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
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)

Definitions

• the present invention relates to novel antibodies to the Streptococcus mutatis bacteria that are naturally found in the mouth, and play a role in the development of dental caries.
• the invention relates to methods of detection of S. mutans using the antibodies of the invention or fragments or derivatives thereof.
• the 20 invention also relates to diagnosing, monitoring, treating and protecting the teeth from dental caries using the antibodies of the invention or fragments or derivatives thereof.
• mutans is one of the primary factors in acid dissolution of the apatite (mineral) component of the enamel then the dentin, or of the cementum then the dentin.
• a strong correlation between the proportion of S. mutans in dental plaque or in saliva relative to other bacterial species and the presence or risk of future outbreaks of dental caries has been documented, (Tanzer, 1997; Krasse, 1988). Therefore, S. mutans in plaque or saliva can serve as an index for both caries activity state and caries risk or susceptibility (Loesche et al.. 1975: Ellen, 1976; Krasse. 1985; Krasse, 1988). These indices play an increasingly important role in the diagnosis and treatment of dental caries (Hume, 1993; Mundorff et al.. 1993; Van Houte. 1993).
• monoclonal antibody based detection methods allow a rapid and accurate, yet economic quantitative measurement of the presence of bacterial cells, and have significant advantages compared to traditional culture sensitivity assays or polymerase chain reaction (PCR) techniques.
• Bacteria produce unique polysaccharide structures of lipoougosaccharide or Hpopolysaccharide or other polysaccharides, among a large range of other chemical components and products, on their cell surfaces.
• Monoclonal antibodies can be raised against these chemical structures, using standard hybridoma techniques (Kohler and Milstein, 1975). The sensitivity and accuracy of this method is largely dependent on the specificity of the monoclonal antibody produced.
• monoclonal antibodies which are species-specific for the desired bacteria, i.e. the monoclonal antibodies ICL 1 1 and ICL12 recognize the 0139 antigen of Vibrio cholerae (Hasan, 1994)).
• these monoclonal antibodies can be linked to various detection systems including, for example, fluorescent reagents, colorimetric reagents or coagglutination reagents.
• the resulting labeled antibodies can specifically bind to the desired bacterium in any sample, and rapidly present the result through the linked detection systems (Harlow and Lane, 1988).
• the invention describes three monoclonal IgG antibodies, referred to as SWLA1, SWLA2, and SWLA3, which appear to recognize a species- specific polysaccharide on the cell surface of S. mutans.
• the invention also describes a rapid method of detection of S. mutans without the need for prior growth of bacteria in culture.
• the invention further describes methods of utilizing these antibodies for rapidly quantitatively detecting S. mutans. These methods are sensitive enough to detect the presence of a single S. mutans bacteria cell. These methods can be widely used in the clinical diagnosis and treatment of dental caries.
• Yet another embodiment of the invention is a diagnostic kit containing the monoclonal antibodies of the invention and reagents for detecting binding of the antibodies to S.
• one aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of Streptococcus mutans and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12559, and which is designated SWLAl.
• Another aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of S. mutans and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12560, and which is designated SWLA2.
• Yet another aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of S. mutans and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12258, and which is designated SWLA3.
• Figure 1 shows the detection of S. mutans SWLAl with fluorescence microscopy; the S. mutans cells (ATCC25175) were labeled as described in Example 1, infra; 1(a) shows a regular phase-contrast light microscope image of a bacteria mixture containing S. rattus, S. gordonii, S. mitis, S. sanguis, E. coll, and S. mutans with FITC conjugated monoclonal antibodies; 1(b) shows the same group of bacteria under fluorescent lighting; the bright spots are all S. mutans, indicating that the SWLAl monoclonal antibody specifically recognizes S. mutans.
• Figure 2 shows flow cytometry analysis of S.
