A SALMONELLA-SPECIFIC ANALYSES REAGENT

Abstract: A purified preparation of Salmonella flagellin is used to obtain fusion partners for the preparation of hybridomas secreting monoclonal antibodies specific for representative Salmonella species. Monoclonal antibodies secreted by those hybridomas are useful in a highly specific and accurate assay for the presence of Salmonella in foods or other organic materials.

Description: A SALMONELLA-SPECIFIC ANALYSIS REAGENT Technical Field 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. Background Art Widespread discomfort, often chronic or acute morbidity and even fatalities, are caused by the presence of contaminating microorganisms in processed or stored food. Even foods processed or stored under aerobic conditions (which would not support botulism-causing bacteria) are susceptible to contamination by organisms which, when ingested, result in ailments ranging from what is commonly known as "food poisoning" to typhoid. The presence of these contaminating organisms is often undetectable by odor or taste. As a result, food preparations which appear to be harmless can turn out to cause nausea, vomiting, diarrhea, and serious disease. To overcome this problem, 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. In addition, 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 Cal well , W. J., et al , J Bacteriol (1966) 9_2 i77; Goepfert, J. M., et al, Appl Microbiol (1969 ) .18 :612; Thompson, B. M., et al , J Food Prot (1981) 4_ :381. Other assays are based on detection of decreased motility resulting from reaction with antibody (Mohit, B., J Bacteriol (1958) 96 ;160) , combinations of antibody reactions with ability to metabolize lactose — i.e., assay for 3-galactosidase (Wilson, C. R., et al, Appl Microbiol (1971 ) _1:346) and detection of antigen/antibody reaction by enzyme mediated methods , e.g., enzyme linked immunosorbent assay (ELISA) (Krysinski, E. P., et al, Appl Environ Microbiol (1977 ) 3_3_:947; Minnich, S. A. , et al , Appl Environ Microbiol ( 1982 ) 3_ ϊ 877 ' Karlsson, K. , et al , Scand J Infect Pis (1980 ) 1^: 41 ) . All of the foregoing have employed polyclonal antibody preparations. All of the foregoing methods suffer from the same basic defect — a lack of adequate specif icity for Salmonella. Generally, 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. A putatively monoclonal preparation from myeloma cell line (MOPC-467), while less cross- reactive with other Enterobacteriacae, was found to be non-reactive with several Salmonella strains (Robison, B. J., Appl Environ Microbiol (1983) 4^:1816). This cell line arose fortuitously, the antigenic determinant against which it is reactive is not an H antigen, and its isolation offers no general method by which antibodies reactive against the remaining Salmonella species can be prepared. Some improvement in specificity exhibited by polyclonal sera by manipulation of the antibody population to remove F c portions was reported in U.S. patent 4,258,130. The lack of correct specificity results in the clearly undesirable result of false positive reactions, requiring the discard of large quantities of food which is, in fact, not contaminated and is harmless, or false negatives, which constitute a clear and present danger to health. Of course, in a clinical context, inadequately specific assays result in mistaken diagnoses and incorrect treatment. 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. Thus, 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. As determined by serotyping, there are many H-antigen types associated uniquely with Salmonella distributed among the many strains. Thus, 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. In other aspects, the invention relates to the hybridomas produced by the foregoing process, and to the monoclonal antibodies secreted by them. In still other aspects, 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. Modes of Carrying Out the Invention A. Definitions As used herein, "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 Collection (ATCC). Recently, immortalizing hybridomas have also been prepared. (See British Patent Publication No. 2,113,715A, 10 August, 1983.) However, any cell line with this property may be used in the invention. "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, Enterobacte , 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 functionalit —i.e., in the case of the hybridoma cell line of the invention, which produce the desired monoclonal antibodies—are included in the definition. B. General Description B.l The