MXPA97006189A - Specific monoclonal antibody for beta peptide - Google Patents

Abstract

The present invention relates to Alzheimer's disease, particularly, with a monoclonal antibody specific for the betaA4 peptide derived from the amyloid precursor protein, cells which produce such an antibody, methods for generating such monoclonal antibodies and methods for using such antibodies in diagnosis and therapy

Description

SPECIFIC MONOCLONAL ANTIBODY FOR PEPTIDE ßA4 FIELD OF THE INVENTION The present invention relates to Alzheimer's disease, particularly, with a monoclonal antibody specific for the ßA4 peptide derived from the Amyloid Precursor Protein, cells which produce such an antibody, methods for generating such monoclonal antibodies, and with methods for using such antibodies in diagnosis and therapy.

BACKGROUND OF THE INVENTION Alzheimer's disease (AD) is a neurodegenerative, progressive, irreversible brain disease. During the course of several years of progress the AD leads to loss of memory, dementia and finally death. Currently, it is the fourth leading cause of death in the United States, contributing approximately 100,000 deaths annually. Typically AD affects mainly the elderly and therefore, with the aging of modern society, this is expected to be a growing health concern in the near future. As soon as the disease manifests, patients require assistance immediately. This REF: 25456 represents a physiological problem as well as a tremendous financial one for our society. Currently, there are no proven means for the diagnosis, prevention, treatment or cure of AD. Neuropathologically, AD is characterized by massive loss of neuronal cells in certain areas of the brain, and by the deposition of proteinaceous material in the brains of patients with AD. These deposits are intracellular neurofibrillary tangles or entanglements and extracellular β-amyloid plaques. The main protein component of the ß-amyloid plaque is the ßA peptide. Sequence analysis of the purified β-amyloid plaque material and mass spectrometry showed that the maximum length of the βA4 peptide is 43 amino acids. Typically, however, peptide species can also end either at position 40 or at position 42 (Miller et al., 1993, Arch. Biochem.Bí ophys 301: 41-52). Similarly, at the N-terminus, a certain inequality can be observed, which leads to various forms of the peptide, starting mainly at position 1, 4 or 11 (Miller, et al, 1993). Molecular cloning revealed that the ßA4 peptide is derived from a much larger precursor protein called "Amyloid Precursor Protein" (APP) (Pang et al., 1987, Na ture 325: 733-736) (Figure 1). The Figure illustrates the Amyloid Precursor Protein (APP), which is a transmembrane protein (Tm = membrane region), where the N-terminal is located extracellularly and the C-terminal is localized intracellularly (cytoplasmically). ßA4 is partially submerged in the membrane, several alternatively spliced isoforms have been described, which undergo extensive post-translational modifications (Selkoe 1994, Ann.Rev. Neurosci. 17: 489-517). The sequence of ßA4 itself is located partially on the extracellular side and extends partially towards the transmembranal region (Figure 2). Figure 2 (SEQ ID NO: 3) illustrates the sequence of ßA4, which shows (circumscribed area) extending with its C-terminal end towards the transmembranal region (Tm, framed area) and the N-terminal end located in the extracellular part . The asterisks indicate the location of familial mutations in the APP gene; they are found either flanking the ßA4 sequence, or are centered in the middle portion of the ßA4 sequence. The three major cleavage sites (a, ß and?) In the APP are indicated. It was therefore postulated that the release of ßA4 occurs through the proteolytic action of one or more proteases on the N-terminal (ß-cut) and on the C-terminal (cut-off) of the peptide (Figure 2) (Selkoe, 1994). The main event during the secretion of the APP is in section a (position 16/17 of ßA4"1-42"). This secreted APP molecule (aAPP) contains the first 16 amino acids of the ßA4 sequence and its carboxyl terminus. The fragments of APP associated with the remaining cell (called C-terminal fragments (CTF)) contain the C-terminal portion of the ßA4 sequence and extend into the cytoplasmic region of the APP. Therefore this proteolytic cleavage results in fragments that can not be processed in such a way that they lead directly or indirectly to amyloidogenic fragments (non-amyloidogenic processing) (Selkoe, 1994). Recently, it was demonstrated that cell lineages expressing large amounts of APP through a stably transfected APP cDNA construct produce high picomolar quantities at low nanomolar ßA4 and release it rapidly into the medium (Shoji et al., 1992, Science 258: 126-129). It has also been found that virtually all primary cell cultures and cell lineages release ßA4 constitutively (Busciglio et al., 1993 PNAS USA 90: 2092-2096). Additionally, healthy controls as well as patients with Alzheimer's disease have been shown to have low nanomolar amounts of ßA4 in serum and CSF (Seubert et al., 1992, Na ture 359: 325-327). Most of the soluble ßA4 species detected in these body sera and conditioned media were ßA4"1- ¡0", which does not truly reflect the total composition found in β-amyloid plaque depositions. The notion that the production and subsequent release of ßA4 is sufficient and therefore responsible for the creation of β-amyloid plaques in the brains of patients with AD could therefore t = > nto not hold more; other factors should contribute to the deposition of ß-amyloid plaques. An open hypothesis is that acute or chronic overproduction of ßA4 causes the increase in amyloid burden observed in AD. The discovery that specific point mutations around the ßA4 region of the APP gene are linked to certain cases of familial Alzheimer's disease (FAD) showed unequivocally that the APP gene is a "disease gene" (Goate). et al., 1991, Nature 349: 704-706, Murell et al., 1991, Science 254: 97-99, Levy et al., 1990, Sci en 248: 1124-1126; Carter et al., 1992, Na ture Geneti cs 2: 255-256). In families in which AD is inherited in a dominant manner with a specific attack gene, point mutations in the APP gene are necessary and sufficient to cause AD. Although the vast majority of cases of Alzheimer's disease are sporadic and probably multifactorial, these familiar APP mutations can teach us much about amyloidogenesis, that is, the generation of the small ßA4 peptide from the larger precursor and its subsequent deposition on the plates β-amyloid. The double mutation in codon 670/671 of the APP (the "Swedish variant", in the N terminal of ßA4 in APP) causes a 5- to 8-fold greater release of ßA4 in cell cultures stably transfected with that cDNA of mutant APP (Figure 2) (Citron et al., 1992; Cai et al., 1993). It is possible that this double point mutation leads to an increase in APP turnover due to increased proteolysis in the β-cut, which in turn leads to a higher level of ßA4 released. The increased amounts of ßA4 monomers, as demonstrated by transfection studies with the "Swedish mutation", may explain the faster kinetics of aggregation of ßA4 to ß-amoiloid plaques in these families. Another FAD mutation is found at the C terminal of ßA4 at position 717 ("London variant") and does not affect the level of ßA4 release in tissue cultures (Figure 2).