• mutans SWLAl as described in example 1, infra; 2(a) shows S. mutans (ATCC25175) without SWLAl and FICT-linked goat-anti-mouse IgG antibody; 2(b) shows S. mutans with SWLAl and FICT-linked goat-anti-mouse IgG antibody; 2(c) shows F. nucleatum (ATCC25586) without SWLAl and FICT-linked goat-anti-mouse IgG antibody; 2(d) shows F. nucleatum with SWLAl and FICT-linked goat-anti-mouse IgG antibody; 2(e) shows T.
• denticola (ATCC33520) without SWLAl and FICT-linked goat-antibody mouse IgG antibody; 2(f) shows T. denticola with SWLAl and FICT-linked goat-anti-mouse IgG antibody; F is the area containing bacterial particles labeled with FITC-linked monoclonal antibody.
• the invention describes three species-specific monoclonal IgG antibodies, referred to as SWLAl, SWLA2, and SWLA3, which recognize a species-specific epitope on the cell surface of S. mutans, and conjugates thereof, which appears to be a species-specific polysaccharide.
• the invention includes methods of using the monoclonal antibodies to detect quantity and presence of S. mutans to monitor the onset and severity of dental caries.
• the monoclonal antibodies SWLAl, SWLA2, and SWLA3 can be prepared by hybridoma fusion techniques or by techniques that utilize EBV- immortalization technologies.
• Hybridoma fusion techniques were first introduced by Kohler and Milstein (see, Kohler and Milstein, (1975); Brown et al., (1981); Brown et al., (1980); Yeh et al., (1976); and Yeh et al, (1982)).
• an immunogen e.g., purified antigen or cells or cellular extracts carrying the antigen
• an animal e.g., a mouse
• a desired immune response i.e., production of antibodies
• S. mutans may be used as the immunogen.
• S. Mutans strain ATCC25175 was used as the immunogen.
• the cells are injected repeatedly, for example, into a mouse and, after a sufficient time, the mouse is sacrificed and somatic antibody-producing cells are obtained.
• mammalian models for example rat, rabbit and frog somatic cells, is also possible.
• the cell chromosomes encoding desired immunoglobulins are immortalized by fusing them with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG).
• a fusing agent such as polyethylene glycol (PEG).
• PEG polyethylene glycol
• Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSI/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), in Rockville, Maryland.
• ATCC American Type Culture Collection
• the resulting cells which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones.
• the supernatants of the hybridomas are screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated.
• a selective medium such as HAT medium
• Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (see, generally, Fink et al., supra, 1984).
• the individual cell line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation.
• the yield of monoclonal antibody can be enhanced by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. Tumors secreting the specific monoclonal antibody produced by the fused cell hybrid develop in the injected animal.
• the body fluids of the animal such as ascites fluid or serum, provide monoclonal antibodies in high concentrations.
• Immunodeficient or nude mice may be used or the hybridoma may be passaged first into irradiated nude mice as a solid subcutaneous tumor, cultured in vitro and then injected intraperitoneally into pristane primed, irradiated nude mice which develop ascites tumors secreting large amounts of specific human monoclonal antibodies.
• chimeric (mouse-human) or human monoclonal antibodies may be preferable to murine antibodies, because patients treated with mouse antibodies generate human antimouse antibodies.
• Chimeric mouse-human monoclonal antibodies reactive with S. mutans can be produced, for example, by techniques developed for the production of chimeric antibodies (Oi et al., (1986); Liu et al., (1987)). Accordingly, genes coding for the constant regions of the SWLAl, SWLA2, or SWLA3 antibody molecule are substituted with human genes coding for the constant regions of an antibody with appropriate biological activity (such as the ability to selectively bind S. mutans).
• Novel antibodies of mouse or human origin can be also made that are analogous to the SWLAl, SWLA2, or SWLA3 antibody and that have the appropriate biological functions.
• These antibodies can have complementarity-determining regions (CDRs) that are identical to one of SWLAl, SWLA2, or SWLA3.