Nature of Salmonella and Its Antigens 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. In traditional classification, 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. The Salmonella classification scheme of Kaufmann-white (Holt, J. G. ed.. The Shorter Bergey's Manual of Determinative Bacteriology, 8th ed., Williams & Wilkins Co., Baltimore, MD, 1977, pp. 102-119) differentiates various serotypes on the basis of these 0, H and Vi antigenic subtypes. There are presently known over 79 subtypes of H-antigen associated with Salmonella flagellin. Flagellin from each serotype contains a typical monomeric unit bearing one or more antigenic domains. Within a culture of a single species, moreover, flagellar antigens may occur in either or both of two forms called Phase I and Phase II. B.2 Necessity for Detection Besides ordinary food poisoning, which is due most commonly to Salmonella typhimurium, there are other, more serious consequences of Salmonella food contamination. Enteric fevers such as typhoid (S. typhi) and paratyphoid (S. paratyphi, S. enteritidis) have been the source of well-publicized epidemics of infections. The case of Typhoid Mary is but the most famous example. In addition, serious septice ias such as meningitis, osteomyelitis, and pneumonia can result from invasion of the blood stream after ingestion of the organisms orally. 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. B.3 Use of Antigen/Antibody Interaction and Detection In general, 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. As to the first, the method of the invention provides a uniquely specific preparation of antibodies which is directed exclusively against antigens associated with Salmonella filamentous flagellar protein. By the use of the antibody preparation of the invention, cross- reaction with antigenic domains included in other bacterial species is avoided. With respect to the second, a number of means for detecting this interaction are well known in the art. Perhaps the most readily perceived methods are those of radioimmunoassay (RIA) or immunofluorescence wherein the antibody is itself labeled with a radioactive component such as 1-125 or with a fluorescent dye which enable detection of. the antigen/antibody complex by monitoring radioactivity or by fluorescence microscopy. The results can be further improved by binding the antibody to an insoluble support. Modifications of this approach include using secondary radioactive or fluorescent labeled antibodies directed against the primary bound antibody which has specifically sought out the antigenic site, or secondary antibodies which immobilize the complex by being bound to a solid support. In either case the result is, in principle, the same: 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 colorimetric 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. Accordingly, methods of analysis using the antibodies of the invention are carried out by means generally known to skilled practitioners; and kits containing the antibodies of the invention comprise, besides these antibodies, the reagents associated with these detection reactions as are known in the art. Thus, as used herein, "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. C. Detailed Description of a Preferred Embodiment " he 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. C.l Preparation of Salmonella Antigen Cultures of Salmonella heidelberg, FDA number 2172H, S.cubana, S.senftenberg and S.typhimurium, ATCC 13311, were grown in tryptic soy broth (DIFCO) at 37°C for 24 hours, and motile organisms selected through several consecutive transfers. These transfers were made in bacto-motility GI medium (GIBCO) by performing inoculation in 60 mm tubes at slightly below the surface, incubating 24 hours at 37°, and transfering into fresh inoculum by removing a 4 mm diameter agar plug from the bottom of the tube. Thus, a migration rate of 50-60 mm in 18-20 hours was selected for. Twenty-four plates of swarm agar (0.8% Brain Heart Infusion, DIFCO) were inoculated with each highly motile Salmonella culture as prepared above. The motility was verified by growth in rays from the point of inoculation. Cells were harvested after 18 hours of incubation at 37°C by scraping the plate surface and washing with 1-2 ml 0.05 N HC1. The pH was adjusted to 1.5, and the scrapings were incubated overnight at 4°C. Flagellin was separated from the bacterial so a by centrifugation at 49,000 g for 60 minutes. The supernatant, which contained monomeric flagellin, was adjusted to pH 7.2, and ammonium sulfate added to 