Recently it was shown that this mutation 717 changes the "1-40 / 1-42" ratio of ßA4 (Suzuki et al., 1994, Science 264: 1336-1340). Although it is not clear when the C-terminal generation of ßA4 occurs, since this part is submerged in the transmembrane region, one hypothesis is that the "London mutation" affects the proteolytic cleavage of APP to ßA4. Possibly, this point mutation interferes with the fidelity of the cleavage of the responsible protease at the site? ßA4 1-40 exhibits, among other things, a dramatic difference in its solubility in aqueous solutions when compared to ßA4 1-42 (Burdick et al., 1992, JBC 267: 546-554). The latter is virtually insoluble in water, while 1-40 is soluble in water up to several mg / ml in vitro. Minor amounts of the larger form 1-42 can increase the presentation of 1-40 in vitro dramatically. A slightly higher proportion of the larger ßA4 1-42 species could explain the initial appearance of ßA4 deposition in iloid plaques in patients with this "London mutation". The proportion of species from 1-4 to the shorter soluble species 1-40 could also be one of the critical factors in sporadic AD cases (ie cases where no genetic predisposition was identified). Monoclonal antibodies that bind specifically to species 1-42, are therefore useful to investigate the production and presence of ßA4 species that end at the amino acid at position 42, and may be useful as a diagnostic indicator of abnormal species present in AD. Recent analyzes with the antibody that recognizes ßA4 that ends at position 40, and an antibody that recognizes the ßA4 species that extend to position 42o beyond, showed that the contribution of the larger ßA4 species can be critical for the attack of the disease (Suzuki et al., 1994). However, the Suzuki monoclonal antibody does not distinguish between ßA4 1- 42, 1-43 and larger ßA4 species. This is also the case for another monoclonal antibody reported 2G9 (Yang et al., 1994, IVeuro Report 5: 2117-2120). Therefore to avoid this cross-reactivity, the antibodies that are specific for ßA4 species ending in position 42 for the exclusion of other forms could be very useful to avoid cross-reactivity with APP terminal fragments associated with the membrane , which are typical cellular products not necessarily associated with ß amyloid plaques. A monoclonal antibody recognizing ßA4 1-42 (Murphy et al., 1994, Am. J. Path. 144: 1082-1088). However, ßA4 1-43 peptide species were not used in those studies, so it is not known what exact specificity of this monoclonal antibody could be the response to peptide 1-43. Competent studies were only conducted with the ßA4 peptides that end at position 40 ("1-40"), and position 44 (• 1-44") and beyond with this antibody. diffuse staining of amyloid, fibrillar amyloid, intraneuronal and extraneuronal neurofibrillary nodes, but not vascular amyloid.An in vitro diagnostic biochemical test for Alzheimer's disease is not available in its primary etiologies as well as means to select individuals at risk The current diagnosis of AD requires a detailed clinical evaluation that can not give clear answers until the significant symptoms of dementia and memory loss manifest In view of the research referred to above, ßA4 1-42 represents a preclinical marker for AD, thereby identifying the level or construction of ßA4 1-42, or another species that ends at residue 42, and how this can progress during the course of the disease, and how it is distributed in the brain, will provide valuable clues to verify the course of how, as well as for the specific diagnosis and possible treatment of AD. It could be useful to prepare diagnostic tests, therapeutic agents for tests to verify AD, to have a monoclonal antibody which, in contrast to the specificity of the antibodies available so far (cross-reactivity with 1-43, which has been reported to have no vascular amyloid), which stains the vascular amyloid and is specific for the ßA4 peptide that ends in residue 42, and therefore extends the diagnostic capabilities of the technique, that is, one that recognizes the free C terminal of ßA4 1- 42 and vat the diffuse and similar amyloid, neurofibrillary and vascular amyloid nodes. Such an antibody is the subject of the present application.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a monoclonal antibody that is specific for the ßA4 peptide, and in particular the C-terminal of ßA4"1-42" and dyes the fibrillar amyloid, neurofibrillary and vascular amyloid nodes. In particular, the present invention provides a monoclonal antibody that is specific for all ßA4 peptides in which the C-terminal is located in residue 42 of the amino acid sequence of ßA4. The present invention also encompasses antibody fragments and constructs thereof that have the same binding specificity. The present invention covers in particular the monoclonal antibody known as "Mab 269.2B" and that is produced by the cell line "369.2B", which has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC) on the 26th. January 1995, and has been assigned access number HB11829. The present invention encompasses the use of the monoclonal antibody of the present invention in diagnosis, purification and therapeutic uses. Thus, one embodiment of the present invention encompasses a monoclonal antibody specific for the ßA4 peptide that ends at position 42, where the antibody binds to diffuse amyloid, fibrillar amyloid, vascular amyloid and neurofibrillary tangles. In a particular embodiment, the present invention provides a monoclonal antibody which is specific for the terminal C amino acids of the ßA4 1-42 peptide. In the most preferred embodiment the present invention encompasses a monoclonal antibody that is identified as 369.2B, and is produced by the cell lineage deposited with the American Type Culture Collection (ATCC) as accession number HB 11829. A preferred embodiment of the present invention it also encompasses a cell which is identified by the accession number ATCC HB 11829. In a further embodiment of the present invention, an immunologically reactive fragment of the monoclonal antibody of the present invention is encompassed, which is capable of effecting the same binding as the monoclonal antibody of the present invention. The present invention also provides methods for generating antibodies specific for ßA4 which recognize the free C terminal residue 42. The present invention also provides methods for detecting the presence of ßA4 peptides terminating at position 42, in tissues, comprising contacting a tissue sample with the monoclonal antibody of the present invention, detecting the presence of the monoclonal antibody in a selective way. The present invention also provides methods for the selective purification of the ßA4 peptides that terminate at position 42, which comprise contacting a sample to be purified with the monoclonal antibody of the present invention, separating the ßA4 peptide from the sample to be purified, and isolate the ßA4 peptide. In a further embodiment, the present invention provides methods for the detection of the ßA4 peptide associated with Alzheimer's disease, comprising contacting a sample to be tested with the monoclonal antibody of the present invention, and detecting the presence of the peptides. ßA4.