• CDRs complementarity-determining regions
• these antibodies can bind an antigen on the surface of S. mutans and can compete at least about 80% as effectively on a molar basis with at least one of SWLAl , SWLA2, or
• SWLA3 as SWLAl, SWLA2, or SWLA3 for binding to the antigen on the surface of S. mutans.
• These antibodies have substantially no reactivity with any of the following bacterial strains: Streptococcus rattus ATCC19645, Streptococcus gordonii ATCC10558, Streptococcus gordonii ATCC13396, Streptococcus mitis ATCC49456, Streptococcus sobrinus ATCC33478, Streptococcus sobrinus 6715, Streptococcus sanguis ATCC10556, Streptococcus sanguis ATCC49295, Streptococcus anginosus ATCC33397, Lactobacillus acidophilus ATCC4356, Lactobacillus casei ATCC4646, Actinobacillus actinomycetemcomitans ATCC 33384, Porphyromonas gingivalis ATCC33277, Prevotella intermedia ATCC
• human monoclonal antibodies may be made by using the antigen, e.g. the portion of the polysaccharide on the cell surface of S. mutans, which binds the antibodies SWLAl, SWLA2, or SWLA3 of the invention, to sensitize human cells to the antigen in vitro followed by EBV-transformation or hybridization of the antigen-sensitized cells with mouse or human cells, as described by Borrebaeck et al. (1988).
• the antigen e.g. the portion of the polysaccharide on the cell surface of S. mutans, which binds the antibodies SWLAl, SWLA2, or SWLA3 of the invention
• the antibodies of this invention were produced via hybridoma techniques. NSL/Ag4.1 mouse myeloma cell line was used as a fusion partner, and whole cells of type c S. muians strain ATCC25175 were used as the immunogen as described in the Example, infra. The hybridomas produced were screened. When this was performed three species-specific monoclonal antibodies against S. mutans were obtained designated SWLAl, SWLA2, and SWLA3. The hybridomas, producing the SWLAl, SWLA2, and SWLA3 antibodies, have been deposited with the ATCC, Rockville, Maryland, and are identified as follows:
• the three monoclonal antibodies were found to be of the IgG subtype.
• Western blot and other biochemical analysis shows that they are against unique polysaccharide on cell surface of S. mutans.
• Western blot techniques are well known by one skilled in the art (Golub, E. S. and D. R. Green, 1991).
• monoclonal antibodies are very different from the monoclonal antibodies found in the prior art. Those antibodies were made against surface proteins that are found on many other bacteria and not the species-specific polysaccharide, which the instant antibodies recognize. This difference makes the prior antibodies acceptable for research purposes but not diagnostic purposes (Carien and Olsson, 1995; Chia et al., 1993; Fukushima et al., 1993; Brady, et al., 1991). Due to the specificity for S. mutans, the monoclonal antibodies of this invention are particularly useful for diagnosis and treatment of human dental caries. Because the monoclonal antibody of the invention are able to detect low numbers of S. mutans cells in small samples they are able to be used to screening for S. mutans cells. These monoclonal antibodies also permit the development of simple and inexpensive dental caries detection methods that could be used for caries risk assessment at a dentist's chairside or in the patient's household.
• the most preferred antibodies will selectively bind to S. mutans and will not bind (or will bind weakly) to non- S. mutans bacteria.
• the antibodies that are particularly contemplated include monoclonal antibodies as well as fragments of monoclonal antibodies containing an S. mutans antigen-binding domain.
• the invention also encompasses antibody fragments that specifically recognize S. mutans.
• an antibody fragment is defined as at least a portion of the immunoglobulin molecule which binds to its target, i.e., the antigen binding region on the S. mutans. This includes Fv, Fab, Fab' and F(ab)' 2 fragments of appropriate specificity.
• the invention further includes a human monoclonal antibody that specifically binds an antigen found on the surface of S. mutans.
• the antigen that is bound is one of those bound by at least one of the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12559 and designated SWLAl, the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12560 and designated SWLA2, and the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12558 and designated SWLA3.
