AU3605700A - Beta-tubulin autoantibody in meniere's disease and other inner ear diseases and a diagnostic method - Google Patents
Beta-tubulin autoantibody in meniere's disease and other inner ear diseases and a diagnostic method Download PDFInfo
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Description
WO 00/50593 PCT/USOO/04795 BETA-TUBULIN AUTOANTIBODY IN MENIERE'S DISEASE AND OTHER INNER EAR DISEASES AND A DIAGNOSTIC METHOD USING SAME 5 CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of co-pending U.S. provisional patent application Serial No. 60/121,549, filed February 25, 1999, which is incorporated herein in its entirety. FIELD OF THE INVENTION 10 The present invention relates to the field of immunology and more specifically relates to the membranous structure of the inner ear and to an immunoassay method for detecting an autoimmune disease of the membranous structures of the inner ear. BACKGROUND OF THE INVENTION Hearing problems can result from a variety of disorders, diseases or traumas of 15 the inner ear. Symptoms of inner ear problems include, but are not limited to, hearing loss, dizziness, vertigo and tinnitus. Several inner ear diseases have recently been classified as autoimmune diseases. These include, but are not limited to, Meniere's disease, progressive bilateral sensorineural hearing loss (PSHL), otosclerosis and sudden hearing loss. 20 Meniere's disease, although idiopathic by definition, has been ascribed to a variety of causes, among which are autoimmune factors. There is evidence to suggest WO 00/50593 PCT/USOO/04795 that antibodies generated against inner ear proteins cause inner ear inflammation and swelling that can result in a complete loss of hearing (Dereby, M. Otolaryngology Head & Neck Surgery 114:360-365, 1996). Symptoms typically associated with Meniere's disease include ringing in the ears, dizziness, a sense of fullness or pressure 5 in the ears, and progressive deafness. These symptoms may be produced by a sudden influx of fluid into the endolymphatic sac, resulting in a rupture of Reisser's membrane in the cochlea. Immunological derangement of the endolymphatic sac or other membranous structures of the inner ear could initiate a cascade of reactions leading to 10 endolymphatic hydrops and presenting as Meniere's disease (Soliman, A. American Journal of Otology 17:70-80, 1996). There are at least four million Meniere's disease patients in the United States, and many more patients report symptoms associated with Meniere's disease but cannot be positively diagnosed. Researchers have attempted to isolate the antigen or antigens responsible for 15 autoimmune inner ear diseases. A protein is considered to be a potential antigen if it is reactive with antibodies produced by -patients exhibiting autoimmune inner ear diseases. For example, antibodies in the sera of patients having inner ear disease have been found to react with protein bands of 58 kD and of 30 kD on Western blots of 20 guinea pig inner ear extracts (Cao, M. et al., Laryngoscope 106:207-212, 1996). The 58 kD band was shown to be nonspecific to the inner ear when antibodies reacted with a 58 kD band on Western blots of guinea pig brain, lung and liver. In contrast, the 30 kD band was specific to the inner ear. Antibodies from patients reacted with a 2 WO 00/50593 PCT/USOO/04795 30 kD band on Western blots of extracts from Corti's organ, the spiral ganglion and the acoustic nerve fiber, but not with extracts from the spinal ligament and the stria vascularis. Antibodies against a 30 kD cochlear protein have been in reported in the serum of some patients with Meniere's disease (Joliat, T. et al., Ann. Otol. Rhinol. 5 Laryngol. 101:1001-1006, 1994 and Cao, M. et al., Laryngoscope 106:207-212, 1996). This 30 kD protein has been identified as the major peripheral myelin protein "PO" and is believed to be associated with acoustic nerve and spiral ganglion (Cao, M. et al., Laryngoscope 106:207-212 (1996)). Antibodies reactive with the 30 kD protein are not specific for Meniere's disease as these antibodies have been found in patients 10 having other autoimmune diseases such as progressive bilateral sensorineural hearing loss (PSNHL), otosclerosis and sudden deafness and in control subjects. (Cao, M. et al., Immunobiology in Otology, Rhinology and Laryngology (eds. Mogi, G., Veldman, J., and Kawauchi, H.) Kugler Publ. Amsterdam/New York, (1994) pp. 263 268). 15 Antibodies against a 68 kD protein in extracts from bovine inner ear have been reported in the serum of PSNHL patients (Harris J. and Sharp P. Laryngoscope 100:516-524, 1990). This 68 kD protein has been identified as a 70 kD heat shock protein that has been implicated in other autoimmune diseases such as Lyme's disease and ulcerative colitis (Billings P. et al. Ann. Otol. Rhinol. Laryngol. 104:181-188, 20 1995). An early diagnosis of autoimmune inner ear disease is critical. Prompt treatment of the disease at an early stage of the illness may preserve any remaining inner ear function. Moreover, the ability to distinguish antigenic epitopes of the inner ear relevant to the pathogenesis of specific autoimmune inner ear diseases will enable 3 WO 00/50593 PCT/USOO/04795 clinical investigation and research on autoimmune inner ear disease, and will further enable the clinical diagnosis of autoimmune inner ear diseases and immunologic therapy. As the availability of human inner ear tissue is extremely limited, there is an 5 on-going need for the identification of disease-specific antigens and for the development of simple, sensitive and reproducible assays for the detection and differential diagnosis of autoimmune inner ear diseases. SUMMARY OF THE INVENTION The present invention provides an isolated Beta-Tubulin antigen present in the 10 membranous structures of the inner ear and an immunoassay method for diagnosing Meniere's disease using the Beta-Tubulin antigen as a target substance for detecting target binding substance in biological fluid from an animal or human having symptoms of a disease of the inner ear. The antigen is a protein or peptide selected from the group consisting of proteins, proteins purified from extracts of membranous 15 inner ear proteins, recombinant proteins or peptides and synthesized proteins or peptides. The antigen is a protein or peptide having a DNA or an amino acid sequence selected from the group consisting of SEQ.ID.NOS.:1l-17 or antigenic variants thereof and mixtures thereof. Sequence ID NOS. 10, 11, and 12 represent one DNA molecule however, due to the size limitation of 50,000 