Thus, the present invention also provides methods for the prevention of aggregation of the βA4 peptide by administration of the monoclonal antibody of the present invention. In a preferred embodiment the monoclonal antibody is identical to 369.2B, or is an immunologically reactive fragment with specific binding specificity thereof. The present invention thus provides means for detecting the presence of the ßA4 peptide comprising an immunologically reactive fragment of the monoclonal antibody of the present invention. As well as means for preventing the aggregation of the ßA4 peptide, comprising an immunologically reactive fragment of the monoclonal antibody of the present invention. The present invention provides means for detecting and verifying the level of ßA4 peptide in tissue or fluid samples (e.g., blood, other body fluids, tissue sections, tissue biopsies, etc.). In a preferred embodiment, the ßA4 peptide that is detected, verified, inhibited or purified is a ßA4 peptide with a free carboxy terminal residual amino acid with residue number 42 of the amino acid sequence of the ßA4 peptide. All references to publications and patent documents in the preceding or subsequent sections are incorporated herein by reference in their entirety. The specific embodiments of the present invention will be more apparent from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more fully understood from the consideration of the following detailed description, taken in conjunction with the drawings, in which: Figure 1 is a diagram showing the ßA4 portion of the Amyloid Precursor Protein (APP) , its location relative to the cell membrane and cleavage sites α, β and β; Figure 2 shows the ßA4 portion of the APP, its position relative to the transmembrane region of a cell, and the three major cleavage sites (a, ß and γ) in the APP; Figure 3 is a diagram of the clone pGK003 which has been used to generalize the ßA4 peptide 1-42; Figure 4A shows SDS-PAGE on 16% Tris / Tricine gel, radiolabelled ßA4 labeled in vitro in a wheat germ system; Figure 4B shows the SDS-PAGE on 16% Tris / Tricine gel, of the radioactively labeled ßA4 translated in vitro from the wheat germ system, immunoprecipitated with MAb 286.8A; Figure 5 is a graph showing the in unoprecipitation of the translated ßA4 (IVT ßA4) with 286 8A; Figure 6 is a diagram of the peptides used to generate the immune response (immunogen) and to select the serum from mice; Figure 7 is a graph showing immunoprecipitations of ßA4 translated in vitro vs. antibody concentration; -O- 286.8A, -? - 369.2B, -D-369.6; Figure 8 is a graph showing the percent of the variations of the ßA4 sequences immunoprecipitated with MAb 369.2; Figure 9 is a graph showing the tracing of the epitope of MAb 369.2 by competitive assays, with -B-being 35-42 (OVA) (Ovalbumin coupled to the ßA4 peptide 35-42), -D- being ßA4 peptide 1-42 , and -f- being peptide βA4 1-40; Figure 10 is a photograph showing the binding of MAb 369.2B to vascular amyloid and other plaques with various morphologies.