• the human monoclonal antibody has substantially no reactivity with any of the following bacterial strains: Streptococcus rattus ATCC 19645, Streptococcus gordonii ATCC10558, Streptococcus gordonii ATCC13396, Streptococcus mitis ATCC49456, Streptococcus sobrinus ATCC33478, Streptococcus sobrinus 6715, Streptococcus sanguis ATCC10556, Streptococcus sanguis ATCC49295, Streptococcus anginosus ATCC33397, Lactobacillus acidophilus ATCC4356, Lactobacillus casei ATCC4646, Actinobacillus actinomycetemcomitans ATCC 33384, Porphyromonas gingivalis ATCC33277, Prevotella intermedia ATCC49046, Bacteroides forsythus ATCC43047, Eikenella corrodens ATCC23834,
• the invention further includes chimeric antibodies, including humanized antibodies.
• SWLA2 or (c) a monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12558 and designated SWLA3.
• chimeric antibodies that have complementarity-determining regions that are identical with the complementarity- determining regions of an antibody that binds an antigen on the surface of S. mutans and can compete at least about 80% as effectively on a molar basis with at least one of SWLAl, SWLA2, or SWLA3 as SWLAl, SWLA2, or SWLA3 for binding to the antigen on the surface of S. mutans.
• chimeric antibodies as described above, have substantially no reactivity with any of the following bacterial strains: Streptococcus rattus ATCC19645, Streptococcus gordonii ATCC10558, Streptococcus gordonii ATCC 13396, Streptococcus mitis ATCC49456, Streptococcus sobrinus ATCC33478, Streptococcus sobrinus 6715, Streptococcus sanguis ATCC 10556, Streptococcus sanguis ATCC49295, Streptococcus anginosus ATCC33397,
• Lactobacillus acidophilus ATCC4356 Lactobacillus casei ATCC4646, Actinobacillus actinomycetemcomitans ATCC 33384, Porphyromonas gingivalis ATCC33277, Prevotella intermedia ATCC49046, Bacteroides forsythus ATCC43047, Eikenella corrodens ATCC23834, Fusobacterium nucleatum ATCC25586, Treponema denticola ATCC33520, Campylobacter rectus ATCC33238, Myxococcus xanthus DZ2, and Escherichia coli HB101.
• chimeric antibodies specifically bind an antigen on the surface of S. mutans and which have at least a portion of the amino acid sequence of the heavy chain or the light chain of a different species origin than the species origin of the complementarity-determining regions.
• at least a portion of the amino acid sequence of the heavy chain or the light chain is of human origin so that the chimeric antibody is a humanized antibody.
• substantially all of the amino acid sequences of the heavy chain and the light chain outside the complementarity-determining regions are of human origin.
• chimeric antibodies according to the present invention may have a non-human antigen-binding site and a humanized effector binding region.
• the non-human antigen-binding portion may include, but is not limited to, a murine, canine, feline or other veterinary model or other mammalian antigen-binding site.
• the invention further includes single-chain binding fragments, known generally as sFv, that have the appropriate specificity for the antigen on the cell surface of S. mutans as defined above.
• Single-chain binding fragments known generally as sFv.
• Methods for preparing such sFv are generally known in the art and are described, for example, in C.A.K. Borrebaeck, ed., "Antibody Engineering” (2d ed., Oxford University Press, New York, 1995), incorporated herein by this reference.
• the specificity of the SWLAl, SWLA2, and SWLA3 antibodies for S mutans antigen make these antibodies excellent markers for screening, diagnosis, prognosis, and follow-up assays, imaging methodologies, and therapeutic methods in the management of dental caries.
• the invention provides various immunological assays useful for the detection of S. mutans and for the diagnosis of dental caries or the risk thereof. This includes various immunological assay formats well known in the art, including, but not limited to, various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzymelinked immunofluorescent assays (ELIFA), and the like.
