Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K16/1235—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Salmonella (G)

Definitions

• This invention relates to the field of detecting contamination by bacteria in organic materials. More specifically, it relates to determining the presence of contaminating microorganisms, specifically Salmonella using specific immunocomplexing reactions.
• processors of food preparations destined for the marketplace are required to conduct tests on their intermediates and products to detect the presence of these microorganisms.
• large institutions which run feeding operations would be well advised to perform such tests were they convenient and adaptable to these needs.
• Bacterial contamination remains a problem in other environments as well. Further, clinical and veterinary detection procedures are needed to diagnose the infections which result. The most important type of organism implicated in causing such contamination is that represented by the various strains of Salmonella. More than 1500 strains of Salmonella have been identified. A number of detection systems based on antibodies formed against Salmonella antigens have been reported in the literature and are used in commerce. Two methods recommended by the Food & Drug Administration utilize either preenrichment and selective plating or immunofluorescence after preenr ichment. Immunofluorescent staining procedures also include those descr ibed by Caldwell , W. J., et al , J Bacteriol (1966) 92:1177; Goepfert, J.
• the antibodies used for these assays are produced as polyclonal mixtures from sera of animals inoculated with Salmonella flagella preparations. These preparations contain a number of contaminants believed responsible for cross reactivity against, mainly, other Enterobacteriacae.
• the sera contain, for example, IgM antibodies with reactivity against O- antigens, which are common contaminants in commercial preparations of Salmonella flagella, as well as IgG antibodies which react against a portion of the flagella (the hook region) which shares immunoreactivity with other organisms.
• the present invention provides a preparation containing monoclonal antibodies which, collectively, react against a wide spectrum of Salmonella strains, but do not cross react with non-Salmonella bacteria.
• the availability of such antibody preparations permits the testing of food batches, clinical samples, and other subject materials, with reliable results. Disclosure of the Invention
• the essence of the present invention rests in the ability to prepare antibodies which are directed specifically against the many strains of Salmonella and which do not cross react with other bacterial species likely to be found in food processing operations or in other samples.
• the antibody preparations of the invention must be capable of reacting with antigens derived from the variety of Salmonella serotypes (on the order of 1500 presently known) while remaining unreactive with other enteric organisms.
• the suitable preparations of specific monoclonal antibodies are obtained by careful screening of hybridomas derived from the fusion of non-secretory myeloma cells and spleen cells isolated from animals previously immunized with highly purified filamentous flagella preparations from various Salmonella strains.
• the filamentous portion of flagella is characterized by the presence of H-antigen.
• H-antigen As determined by serotyping, there are many H-antigen types associated uniquely with Salmonella distributed among the many strains.
• a cocktail of a number of Salmonella-specific monoclonal antibodies may be required for the detection of all Salmonella serotypes.
• the invention then, in one aspect, relates to a process for preparing specific anti-Salmonella monoclonal antibodies.
• This comprises purifying flagella from a specific Salmonella strain, using these filaments to immunize a subject animal, obtaining spleen cells from the subject animal and fusing them with an immortalizing cell line, cloning successful hybridomas, and recovering monoclonal antibodies from appropriate clones.
• the invention relates to the hybridomas produced by the foregoing process, and to the monoclonal antibodies secreted by them.
• the invention relates to a procedure for determining Salmonella contamination in foods and other organic samples using a preparation of the antibodies of the invention, and to analysis kits containing these monoclonal antibodies.
• immortalizing cell line refers to a cell line which has as a component of its genetic structure, elements which operate such that it is capable of perpetual viability through a series of transfers. Further, this line is able to confer this ability upon partners to which it is fused to form hybridomas.
• Immortalizing cell lines used to illustrate the present invention are all of mammalian origin, are often loosely described as tumor cells, and are capable of maintaining not only their own viability, but upon fusion with ordinary, non-immortal cells, of confering immortality in the same sense on the fusion product.
• the most frequently encountered immortalizing cell lines currently in use are mouse myeloma lines. A number of these are available from the American Type Culture
• Preparation of antibodies refers to a preparation which contains at least one, but preferably a mixture of monoclonal antibodies, each of which has a characteristic set of reactivities with respect to particular antigenic sites against which it is reactive. By using more than one such monoclonal antibody in the preparation, a wide spectrum of reactivity against antigens associated with Salmonella is obtained.
• “Specific against Salmonella” when referred to antibodies means an antibody which immunoreacts with an antigen characteristic of Salmonella but fails to react with antigens derived from Serratia, Proteus, Shigella, Enterobacter, Klebsiella, Citrobacter, Eschericia, and other enteric bacteria. It is understood that the flagellar antigens of Salmonella currently appear to be the most specific to them; however, the definition is meant to include any determinants which are capable of characterizing this genus.
• Cells or cell line refers to both individual cells and cultures thereof. It also includes cells derived from the referenced cells, i.e., the progeny or issue thereof. It is understood that spontaneous modification of the chromosomal composition of such cells can occur. Cells and their progeny which have undergone such modification, but have not lost their functionality—i.e., in the case of the hybridoma cell line of the invention, which produce the desired monoclonal antibodies—are included in the definition.