DETAILED DESCRIPTION OF THE INVENTION The depositions of β-amyloid in the brains with Alzheimer's disease consist mainly of ßA4 peptides that show both N and C terminal heterogeneity. The major C-terminal species, identified by peptide sequencing of purified β-amyloid from postmortem brain material, end either in position 40 or 42 of the ßA4 peptide which is at most 43 residues in length. In vitro experiments with synthetic ßA4 peptides that end either in position 40 or 42 indicate profound physicochemical differences. Previously, the distribution of these two species of ßA4 in postmortem tissue as well as their generation in vitro could not be evaluated due to the lack of specific antibodies against the carboxy terminus capable of distinguishing between the subspecies of the ßA4 peptide. Recent evidence suggests that the release of ßA4 is an event norm in virtually all cell cultures. Typically, high picomolar to low nanomolar concentrations of ßA4 can be measured in serum and cerebrospinal fluid (Seubert et al., 1993). This discovery was surprising because it was proposed that the production of ßA4 is a pathological event since ßA4 is massively deposited as ß-amyloid plaques in the cortical and hippocampal regions of the brain of patients with Alzheimer's disease. Detailed sequence analysis of ßA4 released from cell cultures revealed that the main species end at position 40 (Selkoe, 1994). The amyloid plaques purified from postmortem brain show a slightly different picture: the amyloid deposits of congophilic amyloid angiopathy (CAA) are aggregates of ßA4 that surround the blood vessels and are predominantly ßA4 1-40, whereas in contrast the nuclei of the amyloid plaques (APC) are present in the brain parenchyma and are not associated with blood vessels exhibit a N-terminal state (most commonly beginning at residues 1, 4 and 11) and ending mainly at position 40 or 42 (Glenner and Wong, 1984, Bi oche, Bi ophy, Res. Eat, 120: 885-890; Masters et al. , 1985, PNAS USA 82: 4245-4249; Miller et al. , 1993). Occasionally, larger species that end in 43 or that extend even further have also been described (Miller et al., 1993). Because the length of the hydrophobic C terminus is critical to the ability of the peptide to autoaggregate in vitro (Burdick et al., 1992; Jarrett et al., 1993, Biochem. 32: 4693-4697), it is entirely possible that two aggregates Pathologically distinct, APC and CAA and other vascular β-amyloid plaques, can be explained by the different properties of the two species 1-40 and 1-42. This could also be the case for so-called "diffuse plaques" (Selkoe, 1994) which are frequently observed in brains of old humans and are not associated with AD, however, it has been proposed that they are precursors of β-deposits. fibrillar amyloids. A non-fibrillar aggregation of ßA4 has been suggested for these structures. It is therefore of primary importance to determine the tissue-specific production of those larger ßA4 species (that is, those that end in position 42) and their pathological appearance in the brains of patients with AD.