• immunological imaging methods capable of detecting dental caries are also provided by the invention, including but not limited to a colloidal-gold based colorimetric assay, and radioscintigraphic imaging methods using radiolabeled SWLAl, SWLA2, and SWLA3 antibodies (e.g., U.S. Patent No. 4,920,059 issued April 24, 1990; U.S. Patent No. 5,079,172 issued January 7, 1992).
• the antibodies of the invention can be conjugated with other dyes or fluorescent markers and used directly on the tooth to image caries.
• Such assays may be clinically useful in the detection and monitoring of dental caries.
• Such assays generally comprise using one or more of the SWLAl, SWLA2, and SWLA3 antibodies.
• the invention also includes an immunoconjugate comprising a molecule containing the antigen-binding region of the SWLAl, SWLA2, or SWLA3 antibody, or a fragment thereof containing the antigen binding region, joined to for example a therapeutic agent, a diagnostic agent or a cytotoxic agent for treatment of dental caries.
• cytotoxic agents include, but are not limited to, chlorhexidine, fluoride, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, glucocorticoid and radioisotopes.
• SWLAl, SWLA2, and SWLA3 monoclonal antibodies of the invention are useful for diagnostic applications, both in vitro and in vivo, for the detection of dental caries.
• In vitro diagnostic methods are well known in the art (see, e.g., Roth, supra 1986, and Kupchik, supra 1988), and include immunohistological detection of dental caries or serologic detection of S mutans (e.g., in saliva samples or other biological fluids).
• Immunohistological techniques involve contacting a biological specimen, such as a saliva, tartar, or plaque specimen, with the antibody of the invention and then detecting the presence in the specimen of the antibody complexed to its antigen.
• a biological specimen such as a saliva, tartar, or plaque specimen
• the formation of such antibody-antigen complexes with the specimen indicates the presence of the antigen, S mutans.
• Detection of the antibody in the specimen can be accomplished using techniques known in the art, such as the immunoperoxidase staining technique, the avidin-biotin (ABC) technique or immunofluorescence techniques (Ciocca et al., (1986); Helistrom et al., (1986); and Kimball (ed.,), (1986)).
• Serologic diagnostic techniques involve the detection and quantitation of S.
• mutans antigens that have been secreted or "shed” into the saliva or other biological fluids of patients with dental caries.
• antigens can be detected in the saliva using techniques known in the art such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA) wherein an antibody reactive with the "shed” antigen is used to detect the presence of the antigen in a fluid sample (see, e.g., Uotila et al, (1981) and Allum et al, 1986).
• RIA radioimmunoassays
• ELISA enzyme-linked immunosorbent assays
• the antibodies of the invention can be used in most assays involving antigen-antibody reactions.
• assays include, but are not limited to, standard RIA techniques, both liquid and solid phase, as well as ELISA assays, immunofluorescence techniques, and other immunocytochemical assays (see, e.g., Sikora et al. (1984)).
• the antibodies of the invention are also useful for in vivo diagnostic applications for the detection of dental caries.
• One such approach involves the detection of dental caries in vivo by imaging techniques using the antibody labeled with an appropriate imaging reagent that produces a detectable signal when bound to .S mutans.
• Imaging reagents and procedures for labeling antibodies with such reagents are well known (see, e.g., Wensel and Meares, (1983); Colcher et al., (1986)).
• the labeled antibody may be detected by a technique such as radionuclear scanning (see, e.g., Bradwell et al. (1985)).
• the antibody fragments used in the immunoconjugates can include Fv,
• Fab, Fab' or F(ab)' 2 fragments Use of immunologically reactive fragments, such as the Fv, Fab, Fab', or F(ab)' 2 fragments, is often preferable, especially in a therapeutic context, as these fragments are generally less immununogenic than the whole immunoglobulin.