residues, the entire molecule must be 20 broken down into three separate components., The present invention also provides a method of detecting Meniere's disease in an animal or human comprising the steps of: (a) incubating a biological sample from the animal or human with a target substance under conditions sufficient to bind a 4 WO 00/50593 PCT/USOO/04795 target-binding substance in the biological sample to the target substance, wherein the target substance is a Beta-Tubulin antigen or a nucleic acid molecule encoding a Beta-Tubulin antigen of the membranous structures of the inner ear of a mammal and/or recombinant proteins from SEQ.ID.NOS.: 8-17 or antigenic variants thereof; 5 and, (b) detecting the target-binding substance bound to the target substance. The present invention further provides an assay kit for detecting Meniere's disease in a patient comprising: (a) a solid phase having a Beta-Tubulin target substance bound thereto which reacts with a target-binding substance in a biological fluid from an animal or human having symptoms of Meniere's disease; and, (b) means 10 for detecting binding of the target binding substance to the target substance. Accordingly, is an object of the present invention to provide an antigen identified in the membranous structures of the inner ear that reacts specifically with antibodies from the sera of patients having an autoimmune disease of the membranous structures of the inner ear. 15 It is a further object of the present invention to provide a simple, rapid, sensitive and reproducible method for detecting antibodies to antigens from the membranous structures of the inner ear. It is a further object of the present invention to provide an isolated antigen from the membranous structures of the inner ear that reacts specifically with 20 antibodies in the sera of patients having Meniere's disease. It is a further object of the present invention to provide a sensitive blood test for the detection of Meniere's disease in the early stages of the disease. 5 WO 00/50593 PCT/US00/04795 It is a further object of the present invention to provide an immunoassay that can distinguish Meniere's disease from other autoimmune ear diseases. It is a further object of the present invention to provide an immunoassay that can monitor the progression of Meniere's disease or the effects of treatment for 5 Meniere's disease. It is a further object of the present invention to provide an antigen to be used for the immunotherapeutic treatment of Meniere's disease. These and other objects of the present invention will become apparent after reading the following detailed description of the disclosed embodiments and the 10 appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The invention is illustrated in the drawings in which like reference characters designate the same or similar parts throughout the figures of which: Fig. I shows SDS-PAGE of inner ear tissue. 55kD tubulin is seen in the 15 membraneous portion (lane 1) Neural portions are in lane 2 and 3. Fig. 2 shows Western blots of patients sera with inner ear antigens. Note 55 kD beta- tubulin in the membraneous portion in patient I lane. Fig. 3 shows immunohistologic staining of inner ear tissue of guinea pig. The Beta-Tubulin is strongly stained in the spiral ganglion, outer and inner hair cells and 20 pillar cells, stria vascularis and spiral limbus. 6 WO 00/50593 PCTIUSOO/04795 Fig. 4 shows immunohistologic staining of organ of Corti. Note the strong staining of pillar cells and hair cells. Fig. 5 shows immunohistologic staining of endolymphatic sac. Note the presence of Beta-Tubulin in the epithelium of endolymphatic sac. 5 DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS As used herein, the term "Beta-Tubulin antigen" refers to a protein extract or peptide from the membranous structures of the inner ear of a mammal, a protein or peptide having the amino acid sequence (SEQ.ID.NO.: 1) and antigenic variants 10 thereof, a protein or peptide having the nucleotide or amino acid (SEQ.ID.NOS.: 2-7 and antigenic variants thereof and a recombinant Beta-Tubulin protein or peptide results from DNA sequence of (SEQ.ID.NO.:8-13, (SEQ.ID.NO.: 10-12, (SEQ.ID.NO.:14 and, (SEQ.ID.NO.:16 and 17, and antigenic variants thereof. All sequences referred to herein are shown in detail in the Sequence Listing attached 15 hereto and incorporated herein. As used herein, the term "target substance" refers to the Beta-Tubulin antigen or a nucleic acid molecule having a sequence encoding the Beta-Tubulin antigen of the membranous structures of the inner ear of a mammal. As used herein, the "term immune sample" refers to samples having antibodies 20 that interact specifically with the Beta-Tubulin antigen. As used herein, the term "target-binding substance" refers to immune samples and to biological molecules, such as antibodies, which interact specifically with the 7 WO 00/50593 PCT/USO0/04795 Beta-Tubulin antigen. The term "target-binding substance" further include nucleic acid probes that hybridize under stringent hybridization conditions to a nucleic acid molecule having a sequence encoding the Beta-Tubulin antigen of the membranous structures of the inner ear of a mammal. 5 As used herein, the term "preimmune sample" refers to samples not having antibodies that interact specifically with the Beta-Tubulin antigen. As used herein, the term "membranous structures" refers to the basilar membrane, organ of Corti, stria vascularis, spiral ligament and vestibular epithelium of the inner ear. 10 As used herein, the term "neural structures" refers to the spiral ganglion, cochlear nerve in the modiolus and vestibular nerve in the temporal bone of the inner ear. As used herein, the term "antigenic variant" refers to a protein or peptide having an amino acid sequence different from the protein or peptide to which it is 15 compared, but having similar immunologic characteristics such as the ability to bind to one or more antibodies that bind to the protein or peptide to which it is compared. As used herein, the term "antibody" includes, where appropriate, polyclonal antibodies, monoclonal antibodies, antibody fragments and mixtures of the foregoing. DESCRIPTION OF THE PREFERRED EMBODIMENTS 20 An antigen and a diagnostic assay method for detecting antibodies to the antigen in a biological sample are described herein. The antigen is the Beta-Tubulin antigen, as defined above, and the diagnostic assay method is specific for detecting 8 WO 00/50593 PCT/USOO/04795 antibodies to the Beta-Tubulin antigen. The Beta-Tubulin antigen is present