66% saturation (42.3 grams (NH 4 ) 2 Sθ4 P er 120 m total volume). The flagellin which precipitated after overnight storage at 4 β 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. This is an adaptation of the method of Asakura, F., et al, J Mol Biol (1964) 1 :42, and results in the separation of the filamentous material from the somatic antigens and flagellar "hooks" which are antigenically different (Kagawa, H., et al, J Bacteriol (1973) 113:1474). Further purification of the sample was accomplished by depolymerization and repolymerization of the filamentous flagellar proteins. The sample was heated at 60°C for 5 min and centrifuged at 150,000 g for 1 hr to remove debris. Monomeric flagellin was precipitated from the supernatant by adjusting the salt concentration to 0.6 M Na 2 Sθ . τhe precipitated flagellin was pelleted by centrifugation at 150,000 g for 1 hr, resuspended in phosphate buffered saline (PBS), pH 7.4, and stored at 4°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 ug 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. C.2 Formation of Immune Donor Spleen Cells Flagellin prepared as described in paragraph C.l was used to immunize 7-8 week old female mice (Balb/C AmNCr/Br, Charles River), by intraperitoneal injection. Mice were given primary injections of 10- 25 μg flagellin in 100 μl 50% Freunds complete adjuvant in PBS. After one week, serum antibody level was assayed by ELISA. After four weeks, a booster injection of 10- 25 μg flagellin in 100 μl PBS was administered intraperitoneally. One mouse was used per fusion. C.3 Preparation of Candidate Spleen Cells for Fusion Mice were sacrificed 3 days after the booster injection and the spleen removed under asceptic conditions. The removed spleen was washed once in 20 ml GKN (0.2% glucose, 5.37 mM KC1, 0.137 M NaCl, 5 mM aH P04, 10 mM Na 2 HP0 , 0.001% phenol red), placed in 10 rol fresh GKN and disrupted by maceration through a sterile screen. The preparation was further dispersed by pipetting and transferred to a sterile centrifuge tube. Large particulates were allowed to settle for 5 min. The supernatant was centrifuged at 180g for 10 min to recover the spleen cells. The pellet was washed once with GKN and resuspended in 2 ml GKN. C.4 Preparation of Myeloma Non-secreting P3X63-Ag8.653 mouse myeloma cells, Keavey, J. S., et al, J Immunol (1979) 123:1548, 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. C.5 Preparation of Hybridoma Cells A suspension of myeloma cells prepared as in C.4 containing 0.8-1.4 x 10* 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. One ml 50% polyethylene glycol (PEG) was added to the pellet dropwise with gentle mixing over 1 min at 37°C. The mixture was stirred for an additional minute and then was diluted by gradually adding 20 ml GKN over a period of 5 min. The mixture was incubated for 5 min at room temperature and then centrifuged at 180g for 10 min. The pellet was resuspended in 50 ml HAT medium (RPMI-FCS with 13.6 μg/ml hypoxanthine, 0.176 μg/ml aminopterin, 3.78 μg/ml thymidine) and plated in 100 wells of five 24-well tissue culture plates. 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 peri¬ toneal 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, macro¬ phages from 2 mice were required for each spleen used.) The plates were incubated at 37°C in 5% C0 2 at 85% humidity. One to two days after fusion, an additional 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 μg/ml hypoxanthine and 3.78 μg/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 5 wells with visible colonies were tested by solid phase ELISA for the presence of Salmonella-specific antibodies. Cell cultures that were positive by ELISA were expanded to flasks to be frozen and were subcloned by serial dilution in 96-well tissue culture plates over a 0 diluting range of 1:2 to 1:200,000. Positive wells of the highest dilution were further expanded and subcloned until monoclonal cell lines were obtained. C.6 Screening Hybridoma Cultures Hybridoma culture supernatants were assayed for 5 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. o For the preparation of the 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 HC1 and stored overnight at 4°C. The 5 acidified cultures were treated with formaldehyde to a final concentration of 0.3%, titrated to pH 7.2 using ION 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 0 concentration of 0.02% as an antimicrobial agent. The flagellar samples were prepared according to the procedures outlined in 1IC.1, without the heat depolymerization-repolymerization steps. The crude