• These antibodies, as well as unconjugated antibodies, may be useful therapeutic agents naturally targeted to S. mutans cells to kill the cells, thus preventing and or treating dental caries resulting from the accumulation of S. mutans. Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Arnon et al., 1985; Helistrom et al. 1987; Thorpe, (1985); and Thorpe et al., (1982)).
• SWLAl, SWLA2, and SWLA3 antibodies may also be used in methods for purifying S. mutans proteins and peptides and for isolating homologues and related molecules.
• Methods for purification of proteins and peptides using antibodies as capture reagents are well known in the art.
• a method of purifying an S. mutans protein comprises incubating a SWLAl, SWLA2, or SWLA3 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing S. mutans proteins or peptides, under conditions which permit the SWLAl, SWLA2, or SWLA3 antibody to bind to the S. mutans protein or peptides; washing the solid matrix to eliminate impurities; and eluting the S mutans proteins or fragments from the coupled antibody.
• the invention further includes a method for detecting the presence of S. mutans on teeth in a subject or in a saliva, plaque, or tartar sample from a subject, comprising contacting at least one tooth or the sample with the SWLAl, SWLA2, or SWLA3 antibody and detecting the binding of the antibody with the S. mutans on the tooth and or in the sample.
• the antibody can be administered by topical application to the surface of the teeth by means including in a toothpaste, mouthwash, lozenge, gel, powder, spray, liquid, tablet, or chewing gum.
• One can detect the presence of S. mutans by determining the presence of a complex formed between the monoclonal antibodies and S.
• the antibodies of the invention can be labeled so as to directly or indirectly produce a detectable signal.
• the label can for example be selected from the following compounds a radiolabel, an enzyme, a chromophore , a chemiluminescent moiety, a bioluminescent moiety, or a fluorescer.
• a fluorescer When a fluorescer is used the fluorescence can be detected by means of fluorescence microscopy, fluorometer, or by flow cytometry.
• a colloidal gold colorimetric system can also be used to detect the presence of S mutans.
• the colloida, gold system is well known in the art. (J.A.K. Hasan, et al. (1994); and E. Harlow, D. Lane. (1988)).
• the invention also includes a method for diagnosing, in a subject, the early onset of dental caries. This can be accomplished by quantitatively determining on at least one tooth in a subject, or in a saliva, plaque, or tartar sample from a subject, the number of S. mutans present using an antibody of the invention and comparing the number of S. mutans cells so determined to the amount in a sample from a normal control, i.e. a subject frea from dental caries.
• the normal range for S. mutans can be determined using any of the above detection methods (i.e. detecting labeled antibody to S. mutans) and quantifying the amount of S. mutans in a normal subject or subjects free of dental caries.
• a normal range can be 1 cell ml to approximately 1 x 10 5 cells/ml or 1 x 10 " cells/n 1 to 1 x 10 cell/ml. Other ranges are possible. If the subject has a measurably higher amount of S. mutans present that is outside of the normal range it would indicate the early onset of dental caries in the subject.
• the invention also includes a method for monitoring the course of dental caries in a subject.
• the invention further comprises a method of protecting teeth from dental caries by topically applying an SWLAl, SWLA2, or SWLA3 antibody, or a fragment thereof containing the S mutans antigen binding activity, to teeth of a subject.
• the antibody can be applied topically to the surface of the teeth by means of for example, of a toothpaste, mouthwash, lozenge, gel, powder, spray, liquid, tablet, or chewing gum formulated using standard methods.
• the antibody can be linked to a toxic agent that kills the bacteria and applied to the surface of the teeth by, for example, any of the above methods.
• the proper dose of the monoclonal antibodies of the invention can be easily determined using methods which are well known to one skilled in the art (see, generally, Goodman et al. (ed.), 1993).
• kits e.g., U.S. Patent No. 5,141,850 issued August 25, 1992; U.S. Patent No. 5,202,267 issued April 13, 1993; U.S. Patent No. 5,571,726 issued November 5, 1996; U.S. Patent No. 5,602,040 issued February 11, 1997.