in the membranous fraction of the inner ear, but is not present in the neural fraction of the inner ear, facial nerve or brain tissue (Suzuki et al., ORL, 59:10-17, 1997). The molecular size of the 52 kD is solely based on the gel electrophoresis 5 (Suzuki et al., ORL, 59:10-17, 1997). The protein sequence data and as well as DNA sequence data indicates it is actually 55 kD of Beta-Tubulin. Therefore though it appears as 52 kD in our previous experimental setting (Suzuki et al., ORL, 59:10-17, 1997); however, it is actually Beta-Tubulin protein based on both available DNA and amino acid sequence data. 10 Preferably, the Beta-Tubulin antigen has the sequence of SEQ.ID.NO.:1, SEQ.ID.NO.:2-7, or antigenic variants thereof and reacts with a biological fluid from an individual having an autoimmune disease of the membranous structures of the inner ear. More preferably, the 55 kD antigen has the amino acid sequence of SEQ.ID.NO.:l or SEQ.ID.NO.:2-7 or antigenic variants thereof and reacts with a 15 biological fluid from an individual having Meniere's disease. Most preferably, the 55 kD antigen has the amino acid sequence of SEQ.ID.NO.:l and antigenic variants thereof and reacts with a biological fluid from an individual having Meniere's disease. The diagnostic assay for detecting the 55 kD antigen (Beta-Tubulin) of the structures of the inner ear can be, for example, an immunoassay. Such an 20 immunoassay includes, but is not limited to, an ELISA, a Western blot assay, a competitive binding assay, a particle based immunoassay, a dual particle competitive immunoassay, a radioimnunoassay, variants of the foregoing, and any of the other immunoassay methods known to those skilled in the art or developed hereafter. 9 WO 00/50593 PCT/USOO/04795 For example, in a conventional immunoassay, such as an ELISA, an inert solid-phase material, usually a plastic microtiter plate, is contacted with a solution containing the target substance (Beta-Tubulin) so that the target substance binds to, or coats, the solid phase material. The bound target substance is then contacted with an 5 aqueous sample obtained from an individual having symptoms of inner ear disease, which may or which may not contain a target-binding substance (anti - Beta-Tubulin antibody). Unbound target-binding substance is removed, and the amount of reacted target-binding substance is quantitated using any of a number of detection devices known to those skilled in the art. For example, the bound target-binding substance 10 may be detected with a second antibody to which has been attached a detectable label such as an enzyme, radioisotope or fluorescent molecule. The target substance for use in the present invention includes, but is not limited to, protein extracted from the membranous structures of the inner ear, fractions of protein extracted from the membranous structures of the inner ear, and an 15 isolated Beta-Tubulin antigen extracted and purified from the membranous structures of the inner ear. In addition, the target substance for use in the present invention also includes, but is not limited to, a protein or peptide having the amino acid sequence of SEQ.ID.NO.:1 or antigenic variants thereof, a protein or peptide having the amino acid sequence of SEQ.ID.NO.:2-7 or antigenic variants thereof, and a protein or 20 peptide having the DNA or amino acid sequence of SEQ.ID.NOS.: 8-17 obtained using recombinant DNA technology or synthesized by methods known to those skilled in the art of peptide synthesis. It will be understood by those skilled in the art that the term "amino acid sequence of SEQ.ID.NOS.: 1-17" includes antigenic 10 WO 00/50593 PCT/USOO/04795 variants thereof. A mixture of these sequences can also be used as the target substance. The concentration of target substance for use in the present invention can range between approximately I ptg/ml and 100 ig/ml. A preferable range is between 5 approximately 3 pg/ml and 50 pg/ml. A more preferable range is between approximately 5 pg/ml and 30 pg/ml. The target substance is dissolved in an aqueous solution and can be applied to an inert solid-phase support material by dipping, soaking, coating, spotting, spraying, blotting or other convenient means. Preferred methods include coating, spotting, spraying and blotting. More preferred methods 10 include coating and blotting. For example, in an ELISA, a preferred volume for coating is between about 10 pl/well and 200 il/well. A more preferred volume for coating is between about 30 ptl/well and 150 pl/well. A most preferred volume for coating is between about 50 pl/well and 100 pil/well. Determination of the amount of target substance to be used for each method of application is well within the 15 knowledge of one skilled in the art. For example, a standard target substance, target binding substance assay combination can be used to determine the amount of target substance to be applied to the inert solid-phase support material. The solvent for use in the present invention can be any solvent that can solubilize the target-binding substance, and that is sufficiently miscible with water to 20 be completely removed by subsequent thorough rinsing with an aqueous solution. Such solvents include, but are not limited to, phosphate buffered saline (PBS), tris(hydroxymethyl)amino methane (TRIS),
N-
2 -hydroxyethylpeperazine-N'-2 ethanesulfonic acid (HEPES), citric acid-phosphate buffer, carbonate buffer and the like. Such aqueous buffers and their appropriate pHs are well known to those skilled 11 WO 00/50593 PCT/USOO/04795 in the art. Mixtures of solvents may also be used. Preferred solvents include 0.1 M carbonate buffer, pH 9.0, and citric acid-phosphate buffer, pH 5.0. These solvents may contain other chemicals including, but not limited to, SDS, Tween-20, bromphenol blue, glycerol, dithiothreitol, and the like. 5 The solid phase, or inert solid phase support material, for use in the present invention can be in the form of, but is not limited to, a membrane, a bead, a microtiter plate or the like or any other solid-phase support form known to those skilled in the art. Preferred forms include a membrane strip, a membrane well microtiter plate and a plastic well microtiter plate. More preferred forms include a membrane strip and a 10 plastic well microtiter plate. A most preferred form is a plastic well microtiter plate. In addition, the inert solid-phase support material can be placed into a holder, including but not limited to, a membrane sheet holder, a dot-blot apparatus, a microtiter plate, a column, and a filter. Preferred holders include a membrane sheet holder, a dot-blot apparatus and a microtiter plate. 