flagellin was diluted to 10 μg/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 μl of 5 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 0 with PBS. 100 μl of the hybridoma culture supernatant was added to each well and incubated for at least 2 hr at room temperature. The wells were again washed 4X with PBS and then incubated for 2 hr with 100 μl of alkaline 5 phosphatase conjugated goat anti-mouse immunoglobulin (Hyclone) (diluted 1:1000 in 1% BSA/Tris-saline). The anti-mouse immunoglobulin solution was removed and the wells were washed 4X with PBS. 100 μl enzyme substrate solution (2 mg/ml p-nitrophenol phosphate in n diethanolamine buffer, pH 9.8) was added to the wells and incubated for 1 hr at room temperature. After color development, absorbance was measured with a Bio-Tek EIA reader at 405 nanometers. The results obtained are shown, in part, below. 5 Culture supernatants were tested for reactivity against different serotypes of Salmonella as well as against the following non-Salmonella strains: Serratia marcescens Proteus vulgaris Shigella flexneri Enterobacter cloacae Arizona #1 Klebsiella pneumoniae Citrobacter freundii Escherichia coli None of the foregoing strains showed reactivity with the antibodies produced by any of the hybridoma cultures, i.e., no false-positive reactions were observed. However, positive results were obtained against a wide range of Salmonella serotypes. The results are given in Table 1 below. Table 1 Supernatant from Cell Line Serotype C5A1 S5C3 S502 H5B5 S kentucky ++ - ; - ++ S. newington ++ — - ++ S. infantis ++ - , - ++ S. senftenberg ++ ++ ++ _ S montivideo ++ ++ ++ - S. typhi urium ++ - - ++ S. typhimurium ATCC 13311 + - - ++ S. typhimurium var. Copenhagen ++ - .- ++ S. heidelberg ++ - - ++ S. oranienburg - ++ ++ - S. derby + ++ ++ - S. worthington . ++ - - ++ S. molade ++ - - ++ S. minnesota - - - - S. bareilly ++ - - ++ S. cubana ++ - - - S. Chester ++ ++ ++ - S. chameleon - - - - S. braenderup ++ - - + S. typhi - - - + S. blockley ++ — — + S. saint-paul ++ - - + S. brookfield ++ - - + S. rauenchen ++ - - ++ S. bredeney ++ - - ++ S. lexing on ++ - - + S. anaturn ++ - - ++ S. enteriditis + ++ ++ - S. panama ++ - - + S. morocco ++ - — ++ S. apapa + ++ ++ — S. bilthoven - ++ ++ — S. aberdeen ++ - - ++ Serotype C5A1 S5C3 S5D2 H5B5 S. benguella ++ ++ S. dublin + ++ ++ S. budapest ++ ++ ++ S. lansing ++ + S. newport ++ ++ S. tennesee ++ + S. javiana ++ + S. cook s. bioser pullorum ++ s. lille S. tamale ++ s. cairo ++ ++ s. Chicago ++ ++ S. parera + s. gallinarum ++ + s. wentworth ++ ++ s. sara s. paratyphi A S. paratyphi B ++ s. paratyphi C s. hagenbeck ++ - - s. locarno ++ - - s. eilbek ++ - - s. breraen - ++ ++ s. humber - ++ ++ S. drypool - ++ ++ s. raemphis - ++ ++ s. alachua s. flint s. djakarta s. kandla ++ s. good ++ s. welasco ++ s. labadi ++ ++ s. Caracas ++ ++ s. missouri ++ ++ ++ ++ s. basel ++ ++ s. tranorύa ++ s. westphalia s. luton ++ ++ s. taunton ++ ++ s. lawra ++ ++ s. jodhpur ++ s. telaviv + s. bunnik + Serotype C5A1 S5C3 S5D2 H5B5 S. zeist ++ ++ S. quinhon ++ ++ S. ove shie ++ + S. berlin ++ S. urbana S. midhurst + S. agona + ++ ++ S. freetown ++ + S. Vietnam ++ S. mara diphasic ++ S. nairobi S. mia i ++ S. essen ++ ++ S. limete ++ S. uccle ++ ++ S. ma a ++ S. give S. manhattan S. thompson ++ S. degania S. krugersdorp ++ S. illinois ++ S. leipzig ++ + S. betioky ++ + S. saintmarie ++ ++ S. artis ++ + S. milwaukee ++ + S. haardt ++ + S. waycross S. minneapolis ++ S. fischerstrasse ++ S. meleagri is ++ ++ + + S. roggeveld + S. congo ++ ++ ++ S. florida It is seen from Table I that cell lines C5A1, S5C2, S5D3 and H5B5 (arbitrary designations) react with a wide variety of Salmonella serotypes. Cell line C5A1 was deposited at ATCC on 20 June 1984, and given accession number HB8582. The cross-reactivity of the monoclonal antibodies across Salmonella species which are not serotypically related indicated that the antigenic determinant is probably more conserved than the H antigens recognized by serotypic analysis using polyclonal serum antibodies. 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. C.7 Determination of Flagellar Specificity by Immunoblot Analysis Confirmation was obtained that antibodies in the hybridoma supernatants were specific for the flagellar proteins associated with Salmonella using Western blot analysis. The proteins present in S. heidelberg, S. cubana, S. senftenberg and S. typhimurium flagellin preparations (prepared as described in paragraph C.l) were resolved by 12.5% SDS- PAGE and electrophoretically transferred from the gel to nitrocellulose filter paper. The paper was incubated for 30 min in 1% BSA/Tris-saline to block additional protein binding sites. 