• kits include at least one monoclonal antibody of the invention and reagents for detecting the binding of the monoclonal antibody to S. mutans cells present on teeth in or in a sample, e.g. of saliva, taken from a subject.
• the reagents include agents capable of detection, for example by fluorescence and ancillary agents such as buffering agents.
• the kits may also include an apparatus or container for conducting the methods of the invention and/or for transferring samples to a diagnostic laboratory for processing, as well as suitable instructions for carrying out the methods of the invention.
• antibodies of the invention can be conjugated with a regular or fluorescent dye.
• a solution containing such antibodies can be used to rinse a patient's mouth.
• the dyelinked antibodies can bind to the location of the dental caries.
• the dental caries image can be shown on a TV screen through a video or digital micro-camera.
• the monoclonal antibody based detection methods of the invention allows a rapid, accurate, and economic way to quantitatively measure the S. mutans in a subject, with significant advantages compared to current methods.
• These methods especially flow cytometry, are able to rapidly detect the bacterium with high specificity and enumerate it with high accuracy. With these methods, it will be possible to process a large number of saliva samples in a short period of time at low cost.
• Such assays can consists of monoclonal antibodies linked to a colloidal gold colorimetric system on test strips.
• the invention includes the use of a test system for rapid and simple assay of S. mutans by color change with simple immersion in fresh saliva. Such a method is suitable for use at a dentist's chairside as well as in the patient's household to assess dental caries risk.
• the accurate and objective assessment of dental caries risk state and/or caries activity state with any of these or with similar technologies will permit targeted preventive and curative treatment, thereby significantly improving human dental health.
• Bacterial strains used are listed in Table 2. S mutans was grown in Brain-Heart Infusion (BHI) medium (Difco) with supplementations of haemin (5 ⁇ g/ml). Other bacteria were grown in various media as suggested by the American Type Culture Collection (ATCC). The anaerobic bacteria were grown in an atmosphere of 80% N 2 , 10% CO 2 and 10% H 2 at 37°C.
• BHI Brain-Heart Infusion
• ATCC American Type Culture Collection
• Type c S mutans strain ATCC25175 were grown to log phase in BHI medium and washed twice with phosphate buffered saline pH 7.2 (PBS) by centrifugation at 3000 x g for 5 min. The pellet was resuspended in 1% formalin/0.9% NaCl, mixed at room temperature (RT) for 30 min and washed twice with 0.9% NaCl.
• BALB/c mice (8-10 weeks) were immunized intraperitoneally with 100 ⁇ l of the antigen containing approximately 10 whole cells of formalinized intact S. mutans bacteria emulsified with Freund's incomplete adjuvant (FIA). After 3-5 weeks they received a second dose of antigen (10 whole cells of bacteria in FIA). Three days prior to fusion, the mice were boosted intravenously with 10 whole cells in saline.
• the standard tissue culture media was RPMI 1640 (Gibco) medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES and containing 100 ⁇ g/ml penicillin and 100 ⁇ g/ml streptomycin with 10% fetal calf serum.
• Hybrids were selected in media containing HAT (100 ⁇ g Hypoxanthine, 0.4 ⁇ M Aminopterin; 16 ⁇ M Thymidine).
• HAT 100 ⁇ g Hypoxanthine, 0.4 ⁇ M Aminopterin; 16 ⁇ M Thymidine
• HT 100 ⁇ g Hypoxanthine, 16 ⁇ M Thymidine
• OPI (1 mM oxaloacetate, 0.45 mM pyruvate and 0.2 U/ml bovine insulin) was added as additional growth factors to the tissue culture during cloning of hybridomas. Hybridomas were raised according to the procedure reported by Kohler & Milstein (1975). The NSI/Ag4.1 mouse myeloma cell line was used as the fusion partner and grown in spinner cultures in 5% CO 2 at 37°C and maintained in log phase of growth prior to fusion.
• the following approach was used for screening for species-specific monoclonal antibodies against S mutans.
