248l7l l999-0l-29 ..
Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Ser Ser Ala Arg Ser Arg Ile Glu Asp Ala (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Arg Phe Asp Ser Ala Ile Thr Asn Leu Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Ala Arg Ser Arg Ile Glu Asp (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Phe Ile Ser Glu Ala Ile Ile His Val Leu His Ser Arg Ser Ser Ala Arg Ser Arg Ile Glu Asp CA 02248l7l l998-l0-22 C 7482 (V) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Ala Arg Ser Arg Ile Glu Asp (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Phe Ile Ser Glu Ala Ile Ile His Val Leu His Ser Arg Ser Ser Ala Arg Ser Arg Ile Glu Asp _ _ ... .
Claims (36)
1. An immunoglobulin molecule or antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains.
2. A polyclonal or monoclonal antibody according to claim 1.
3. An immunoglobulin molecule or antigen-binding variant thereof according to claim 1 or 2, which binds to an epitope present within amino acid residues 418 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains.
4. An immunoglobulin molecule or antigen-binding variant thereof according to any one of claims 1, 2 or 3 which binds to an epitope present within amino acid residues 427 to 436 of the flagellin protein of Salmonella typhimurium PIR
accession No. A24262, or the equivalent portion from other Salmonella strains.
accession No. A24262, or the equivalent portion from other Salmonella strains.
5. An immunoglobulin molecule or antigen-binding variant thereof according to any one of the preceding claims, which binds to the amino acid sequence RFDSAITNLG and/or RFNSAITNLG.
6. A monoclonal antibody according to any one of the preceding claims, obtainable from the hybridoma 5836.1.
7. An immunoglobulin molecule or antigen-binding variant thereof according to any one of claims 1, 2 or 3, which binds to an epitope present within amino acid residues 444 to 451 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion from other strains.
8. An immunoglobulin or antigen-binding variant thereof molecule according to any one of claims 1, 2 or 3, which binds to the amino acid sequence SARSRIED.
9. A monoclonal antibody according to claim 7 or 8, obtainable from the hybridoma 6035.1.
10. A monoclonal antibody according to any one of the preceding claims, present in a cell culture supernatant, ascites fluid or as a purified composition.
11. A host cell capable of producing an immunoglobulin molecule or antigen-binding variant thereof according to any one of the preceding claims.
12. A host cell according to claim 11, wherein the cell is an immortalised mammalian cell line.
13. A host cell according to claim 10 or 11, wherein the host cell is a hybridoma.
14. A host cell according to any one of claims 11, 12 or 13, being the hybridoma 5836.1 or 6035.1.
15. A nucleic acid sequence encoding an immunoglobulin molecule or antigen-binding variant thereof in accordance with any one of claims 1 to 10.
16. A nucleic acid sequence according to claim 15, obtainable from the hybridoma 5836.1 or 6035.1.
17. A method of producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising immunising a mammal with an immunogen which comprises at least one B-cell epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella strains, said B-cell epitope being in isolation from Salmonella flagellin residues outside the portion 403 to 452.
18. A method according to claim 17, to produce a monoclonal antibody according to any one of claims 1 to 10.
19. A method according to claim 17 or 18, wherein the immunogen is a synthetic peptide.
20. A method of producing an immunoglobulin molecule or antigen-binding variant thereof, the method comprising the steps of: obtaining a library of nucleic acid sequences encoding immunoglobulin molecules or antigen-binding variants thereof; screening the library with a screening molecule which comprises at least a portion of amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A 24262, or the equivalent portion of flagellin from other Salmonella strains, said portion being in substantial isolation from Salmonella flagellin residues outside the portion 403 to 452; selecting those nucleic acid sequences which encode an immunoglobulin or antigen-binding variant which binds to the screening molecule; and causing expression of the selected nucleic acid sequences to obtain the desired immunoglobulin or antigen-binding variant thereof.
21. A method for detecting the presence of Salmonella organisms in a sample under test, comprising contacting the sample with an immunoglobulin molecule or antigen-binding variant thereof which binds to an epitope present within amino acid residues 403 to 452 of the flagellin protein of Salmonella typhimurium PIR accession No. A24262, or the equivalent portion of flagellin from other Salmonella strain; and monitoring binding of the immunoglobulin molecule, or antigen-binding variant thereof, to the sample.
22. A method according to claim 21, comprising use of an immunoglobulin or antigen-binding variant thereof in accordance with any one of claims 1 to 10.
23. A method according to claim 21 or 22, wherein the anti-Salmonella immunoglobulin or antigen-binding variant thereof is labelled.
24. A method according to claim 21 or 22, comprising use of a second immunoglobulin or antigen-binding variant thereof to monitor binding of the anti-Salmonella immunoglobulin or antigen-binding variant to the sample under test.
25. A method according to claim 24, wherein the second immunoglobulin or antigen-binding variant has binding specificity for the anti-Salmonella immunoglobulin or variant thereof.
26. A method according to claim 24, wherein the second immunoglobulin or antigen-binding variant has anti-Salmonella binding specificity.
27. A method according to any one of claims 21-24, comprising use of an immunoglobulin molecule or antigen-binding variant thereof in accordance with claim 4, and use of an immunoglobulin molecule or antigen-binding variant thereof in accordance with claim 7.
28. A method according to any one of claims 21 to 27, comprising use of a label selected from the group consisting of: radioactive labels; enzyme labels; biochemical labels;
and coloured latex particles.
and coloured latex particles.
29. An assay device for detecting the presence of Salmonella organisms in a sample under test, the device comprising an immunoglobulin molecule or antigen-binding variant thereof in accordance with any one of claims 1 to 10.
30. A device according to claim 29, for performing the method of any one of claims 21 to 28.
31. A molecule comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S.
typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence.
typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence.
32. A molecule according to claim 31, comprising residues 427 to 436 of the flagellin protein of S. typhimurium PIR
accession No. A24262, or the equivalent portion from other Salmonella strains.
accession No. A24262, or the equivalent portion from other Salmonella strains.
33. A molecule according to claim 31 or 32, comprising residues 444 to 451 of the flagellin protein of S.
typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains.
typhimurium PIR accession No. A24262, or the equivalent portion from other Salmonella strains.
34. A method of detecting the presence of Salmonella organisms in a sample under test, comprising use of a molecule comprising at least part of amino acid residues 403 to 452 of the flagellin protein of S. typhimurium PIR
accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence.
accession No. A24262, or the equivalent portion from other Salmonella strains, in substantial isolation from other portions of the flagellin sequence.
35. An assay device for detecting the presence of Salmonella organisms in a sample under test, the device comprising a molecule in accordance with any one of claims 31, 32 or 33.
36. An immunoglobulin molecule as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP97308838 | 1997-11-04 | ||
| EP97308838.8 | 1997-11-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2248171A1 true CA2248171A1 (en) | 1999-05-04 |
Family
ID=29414892
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2248171 Abandoned CA2248171A1 (en) | 1997-11-04 | 1998-10-22 | Improvements in or relating to detection of salmonella |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2248171A1 (en) |
-
1998
- 1998-10-22 CA CA 2248171 patent/CA2248171A1/en not_active Abandoned
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