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/T is-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. After incubation with gentle shaking for 3 to 4 hr at room temperature, the strips were washed 3X for 10 min with wash buffer, and 3 ml substrate solution (PBS with 6% (v/v) chloronaphthol stock solution (0.3% w/v in methanol), and 0.4% (v/v) 30% H2O2) was added. Strips were incubated for 15 min in the dark at room temperature to obtain the purple bands indicative of the presence of the antigen-antibody complex. C.8 Isotype Analysis of Monoclonal Antibodies Commercially available 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/BSA- saturated 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 μl of a subclass- specific rabbit anti-sera. The wells were again washed 4 times with PBS and then filled with 100 μl of peroxidase- conjugated goat anti-rabbit Ig's diluted 1:500 in 1% BSA/Tris-saline. After 1 hr incubation, the wells were washed and incubated with 100 μl of the peroxidase substrate solution (0.1 M sodium acetate, 0.05 M sodium dihydrogen phosphate, 0.01%, thimerosal,.2.5 mM H 2 θ2 r and 2 mM ABTS). A dark green color was indicative of a positive result. When thus tested, H5B5, S5C3 and S5D2 cell lines contained IgGl heavy and light chains; C5A1 showed IgG2b heavy and K light chains. C.9 Use of Monoclonal Antibodies in Detection of Salmonella The following represents a prototype testing procedure for detecting Salmonella. As indicated in paragraph B.3 above, a variety of configurations are possible and the invention is by no means limited to the use of the protocol described below. Two types of plastic carrier supports 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 j paragraph C.7 above. This system was tested using S. heidelberg flagellin diluted 10 μg/ml in carbonate buffer as the sample. Antibodies used were as either DEAE-column purified or unpurified cell supernatants. In all cases, the procedure succeeded in detecting S. heidelberg.

Claims: Claims 1. A method for preparing a hybridoma cell line capable of secreting antibodies specifically reactive against Salmonella which method comprises: (a) isolating the flagellin from the filamentous portions of flagella from a strain of Salmonella; (b) using flagellin to immunize a mammalian host; (c) preparing spleen cells from the immunized host; (d) fusing the spleen cells to an immortalizing line; (e) culturing the resulting hybridomas; (f) subcloning successful cultures to obtain single clones. 2. Hybridoma cells prepared by the method of claim 1. 3. A hybridoma cell line which secretes antibody specific against Salmonella. 4. The hybridoma cell line of claim 3 which is selected from the group consisting of C5A1, S5C3, S5D2, and H5B5. 5. Monoclonal antibodies secreted by the hybridoma of claim 2. 6. Monoclonal antibodies specific, against Salmonella. 7. The monoclonal antibodies of claim 6 which are specific against Salmonella flagellar protein. 8. A method for prepar ing monoclonal antibodies specific for Salmonella, which method comprises : (a) isolating the flagellin from the filamentous portions of flagella from a strain of Salmonella; (b) using flagellin to immunize a mammalian host; (c) preparing spleen cells from the immunized host; (d) fusing the spleen cells to an immortalizing line; (e) culturing the resulting hybridomas; (f) subcloning successful cultures to obtain single clones; (g) recovering antibodies secreted by at least one of said clones. 9. Monoclonal antibodies specific against Salmonella prepared by the process of claim 8. 10. A method of detecting Salmonella in organic matter which comprises reacting a sample of the organic matter with the monoclonal antibodies of claim 5. 11. A method of detecting Salmonella in organic matter which comprises reacting a sample of the organic matter with the monoclonal antibodies of claim 6. 12. An analysis kit suitable for detecting Salmonella contamination in organic matter which comprises the monoclonal antibodies of claim 5. 13. An analysis kit suitable for detecting Salmonella contamination in organic matter which comprises the monoclonal antibodies of claim 6.

Inventors: CHING LYNN W, MASON THOMAS L, SOLBERG MYRON, WISSEMANN KIMBERLY W, CHING, LYNN, W., MASON, THOMAS, L., SOLBERG, MYRON, WISSEMANN, KIMBERLY, W.
Applicants: ADVANCED BIOTECH ASS, ADVANCED BIOTECHNOLOGY ASSOCIATES, INC.
Application year: 1985
Main classification: G01N33
IPC codes: C07K16