• the initial screening was performed using an ELISA assay, which selects for the culture supematants containing antibodies that bind to S. mutans.
• a volume (100 ⁇ l) of mature hybridoma supematants were added to the appropriate wells of the antigen plates, incubated for 1 hr at room temperature, washed 3 times with PBS-0.05% Tween 20, and bound antibody was detected by the addition of polyvalent goat-anti-mouse IgG antibody conjugated with alkaline phosphatase diluted 1 : 1000 with PBS- 1 % fetal calf serum.
• a volume (10 ⁇ l) of bacterial solution was mixed with 5 ⁇ l of culture supernatant and incubated at room temperature for 20 seconds, then 1 ⁇ l fluorescein isothiocyanate (FITC) linked goat-anti-mouse IgG antibody (Sigma) was added to the solution.
• FITC fluorescein isothiocyanate
• the mixture was placed onto a Hausser bacterial counting chamber (Bright- line 1475) and observed with a fluorescent microscope (Zeiss Axiophot) for enumeration of the bacteria.
• the bacteria were labeled with FITC molecules in the same way described above.
• the mixture was washed twice with PBS by centrifugation to remove the excess FITC-linked goat-anti-mouse antibody in solution.
• the pellet was then resuspended in PBS solution and put into a fluorometer (TD700, Turner Designs, Sunnyvale , CA) to measure the FITC fluorescent dyes bound to S. mutans, which reflects the bacterial concentration in the sample.
• the bacteria were labeled with FITC molecules in the same way described above and analyzed with a Fluorescence- Activated Cell Sorter (FACS) (COULTER EPICS elite flow cytometer, Coulter Corp. Miami, FL).
• FACS Fluorescence- Activated Cell Sorter
• the FACS machine detects every particle in a solution and separates them based on their fluorescent intensity, with a capacity of 10,000 cells per second with an accuracy of 99.99%.
• FITC-linked monoclonal antibodies against S. mutans the bacteria can be easily detected and enumerated by the FACS machine in any bacterial mixture.
• mice Three BALB/c mice were immunized with formalinized S. mutans and used for production of monoclonal antibodies.
• the No. 2 mouse was selected because its serum showed the strongest positive reactivity with the bacterium and because 1835 mature hybridomas were obtained from this mouse.
• SWLAl, SWLA2 and SWLA3 antibodies were used to specifically label S. mutans in a mixture of bacterial cells.
• the monoclonal antibody-labeled S. mutans cells were then treated with FITC linked goat-anti-mouse IgG antibody to bind the monoclonal antibody and consequently label S. mutans cells with FITC.
• the FITC labeled S. mutans cells could be viewed and enumerated directly using a fluorescence microscope.
• Fig. 1(a) shows a microscopic image of a bacterial mixture containing E rattus, S gordonii, S. mitis, S. sanguis, E. coli as well as S. mutans.
• phase contrast lighting was shifted to fluorescent lighting, only S. mutans cells exhibited a fluorescent image due to bound FITC molecules, while other bacteria did not (Fig. 1(b).). In this way, the number of S. mutans in the mixture was easily recognized and enumerated.
• S. mutans cells in a solution can be fluorescence- labeled with the monoclonal antibodies of the invention plus FITC-linked goat-anti- mouse IgG antibody, and the amount of fluorescence can be measured with a fluorometer.
• a linear correlation was observed between the amounts of fluorescence and concentrations of S mutans in the solutions assayed by the conventional colony counting method (the colony counting was performed by diluting bacteria with PBS and plating on BHI plates.) (see Table 4).
• the FITC labeled S. mutans cells were detected and enumerated with FACS.
• Figure 2 shows that the FITC-linked monoclonal antibodies which specifically bound S. mutans were effectively detected by FACS while other oral bacteria, such as F. nucleatum or T. denticola labeled with the same antibodies, showed no fluorescence, i.e. the antibodies did not bind to the other oral bacteria and were specific to S mutans.

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