15 The blocking buffers for use in the present invention to prevent non-specific binding can be any suitable blocking buffer including, but not limited to, goat serum, fetal calf serum, gelatin, low fat milk, and Tween-20 at various dilutions in an aqueous solution. The washing solution for use in the present invention can be any suitable 20 aqueous buffer including, but not limited to, phosphate buffered saline (PBS), tris(hydroxymethyl)amino methane (TRIS) and N- 2 -hydroxy-ethylpeperazine-N'-2 ethanesulfonic acid (HEPES). Such aqueous buffers and their appropriate pHs are well known to those skilled in the art. 12 WO 00/50593 PCTIUSOO/04795 The target-binding substance for use in the present invention is a substance which binds specifically to the target substance. Examples of target-binding substances include, but are not limited to, antibodies (including polyclonal and monoclonal antibodies, and antibody fragments and mixtures of the foregoing). 5 Preferred target-binding substances are antibodies to proteins of the membranous structures of the inner ear. More preferred target-binding substances are antibodies to a Beta-Tubulin antigen of the membranous structure of the inner ear in serum from individuals having inner ear disease. Most preferred target-binding substances are antibodies to a Beta-Tubulin protein of the membranous structure of the inner ear in 10 the serum of individuals having Meniere's disease. Any convenient indicator method can be used to detect binding of a target binding substance to a target substance. Such methods include, but are not limited to, the use of enzymes, enzyme cofactors, enzyme effectors, chromogenic substances, fluorogenic substances, chemiluminescent substances, bioluminescent substances, and 15 labeled (for example, radiolabeled) antibodies. Preferred indicator methods are the peroxidase-labeled antibody method and the alkaline phosphatase-labeled antibody method. The present invention further comprises an assay kit for detecting target binding substance in a biological sample comprising an inert solid-phase support 20 material having target-binding substance immobilized thereon and may further contain reagents and a holder for the inert solid-phase support material. Such a kit may additionally contain equipment for safely containing the samples, a vessel for containing the reagents, a timing means, and a colorimeter, reflectometer, or standard against which a color change may be measured. The reagents, including the target 13 WO 00/50593 PCTIUSOO/04795 substance coated particle and the detectable particle are preferably lyophilized. Most preferably, the coated particle, and the detectable particle are provided in lyophilized form in a single container. For example, an immunoassay kit useful for measuring the target-binding 5 substance in a biological sample can involve a "sandwich immunoassay." Such a kit contains a particle coated on its surface with the binding substance, a detectable particle capable of binding to the target-binding substance and a porous membrane having a pore size that prevents passage of the coated particle and allows passage of the detectable particle. The first step in the immunoassay is a binding step, and the 10 second step is a detection step. In the binding step, a solid phase particle or sphere coated with the target substance is combined in a solution with a sample containing the target-binding substance and reacted for a sufficient amount of time to allow the target substance and the target binding substance to interact. In the detection step, a detectable particle, 15 such as a colored bead, coated with a substance that binds readily to the target binding substance, such as protein A, protein G, a second antibody reactive to the target-binding substance, or a small synthetic affinity ligand is added to the suspension. The detectable particle binds to the target-binding substance complexed to the target substance coated particle. The reaction mixture is then placed on a 20 membrane having a pore size of sufficient dimension to exclude passage of target substance coated particle which have bound target-binding substance and, therefore, bound detectable particle. Those components which are complexed as target substance particle plus target-binding substance plus detectable particle are retained on the membrane while the other components pass through the pores. 14 WO 00/50593 PCT/USOO/04795 The complex is detected either visually with the naked eye or using a conventional detector, such as a colorimeter or reflectometer, or other detection device well known to those skilled in the art. In this sandwich immunoassay, the presence of detectable particles indicates the presence of target-binding substance in 5 the sample. The present invention will be further described in connection with the following Examples, which are set forth for purposes of illustration only. Parts and percentages appearing in such Examples are by weight unless otherwise stipulated. Example 1: Cloning gene for inner ear antigens 10 Meniere's disease is a chronic ear disease with unknown etiology. It has recently been shown that Meniere's disease serum contains antibodies against a 30 kD cochlear protein antigen in addition to type II and type IX collagen. On the basis of animal and human studies, an underlying immune dysfunction is now thought to contribute in whole or in part to the pathogenesis of several of these previously ill 15 defined diseases, including sudden hearing loss, Meniere's disease chronic progressive sensorineural hearing loss (CPSNHL) and otosclerosis. Animal studies have associated laboratory inoculation of cochlear components with the development of auto-antibodies, cochlear lesions and sensorineural hearing loss. Furthermore, experimental induction of CPSNHL produced by immunization of animals with 20 purified Type II collagen, gave rise to specific anti-type II collagen antibodies and inner ear lesions histologically similar to those seen in CPSNHL and Meniere's disease. 