• Salmonellae are Gram negative, motile aerobic rods which fail to ferment lactose and are pathogenic for humans and animals when ingested orally.
• the various species are closely related antigenically, though a number of subtypes of antigens are known.
• antigens are considered to derive from the flagella (H-antigens), the cell wall (O-antigens), and from the capsule (the Vi-antigens).
• the method of the invention utilizes the H-antigens since they are the most characteristic of Salmonellae as opposed to other types of bacteria.
• the flagella are entirely protein, and are rooted in the cell membrane by a complicated system of hooks and rings which permit the controlled rotation of the filamentous portion of the flagellum. It appears that the filamentous region is, in turn, more characteristic of the genus Salmonella than is the hook portion.
• the filament is composed entirely of a monomeric protein, flagellin, which is aggregated into polymeric units in constructing the filament. The polymerized flagellin can be decomposed into monomeric units by treating with heat or acid.
• Contaminations of food preparations by Salmonella can be traced to clinical carriers, feces of unsuspected subclinical cases, contaminated water supplie-s in which shellfish or other fish are grown, eggs from infected fowl and numerous other sources.
• the ubiquity of possible sources of infection and the severity of the consequences of failure to prevent its spread has made reliable detection of Salmonella contamination and accurate diagnosis of its transmission to human and animal subjects a necessity.
• the utility of the antigen/antibody interaction as a means for detecting and measuring the amount of an antigenic suspected contaminant rests on two characteristics: the specificity of the antibody/antigen reaction and the ability to make such an interaction result in a detectable characteristic.
• the method of the invention provides a uniquely specific preparation of antibodies which is directed exclusively against antigens associated with Salmonella filamentous flagellar protein.
• the antibody preparation of the invention By the use of the antibody preparation of the invention, cross- reaction with antigenic domains included in other bacterial species is avoided.
• the amount of antigen can be directly determined by the quantity of detectable material which is insolubilized, or indirectly determined by the amount which is left behind in solution. More recently, systems wherein the interaction is detected by means of an enzyme mediated color imetric or otherwise measurable reaction have been employed. These techniques, such as the enzyme linked immunosorbent assay (ELISA), utilize the generation of an enzymatic reaction product by virtue of enzyme bound directly to the reacting or secondary antibody. In any event, such means of detecting and measuring the antigen/antibody specific reaction are well known in the art. They are employed by, but not a part of, the invention.
• ELISA enzyme linked immunosorbent assay
• kits containing the antibodies of the invention comprise, besides these antibodies, the reagents associated with these detection reactions as are known in the art.
• analysis kit refers to a packaged entity containing reagents and possibly associated containers or supports useful in the detection and measurement of the specific antigen/antibody reaction which is associated with the monoclonal antibodies of the invention.
• the following example illustrates the method of the invention for preparing the hybridoma cell lines and desired monoclonal antibodies of the invention, as well as their use. It is not intended to limit the invention; indeed, specific monoclonal antibodies other than those exemplified are prepared using this method so as to provide immunoreactivity against the entire spectrum of Salmonella.
• the flagellin which precipitated after overnight storage at 4°C was harvested by centrifugation at 10,000 g for 15 min.
• the pellet containing polymeric flagellin was suspended in about 0.2 ml distilled deionized water , dialyzed overnight against distilled deionized water at 4°C for 16 hr, and then against barbital buffer (0.02 M, pH 8.4) for 4 hours at 4°C.
• the purified antigen was stored at -20°C.
• Protein concentration of the sample was determined using Bradford reagent (Bio-Rad Protein Assay, Bio-Rad). Purity of the preparation was assured by subjecting 10 ⁇ g to polyacrylamide gel electrophoresis in 12.5% slab gels containing SDS, and staining with Coomasie blue. The peptides showed a molecular weight range of 45,000-50,000 daltons and were at least 95% pure.
• Flagellin prepared as described in paragraph C.1 was used to immunize 7-8 week old female mice
• 25 ⁇ g flagellin in 100 ⁇ l PBS was administered intraperitoneally.
• One mouse was used per fusion.
• Non-secreting P3X63-Ag8.653 mouse myeloma cells were recovered from frozen stocks and grown to early log phase in RPMI 1640 with 20% heat-inactivated fetal calf serum (RPMI-FCS). Cells were harvested and centrifuged at 180g for 10 min. The pellet was resuspended in 2 ml GKN and viable cells (as indicated by trypan blue exclusion) were counted using a hemocytometer.
• a suspension of myeloma cells prepared as in C.4 containing 0.8-1.4 x 10 8 cells was added to 2 ml of the spleen cell preparation in paragraph C.3, diluted to 45 ml with GKN and centrifuged at 180g for 10 min.
• 0.5 ml of macrophage suspension was added to each well to bring the final volume to 1 ml.