Recently three reports have been published in which antibodies have been described, which distinguish species 1-42 and 1-40 from ßA4 (Suzuki et al., 1994, Murphy et al., 1994; Yang et al. , 1994). Unlike the antibodies of the present invention, the antibodies reported by Suzuki et al. , and Yang et al. , cross-react to a significant degree with both species 1-43 and 1-42 of the ßA4 peptide.

The Murphy et al. Antibody, although not tested for binding to species 1-43 of the ßA4 peptide, exhibits a different tissue binding pattern than the antibodies of the present invention, and thus can recognize a different epitope or modified to that recognized by the antibodies of the present invention. The 29 to 42 positions of the ßA4 peptide are entirely within the putative transmembrane region of the Amyloid Precursor Protein and are hydrophobic in nature (Miller et al., 1993). Synthetic peptides for C-terminal sequences in this region must overcome the ability of sequence 34-42 to form an unusually stable β structure that is virtually insoluble in water and strong denaturing agents (Halverson et al., 1990 Bi ochem 29: 2639 -2644) if it is to be used to produce good immune responses against soluble ßA4. We designed hydrophilic separators of five residues in length that could overcome those problems of solubility and also extend the presumed epitope away from the proximity of the carrier. To reduce the likelihood of cross-reactivity with shorter but larger ßA4 species, 1-40, we chose a minimum peptidyl epitope of 8 residues corresponding to positions 35-42 of the ßA4 sequence. The entire synthetic sequence designated in this manner was coupled by a free sulfhydryl group on a N-terminal cysteine residue to KLH (keyhole limpet hemocyanin).