15 WO 00/50593 PCT/USOO/04795 It has been previously demonstrated that serum from Meniere's disease patients is capable of binding to a 30 kD cochlear protein antigen in addition to type II and type IX collagen. On the basis of animal and human studies, an underlying immune dysfunction is now thought to contribute in whole or in part to the 5 pathogenesis of several of these previously ill-defined diseases, including sudden hearing loss, Meniere's disease, chronic progressive sensorineural hearing loss (CPSNHL) and otosclerosis. Animals studies have associated laboratory inoculation of cochlear components with the development of auto-antibodies, cochlear lesions and sensorineural hearing loss. 10 Furthermore, experimental induction of CPSNHL produced by immunization of animals with purified Type II collagen, gave rise to specific anti-type II collagen antibodies and inner ear lesions histologically similar to those seen in CPSNHL and Meniere's disease. It has been previously demonstrated that serum from Meniere's disease 15 patients is capable of binding to a 30 kD protein derived from human inner ear (Joliat et al., Annals of Otol. Rhinol. & Laryngol., 101:1001-1006, 1992). This also shows that these sera react with guinea pig inner ear proteins. This is similar to the results of a study with a panel of inner ear disease patients and guinea pig inner ear antigens ( Cao et al., Mol. Cell. Biochem. 146:157-163, 1995). These sera were used to isolate 20 the relevant (auto)antigens. This enabled the role and extent of autoimmunity to these proteins in the pathogenesis of inner ear diseases in general and in Meniere's disease in particular to be clarified. This problem was approached by utilizing the Meniere's patient serum to immunoscreen expression cDNA libraries. 16 WO 00/50593 PCT/USOO/04795 Sera Preparation Sera with autoantibody to the 30kD protein from the Meniere's disease patients (#1 and #8, Joliat et. al., 1992) were prepared from 50 mL peripheral blood. The red blood cells were separated in 5mL 6% dextran in 0.9% NaCl. The mixture 5 was incubated in 37'C for 1 hour and spun at 2,000 rpm for 10 min. Anti-E. coli antibodies were removed prior to use in screening the libraries using two different procedures. For the two libraries (human and rat) screened first, the sera were pseudoscreened as in Molecular Cloning; A Laboratory Manual, Sambrook, Fritsch and Maniatis eds. (hereafter referred to as MCALM). 10-137mm plates of 10 nonrecombinant Unizap vector semiconfluently lysed XL1-Blue MRF lawns were transferred to supported nitrocellulose filters and incubated with a 1:10 dilution of the sera in TNT plus 3% bovine serum albumin (BSA) overnight at 4*C . For the screening of the guinea pig plasmid library, a slight modification of a newer method was used (Gruber, A., and Zingales, 1995). Two 500 mL cultures of XL1-Blue 15 bacteria were grown overnight in Terrific Broth (TB) (see MCALM A.2) + 12.5 ug/mL tetracycline. Both cultures were spun down at 4K rpm for 20 minutes and washed twice with PBS. One was treated with 0.5% w/v paraformaldehyde in PBS for 2 hours at 4'C with vigorous shaking. The other was autoclaved at 121 C for 20 minutes. The two suspensions were mixed, centrifuged at 4,000 x g and washed twice 20 with PBS. The pellets were aliquoted into 16 50 mL centrifuge tubes and stored at 20*C until used. The antisera was diluted to 1:10 in TNT plus 3% BSA, and 15 mL was absorbed to one of the pellets at 4*C for two hours. The bacterial debris were removed by centrifugation, and the sera absorbed to a fresh pellet as above. A total of 17 WO 00/50593 PCT/USOO/04795 four cycles of absorption were used. The sera were stored with 0.01 % sodium azide at 4*C until used. The Libraries for Screening The cDNA library from human fetal cochlear tissue was generously supplied 5 by Dr. Morton's group (Brigham and Women's Hospital, Harvard Medical School). This is one of a very few human cochlear libraries constructed to date. Briefly, this cDNA library was constructed from 173 human fetal (16 to 22 weeks) cochleae. Poly(A)+ RNA was selected from approximately 500 mg total cochlear RNA. The cDNA were generated with oligo (dT) and directionally packaged into Uni-ZAP TM 10 XR (Stratagene). 3.8 million primary plaques were obtained with less than 5% nonrecombinants. A random selection of 106 clones suggested that 37% of the clones have <0.5 Kb inserted cDNA, 57% ranging between 0.5Kb and 1.0 Kb, and 6% greater than 1 Kb. Analysis by the inventor by PCR of insert sizes indicated fairly small (< 500) average insert sizes indicating a low probability of finding full length or 15 nearly full length clones. This is important because in an oligo dT primed library, the 3'untranslated region is copied first. This short insert size greatly diminishes the chance of finding and expressing coding regions. Two additional based cDNA libraries were generously donated by Dr. Kirk W. Biesel. These are a mouse inner ear library and a rat cochlear library. Both were 20 constructed in ZAP XR (Stratagene) vectors with directional cloning of oligo(dT) primed inserts in the EcoRI and Xhol sites. The rat library is believed to be more specific since the mouse library included the entire tympanic bulbae. The inventor's 18 WO 00/50593 PCT/USOO/04795 PCR analysis of average insert size indicated both libraries have reasonable average insert sizes of 800-1000 bases. Both libraries had titers at 3-8 x 10 pfu/mL. The fourth cDNA library is plasmid based with origins in mRNA from microdissected guinea pig organ of Corti (Hearing Res. 62:124-126). This library was 5 generously donated by Dr. E. R. Wilcox. The average insert size as tested by PCR r appears to be >1000 bases. It appears to be one of the highest quality cDNA libraries the inventor has worked with. Immunoscreening of the human Unizap and rat ZAP XR cDNA libraries. The procedures for the immunoscreening were based on cDNA libraries with 10 patients' sera was based upon MCALM and was the following: A single colony of E. coli XL 1-Blue MRF' was grown in NZYM medium (pH 7.5) supplemented with 12.5 ug/mL tetracycline, overnight. The cells were spun down at 4K rpm for 15 minutes and then resuspended in 0.5 volume of 10 mM MgCl 2 . 