• Peritoneal macrophages were obtained from ether-killed mice by flushing the peritoneal cavity with 7-10 ml 0.34 M sucrose, asceptically removing the sucrose solution and centrifuging at 180g for 10 min. The suspension referred to was of the resulting pellet in 50 ml HAT medium. Typically, macrophages from 2 mice were required for each spleen used.
• the plates were incubated at 37°C in 5% CO 2 at 85% humidity. One to two days after fusion, an additional
• HAT medium 0.5 ml HAT medium was added to each well; cells were then fed every 3-4 days by replacing the media; after 10 days, HT medium (RPMI-FCS with 13.6 ⁇ g/ml hypoxanthine and 3.78 ⁇ g/ml thymidine) was used instead of HAT. After 19- 20 days, the cells were maintained in RPMI-FCS.
• Colonies of hybrid cells become visible approximately 2 weeks after the fusion. Supernatants of wells with visible colonies were tested by solid phase ELISA for the presence of Salmonella-specific antibodies.
• Hybridoma culture supernatants were assayed for antibody species specificity using ELISA.
• Various antigens were used in the screening procedure including acidified culture samples of various Salmonella and non- Salmonella species as well as flagellar preparations of S.heidelberg, S.typhimurium, S.cubana and S.senftenberg.
• acidified culture samples bacteria were grown at 37°C for 18-24 hrs in 100 ml M Broth after repeated culturing in bacto-motility GI medium and/or in M Broth. The cultures were acidified to pH 1.5 with HCl and stored overnight at 4°C.
• the acidified cultures were treated with formaldehyde to a final concentration of 0.3%, titrated to pH 7.2 using 10N NaOH and centrifuged at 400g for 5-6 minutes to remove cellular debris.
• the supernatant was mixed with an equal volume of carbonate buffer and NaN 3 was added to a final concentration of 0.02% as an antimicrobial agent.
• the flagellar samples were prepared according to the procedures outlined in ⁇ C.1, without the heat depolymerization-repolymerization steps.
• the crude flagellin was diluted to 10 ⁇ g/ml in carbonate buffer, pH 9.6 for application to the plastic support.
• Antigen was bound to wells of 96-well polyvinyl chloride (PVC) microtiter plates by incubating 100 ⁇ l of antigen solution in each well for 2 hr at room temperature. The solutions were, removed and additional protein binding sites on the PVC were blocked with 1% BSA/Tris-saline (20 mM Tris, 0.9% NaCl, pH 7.2) for 30 min at room temperature. The wells were then washed 4X with PBS.
• PVC polyvinyl chloride
• cell lines C5A1, S5C2, S5D3 and H5B5 react with a wide variety of Salmonella serotypes.
• Cell line C5A1 was deposited at ATCC on 20 June 1984, and given accession number HB8582.
• a mixture of the antibodies from C5A1, S5C3 and H5B5 is capable of detecting 83% of the Salmonella serotypes tested thus far. Additional preparations of hybridoma cell lines following the methods outlined above will broaden the spectrum of cross reactivity against Salmonella species.
• the filters were cut lengthwise into 0.3 cm strips and incubated overnight at 4°C with 1 ml culture supernatant and 3 ml 1% BSA/Tris-saline. After washing 3X at 15 min intervals with wash buffer (0.1% BSA/Tris-saline with 0.05% NP-40), the strips were incubated in 4 ml peroxidase conjugated rabbit anti-mouse immunoglobulins diluted 1:500 in 1% BSA/Tris-saline.
• rabbit anti-sera each with a specific immunoreactivity against IgA, IgGl, IgG2a, IgG2b, IgG3, and IgM heavy chains, and against kappa and lambda light chains were used to characterize the isotypes of the antibodies produced.
• Hybridoma culture supernatants were added to antigen-coated/BSAsaturated microtiter plates and allowed to incubate 2 hours or longer at room temperature. The wells were washed 4 times with PBS and incubated for at least 2 hours at room temperature with 100 ⁇ l of a subclassspecific rabbit anti-sera.
• plastic carrier supports Two types were used: a PVC-coated microtiter well (Falcon) or Falcon "FAST" system microsticks.
• the support was treated with a series of reagents as outlined below. After treatment and incubation with each reagent, the support was washed 4X with PBS prior to treatment with the next reagent.
• the support was first coated with an antibody of a first subclass, e.g., IgGl, obtained from the supernatant of a hybridoma prepared as described above. Remaining binding sites on the support were blocked with 1% BSA/Tris-saline before incubating with the test sample containing flagellin. This was followed by incubation with an antibody from a second hybridoma of a different subclass from the first, e.g., IgG2b. The resulting sandwich of two antibody subclass types attached to the sample to be detected was then treated with rabbit anti- mouse Ig specific to the second subclass of antibody (in this case, IgG2b) and then with a peroxidase conjugated goat anti-rabbit immunoglobulin. Finally, the support was incubated with a substrate solution permitting the development of a color reaction in response to the peroxidase. The concentrations and amounts of materials used in each incubation correspond to those outlined in paragraph C.7 above.

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• 1985
  • 1985-07-24 EP EP19850903935 patent/EP0188603A1/de not_active Withdrawn
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