0.2mL of the cells were infected by 5 x 10 4 pfu of cDNA library in 0.1 mL SM (100 mM NaCl, 10m M 15 MgC1 2 , 20 mM Tris-HCI pH = 7.5, 0.01% gelatin) at room temperature for 15 min and then at 37 0 C for 15 min. 7.5 mL of NZY soft agar (pH 7.5) were added to the infected culture and plated on 150 mm LB plates. For the human library, 20 plates were screened. For the rat library, with its higher quality and apparent complexity 40 plates were screened. The plates were incubated at 42'C for 3.5 hours. The plates 20 were overlaid with a pre-autoclaved, supported nitrocellulose membrane disk (Hybond CR, Amersham) which has been saturated previously in 10 mM isopropyl B-D-thiogalactopyranoside (IPTG) in water and the plates and filters were incubated at 37*C for 4 hours. The antigen-bound membrane was floated in three changes of TNT at room temperature for 20 min. each time. 19 WO 00/50593 PCT/USOO/04795 The non-specific binding sites were saturated with 15mL protein blocking agent (1% bovine serum albumin, 1% gelatin for the human library; 3% BSA for the rat library (in TNT) at room temperature for 30 min. The membrane was incubated with the preabsorbed patient sera (1:50 dilution in TNT buffer, 15 mL) at 4'C 5 overnight. The primary antibody-bound membranes were washed with 15 to 20 mL TNT buffer plus 0.1% BSA (TNTB) then TNTB plus 0.1% Nonidet P-40 and finally with TNTB for 30 min each at room temperature to remove the non-specific antibody binding. After removal of the final wash solution, 15 mL of HRP conjugated goat anti-human IgG,A,M (1:1000 dilution, Sigma, St. Louis MO) was added with 10 incubation at room temperature for 1.5 hours. The filters were then sequentially washed at room temperature in TNTB, TNTB plus 0.1% Nonidet P-40, TNTB and TN for 10 min each. The color reaction was incubation for 15-20 min at room temperature in 1OmL of mix: [60 mg of 4-chloro- 1 -naphthol in 20 mL of ice cold methanol] plus [60uL of H 2 0 2 in 100 mL of TN] scaled appropriately to number of filters. 15 An agar plug containing phage particles from the region of the plate corresponding to the signals on the membrane was picked up and incubated with I mL of ^ dilution buffer (10 mM Tris-HCL pH 7.5 and 10 mM MgC 2 ) for 1 hour. The phage particles were replated and a second screening procedure as shown above applied to the secondary plates. The primary screen of the human library yielded 18 potential 20 positives but none were positive on secondary screen. Unfortunately, this was also the case for the rat library where 36 potential plaques turned up negative upon secondary screen. The problem with both library screening was an excessive positive signal from all plaques which made it difficult to pick up true positives. Rescreening was done 20 WO 00/50593 PCT/US0O/04795 using a more thorough anti-E. coli antibody depletion method-a combination of the psuedoscreen method and the paraformaldehyde autoclaved. RESULTS Screening of the libraries 5 The primary screen of the human library yielded 18 potential positives, but none were positive on secondary screen. Unfortunately, this was also the case for the mouse and rat libraries where 23 and 36 potential plaques turned up negative upon secondary screen. The problem with both library screenings was an excessive positive signal from all plaques which made it difficult to pick up true positives. 10 Screening of the plasmid based guinea pig organ of Corti cDNA library was considerably more successful. 36 filters were screened producing 60 potential positives on a very low background. From this group came 14 positive colonies that survived secondary and tertiary screens. They have been designated 647, 6D1, 7A1, 7
A
2 , 7A3, 7BI, 7Cl, 9B1, 9B2, 1 1Al, 1iB, 11B2,20E1, 21A4. 15 Insert sizes were estimated by PCR amplification of the plasmid by two oligomers, BV40 and BV41 that flank the polylinker cloning sites; the method for this is described below in Approach II section 2. The following are the estimated sizes of the inserts, not including polylinker (±50 bp): Clone Insert Size Clone Insert Size Clone Insert Size 647 850 7B1 900 11B 900 6D1 1050 7C1 1500 11B2 900 7A1 1000 9B1 1550 20E1 670 21 WO 00/50593 PCT/USOO/04795 7A2 1000 9B2 1500 21A4 1800 7A3 2000 1lAl 1500 Each of these clones other than 20E1 has been partially sequenced. The sequences of each clone showed substantial open reading frames. Comparison of the partial sequences in hand with the non-redundant database of the Blastn server at the 5 NIH has enabled the assignment of identities to some of the clones. Clone 7Al had high similarity to a Line repeat sequence - a retroviral-like repeat sequence found in many mammalian species; some Line repeats do produce protein products, however, the relevance of this clone to inner ear autoimmune pathology is highly questionable. Clone 21A4 proved to be the cDNA of the 55 kD b-tubulin. The sequence study of 10 clone 21A4 showed that it matched the sequences of four human Beta-Tubulin; the perfect matches with four human Beta-Tubulin genes (gene bank accession numbers AF141349.1, AF070600, AF070593 and AF070561 (SEQ.ID.NOS.: 3-17. These four genes consist of the same DNA sequence and are similar, not identical, to the human Beta-Tubulin I gene (clone m40 and gene bank accession number J00314) 15 (SEQ.ID.NO.:2) The clone m40 is translated to a protein containing the peptide sequence, EIVHIQAG (SEQ.ID.NO.: 1). In humans, Beta-Tubulin I is mainly expressed in the brain and the thymus and at the low level in lung, spleen, heart, kidney, liver, muscle, stomach and testis (Luduena RF (1998) International Review of Cytology, 178:207-275). 20 Example 2: Isolation and Identification of 55 kD Protein from the Membranous Fraction of Cochlear Tissue Protein Extraction 22 WO 00/50593 PCT/US0O/04795 Proteins are extracted from the membranous fraction of cochlear tissue in accordance with the method of Suzuki, M. et al. ORL, 59:10-17, 1997 as follows. Briefly, forty Harley strain male guinea pigs (Sasco Co., Wilmington, MA) are anesthetized by intraperitoneal injection of a mixture of ketamine (70 mg/kg) and 5 xylazine (70 mg/kg) and perfused with 0.01 M phosphate buffered saline (PBS), pH 7.4, through the left ventricle. Both temporal bones are removed and placed in crushed ice. The membranous portion of the inner ear, including basilar membrane, organ of Corti, stria vascularis, spiral ligament and vestibular epithelium are dissected under 10 a microscope. The neural portion of the inner ear, including the spiral ganglion, cochlear nerve in the modiolus, and vestibular nerve in the temporal bone, the facial nerve and brain tissue are dissected under a microscope. The membranous tissue and the neural tissue are each sonicated for 20 seconds in lysis buffer (100 mM NaCl, 10 mM Tris buffer (Sigma Chemical 15 Company, St. Louis, MO), pH 7.6; 1 mM ethylenediaminetetraacetate (EDTA; Sigma Chemical Company, St. Louis, MO), pH 8.0; a surfactant including, but not limited to, 0.1% sodium dodecyl sulfate (SDS, Sigma Chemical Company, St. Louis, MO) and 1% Nonidet P-40 (Sigma Chemical Company, St. Louis, MO; 2 pg/ml aprotinin; and, 100 mg/ml phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Company, 20 St. Louis, MO) or mixtures thereof. The extracted membranous proteins and the extracted neural proteins are incubated at 0 0 C for 30 minutes and centrifuged at 10,000 rpm for 10 minutes. Each supernatant is filtered through a 0.22 pm filter (Millipore Co., Bedford, MA), boiled for five minutes in a boiling water bath and stored at -80*C. 23 WO 00/50593 PCT/USOO/04795 Protein concentration is determined after electrophoresis in one-dimensional 12% SDS-polyacrylamide gels (SDS-PAGE) using molecular weight standards (Life Technology, Inc., Grand Island, NY.). A range of I gg/ml to 10 gg/ml of standard is loaded on the same gel and is compared with inner ear protein extract and with Raf-1 5 protein. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Samples are fractionated by SDS-PAGE using a 12% running gel and a 5% stacking gel in accordance with the method of Laemmeli et al. Samples of membranous proteins, of neural proteins and of Beta-Tubulin protein are each mixed 10 1:1 with 100 mM Tris, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol and 200 mM dithiothreitol and heated at 100'C for three minutes. The gels are electrophoresed in a vertical electrophoresis apparatus (Life Technologies, Inc., Grand Island NY) at 90 volts for seven hours. The gels are fixed and stained with Coomassie brilliant blue (BioRad, Melville, NY) and are destained with 45% 15 methanol, 10% acetic acid and 45% distilled water. Apparent molecular weights of the separated components are calculated by comparison with prestained molecular weight markers (Life Technologies, Inc., Grand Island, NY) electrophoresed in the same gel. Western Blotting 20 Proteins, separated in 12% one-dimensional SDS-PAGE as described above, are electroblotted onto polyvinylidene difluoride (PVDF) membrane (BioRad, Melville, NY) using a BioRad Semi-Dry Transblot Cell (BioRad, Melville, NY) for 1 24 WO 00/50593 PCT/US0O/04795 hour. The PVDF membrane is washed one time with 20 mM Tris, pH 7.5, 500 mM NaCl containing 0.025% Tween-20 (TTBS). Amino Acid Sequencing Samples separated in SDS-PAGE and are electroblotted onto PVDF 5 membrane as described above. The band corresponding to the molecular weight of Beta-Tubulin is excised from the PVDF membrane and is microsequenced using automated Edman degradation (Applied Biosystems, Red Wood City, CA). Ten amino acids, having the sequence EIVHQAG (SEQ.ID.NO.: 1), are identified. Sequence comparison with the Swiss Protein Database shows the 10 microsequence of the Beta-Tubulin membranous inner ear protein to have 100% identity, amino acid residues (SEQ.ID.NO.:3-7) from B-Tubulin I gene clone m40 gene bank accessment number J00314. Example 3: Determination of Beta-Tubulin Antibodies in Sera From Meniere's Patients 15 Proteins extracted from the membranous portion of the inner ear and recombinant Beta-Tubulin protein are electrophoresed in SDS-PAGE and electroblotted onto PVDF membrane as in Example 2 for immunochemical analysis as in Figure 2. Sera from patients with symptomatic Meniere's disease are provided by Drs. J. 20 Bernstein (Department of Otolaryngology, State University of New York, Buffalo, NY) and Y. Yazawa (Department of Otolaryngology, Shiga University of Medical 25 WO 00/50593 PCTIUSOO/04795 Science, Seta, Japan). The diagnosis of Meniere's disease is based on the AAO-HNS criteria. Sera are stored at -20 0 C. Figure 2 shows that sera from Meniere's disease patients react with the 55 kD antigen (SEQ.ID.NO.: 1) extracted from the membranous portion of the inner ear of 5 the guinea pig. Figure 2 shows that sera from the same Meniere's disease patients reacts with the Beta-Tubulin (SEQ.ID.NO.: 1). Example 4: Presence of Beta-Tubulin (SEQ.ID.NO.: 1, SEQ.ID.NO.:2) In Neural Inner Ear Proteins, Facial Nerve Proteins and Brain Tissue Proteins extracted from the membranous portion of the inner ear, the neural 10 portion of the inner ear, the facial nerve and the brain are electrophoresed and electroblotted onto PVDF membrane as in Example 2. Anti-Beta-Tubulin specific monoclonal antibody is obtained from Transduction Laboratories (Lexington, KY). This anti-tubulin antibody recognizes tubulin protein (SEQ.ID.NO.:15). For use with anti-Raf- 1 as target-binding substance 15 (1st antibody), nonspecific binding is blocked using 5% nonfat dry milk in 10 mM Tris, pH 7.5, 100 mM NaCl and 0.1% Tween-20 for one hour at room temperature. The PVDF membrane is incubated in anti-Raf- 1 diluted 1:1000 in 5% nonfat dry milk in 10 mM Tris, pH 7.5, 100 mM NaCl and 0.1% Tween-20 overnight at room temperature. The membrane is then washed three times in TTBS and incubated with 20 peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO). Immunoreactive bands are visualized using 0.05 M Tris-HCl, pH 7.6, containing 0.02% 3,3'-diamino-benzidine (Chemicon International, Inc., Temecula, CA) and 0.01% hydrogen peroxide. 26 WO 00/50593 PCT/USOO/04795 Anti-tubulin monoclonal antibody recognizes Beta-Tubulin in extracts from the membranous portion of the inner ear. Anti-Beta-Tubulin monoclonal antibody recognizes the Beta-Tubulin protein (SEQ.ID.NO.:1) in extracts from the membranous portion of the inner ear, and in extracts from the neural portion of the 5 inner ear, the facial nerve and the brain. Example 5: Reactivity of Sera From Meniere's Disease Patients with Beta-Tubulin Sera from 113 Meniere's disease patients, are assayed for antibodies against Beta-Tubulin in ELISA. One hundred microliters of Beta-Tubulin containing 5 g/l in 0.1 M carbonate 10 buffer, pH 9.6, are dispensed into each well of a polystyrene microtiter plate (Costa, Cambridge, MA) and incubated overnight at 4C. The antigen coated plates are washed three times in PBS-0.05% Tween buffer and incubated with patient's sera (1:40, 1:80, or 1:160 dilution) or with 0.1 M carbonate buffer, pH 9.6, as a control, overnight at 4*C. The plates are washed five times with PBS-0.05% Tween buffer and 15 incubated overnight with c-chain specific anti human IgG antibodies (Sigma Chemical Co., St. Louis, MO) at 4C. The plates are washed five times with PBS 0.05% Tween buffer and citric acid-phosphate buffer, pH 5.0, containing 0.15 mg/ml of o-phenylenediamine (Sigma Chemical Co., St. Louis, MO) is added. The color is developed at room temperature and the reaction is stopped by 2.5 M sulfuric acid. The 20 color is measured at 492 nm. 67 of 113 sera obtained from Meniere's patients (59%) recognize Beta Tubulin in ELISA at dilutions of 1:40, 1:80 and 1:160. Seven of these sera are tested 27 WO 00/50593 PCT/USOO/04795 in Western blot analysis and show positive reactivity with the 55 kD band in membranous inner ear extracts of the guinea pig. While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be 5 understood that the practice of the invention encompasses all of the usual variations, adaptation, modification or deletions as come within the scope of the following claims and their equivalents. Table I 10 Patients' diagnoses and Western blot immunochemistry of guinea pig inner ear protein probed from patients with various inner ear diseases n Positive Western blot n % Meniere's disease 25 15 60 Otosclerosis 6 3 50 Hearing loss and tinnitus (diagnosis undetermined) 6 3 50 PSNHL 2 2 100 Cogan's syndrome 1 0 0 Sudden deafness 1 0 0 Strial atrophy 2 0 0 Hereditary hearing loss 1 0 0 28 WO 00/50593 PCT/USOO/04795 Syphilitic labyrinthitis 1 1 100 Control 10 0 0 Among the positively reactive bands detected, a 52 kD band is the most common positive band found in sera from Meniere's disease (Suzuki et al., ORL, 59:10-17, 1997). The 52 kD protein of Suzuki et al. (ORL, 59:10-17, 1997) is found 5 to be 55 kD Beta-Tubulin of this application. 58 kD of Cao et al (Cao, M. et al., Laryngoscope 106:207-212, 1996) is likely to be Beta-Tubulin. As shown in Table 2 of 24 patients who show at least one positively reactive band, 10 show a positive reaction only in the membranous part, 1 only in the neural part and 13 in both parts. 10 Table 2 Differences in Western blotting results between the membranous part and the neural part of the inner ear 25 28 30 32 40 42 46 52 67 79 Membranous part 4 9 1 1 1 2 1 7 6 2 Neural part 0 0 2 1 1 2 1 6 4 0 Example 6: Presence of Beta-Tubulin (SEQ.ID.NO.: 1, SEQ. ID.NO.:2) In Neural 15 Inner Ear Proteins, Facial Nerve Proteins and Brain Tissue. 29 WO 00/50593 PCT/USOO/04795 Proteins extracted from the membranous portion of the inner ear, the neural portion of the inner ear, the facial nerve and the brain are electrophoresed and electroblotted onto PVDF membrane as in Example 2 (Figure 1). Although only a few exemplary embodiments of this invention have been described in 5 detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims. It should further be noted that all patents, applications, documents 10 or publications referred to herein are incorporated by reference in their entirety. 30
Claims (15)
1. An antigen of the membranous structure of the inner ear protein, wherein the antigen is reactive with antibodies from patients having Meniere's 5 disease. 2 The antigen of claim 1, wherein the antigen is a protein or peptide selected from the group consisting of proteins, proteins purified from extracts of membranous inner ear proteins, recombinant proteins or peptides and synthesized proteins or peptides. 10
3. The antigen of claim 1, wherein the antigen is a protein or peptide having a DNA or an amino acid sequence selected from the group consisting of SEQ.ID.NOS.: 1-17, or antigenic variants thereof and mixtures thereof.
4. The antigen of Claim 1, wherein the antigen is Beta-Tubulin. 31 WO 00/50593 PCT/USOO/04795
5. A method of detecting Meniere's disease in an animal or human comprising the steps of: (a) incubating a biological sample from the animal or human with a target substance under conditions sufficient to bind a target-binding substance in the 5 biological sample to the target substance, wherein the target substance is a Beta Tubulin antigen or a nucleic acid molecule encoding a Beta-Tubulin antigen of the membranous structures of the inner ear of a mammal and/or recombinant proteins from SEQ.ID.NOS.:
8-17 or antigenic variants thereof; and, (b) detecting the target-binding substance bound to the target 10 substance. 6. The method of claim 5, wherein the target substance is a protein or peptide having a DNA or an amino acid sequence selected from the group consisting of SEQ.ID.NOS.: 1-17 or antigenic variants thereof. 7. The method of claim 5, wherein the target-binding substance is an 15 antibody. 8. The method of claim 5, wherein the target-binding substance is a nucleic acid probe.
9. The method of claim 5, wherein the amount of target-binding substance in the biological sample is quantitated. 20
10. The method of claim 5, wherein the biological sample is a biological fluid from an animal or human having symptoms of an autoimmune inner ear disease. 32 WO 00/50593 PCT/USOO/04795
11. The method of claim 5, wherein the biological sample is a biological fluid from a patient having symptoms of Meniere's disease.
12. The method of claim 5, wherein the biological fluid is serum.
13. The method of claim 5, wherein said target substance is present in an 5 amount of from about 1 to about 100 pg/ml.
14. The method of claim 5, wherein said target substance is present in an amount of from about 3 to about 50 pg/ml.
15. The method of claim 5, wherein said target substance is present in an amount of from about 5 to about 30 pg/ml ml. 33 WO 00/50593 PCT/USOO/04795
16. An assay kit for detecting Meniere's disease in a patient comprising: (a) a solid phase having a Beta-Tubulin target substance bound thereto which reacts with a target-binding substance in a biological fluid from an animal or human having symptoms of Meniere's disease; and, 5 (b) means for detecting binding of the target binding substance to the target substance.
17. The assay kit of claim 16, wherein the target substance is a protein or peptide selected from the group consisting of extracts of membranous inner ear proteins, fractions of extracts of membranous inner ear proteins, proteins or peptides 10 purified from extracts of membranous inner ear proteins, recombinant proteins or peptides and synthesized proteins or peptides.
18. The assay kit of claim 16, wherein the target substance is a protein or peptide having a DNA or an amino acid sequence selected from the group consisting of SEQ.ID.NOS.: 1-7 or antigenic variants thereof and mixtures thereof, and 15 recombinant protein selected from the gene sequence group consisting of SEQ.ID.NOS.: 8 or antigenic variants thereof and mixtures thereof. 34
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US6306615B1 (en) * | 1999-08-19 | 2001-10-23 | Tularik Inc. | Detection method for monitoring β tubulin isotype specific modification |
EP1387888A2 (en) * | 2000-12-21 | 2004-02-11 | Incyte Genomics, Inc. | Nucleic acid-associated proteins |
US20150044271A1 (en) | 2012-02-27 | 2015-02-12 | O-Ray Pharma, Inc. | Solid drug implants for intracochlear delivery of therapeutics for the treatment of otic disorders |
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US5885783A (en) * | 1997-07-02 | 1999-03-23 | Yoo; Tai-June | Autoimmune inner ear disease antigen and diagnostic assay |
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