The successful use of hydrophilic separators and residues in the production of antipeptide antibodies is well documented as the use of hydrophilic structures to put insoluble haptens in solution for conjugation (McMillan et al., 1983, Ceii 35: 859-863; Makela and Seppala, 1986, in Handbook of Experimental Immunology, Volume I: Immunochemistry, ier, DM, editor Black ell Scientific Publications, Oxford, pp 3.1-3.13). The success of this method in the production of specific antibodies can at least in part be attributed to the presence of a free charged carboxyl terminal, especially in the context of a hydrophobic sequence, as terminal residues on peptide antigens that give rise to significant proportions of antibodies antipeptide (Gras-Masse et al., 1985, in Synthetic Peptides in Biology and Medicine, Alitalo, K. et al., eds. Elsevier, Amsterdam, p.105). This, together with the novel selective use of a minimal ßA4 sequence used as an immunogen, maximizes the possibility of producing an antibody that could distinguish between ßA4 species that end at positions 42 with those that do not. Although peptide competence studies do not re fl ect the map of antigenic determinants, other ßA4 sequences other than 1-42 were not effective in inhibiting binding. The fact that 1-42 does not compete fully with the in vitro translated ßA4 labeled with 35S-methionine may be due either to the particular properties of the molecule itself or to the fact that the peptide immunogen 35-42 was present in the context of a separate and / or specific carrier, or that an excess of 200-1000 times of unlabeled peptide is not sufficient to stop the signal. The non-specific effects of N-terminal residues on antigenic activity are also well documented (Benjamini et al., 1968, Bi ochem 7: 1261-1264). The intriguing discovery that 25-35 actually increases the ability of 369.2B and other antibodies to bind to ßA4 may be due to a particular interaction between the subtracted peptide and the sequence of full length ßA4 itself. It is believed that residues 26-33 exist as a random helix in aqueous solution (Halverson et al., 1990) and may be able to interact with the soluble ßA4 in such a way that the C-terminal becomes more accessible to the binding sites on the antibodies. The highly specific antibody of the present invention, of which 369.2B is a particular example, was raised against a synthetic ßA4 peptide having residues 35-42, and does not recognize shorter ßA4 species 1-40 in solution or in a solid phase. In addition, both 1-40 and 1-43 were unable to absorb the antibody when used immunohistochemically. A secondary selection method with full-medium capacity was applied for the selection of hybridoma supernatants using the radiolabelled translated in vitro ßA4 so that the antibodies chosen from the primary selection could be further selected for their ability to immunoprecipitate soluble ßA4 . This method can be easily adapted to other proteins / antibodies of interest. The resulting MAb 369.2B represents a superior tool for investigating the role of ßA4 peptides that end at position 42 in situ, postmortem tissue, transgenic animals and in vitro generation of ßA4 peptides in established cellular ßA4 production models, to be used in diagnosis and for therapeutic agents. The monoclonal antibody of the present invention represents an important tool necessary to establish a diagnostic test kit. Allow one to measure / quantify the amount of ßA4 1-42 or derivatives thereof (eg species 4-42, and other forms truncated with the carboxy terminus "42") in human body fluids (CSF, serum, urine, etc.) or tissues. It can also be used to study the distribution pattern of 1-42 or ßA4 species that end at residue 42, in brains with AD compared to healthy controls. His exceptionally high avidity makes him a superior and novel tool for such a test. The monoclonal antibodies described herein can also be used in biological model systems such as cell culture models or transfected animals (transgenic mice), designed to measure and / or influence the presence and / or production of ßA4 species that end in the amino acid 42. These model systems represent means to identify selective modulators of ßA4 production that end at the amino acid at position 42 of ßA4 in biological systems. The antibodies of the present invention provide methods for preventing aggregation of the ßA4 peptide because the specificity of the antibody will allow specific interference with the free C-terminal residue, thereby interfering with and disrupting the aggregation that may be pathogenic in the AD . Surprisingly, the antibody of the present invention differs from the prior art in that it has diffuse fibrillar amyloid, neurofibrillary and vascular amyloid nodes and is specific at the same time for residue 42 C terminal free of the ßA4 peptide. This unique binding pattern shows that the antibody of the present invention recognizes an epitope different from that of the prior art, and that the tissue distribution or accessibility of the ßA4 peptide recognized by the antibody of the present invention is also different. In addition, the present invention provides the monoclonal antibody that can precipitate the βA4 peptide out of a solution, which was not demonstrated by the prior art antibodies. Thus, the present invention provides unique monoclonal antibodies that recognize a unique subset of ßA4 species that has a distinct tissue distribution that is most likely a better diagnostic indicator than previously available and a unique target for therapeutic intervention. Thus the present invention provides the antibodies, antibody fragments and constructs thereof which are specific for ßA4 peptide species wherein the C terminus ends at residue 42. The present invention also provides for the use of such antibodies, fragments and binding constructs thereof in diagnostic, therapeutic analytical and biochemical purification methods, which employ the binding specificity of the monoclonal antibodies herein and their use within pharmaceutical formulations. The following examples will better explain the present invention and are shown by way of illustration, not by limitation. The following examples illustrate certain aspects of the methods and compositions identified above as well as the advantageous results.

Example 1: ßA4 Peptide Expression System Preparation of plasmid pGK002 General molecular biology and cloning procedures are found, for example, in Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning 2nd edition, Cold Spring Harbor Lab Press. Plasmid pMTI-26, which is a Bluescript KS clone containing 2 415 kb (pairs' of kilobases) of the APP sequence with a high TAG codon followed by a Ba HI site placed in frame by site-directed mutagenesis site after the codon of amino acid 42 of the ßA4 region, was modified by cleaving a 1.8 kb Xba I / Bgl II fragment and reattaching the plasmid after filling the ends. The resulting construct, designated pGK002, places the consensus that the start codon of the ßA4 sequence has immediately downstream of the Bluescript T7 promoter.

Preparation of plasmid pGK003 The plasmid pGK003 (Figure 3), used in all the translations is in vitro in wheat germ of ßA4 has to be described below, was done subcloning a fragment Notl / Xho I of 590 bp (base pairs) of pGK002 containing all sequences of human ßA4 with high / Ba Hl mutant in a vector pSP64 polymerization A (Promega Corp.). In the preparation of this plasmid, pGK002 was digested with Not I and Xho I and the 590 bp fragment was filled with Klenow, isolated and ligated with pSP64polyA linearized with Sma I. Figure 3 is a diagram of the Clone pGK003. The open reading frame of ßA4 1-42 was expressed in vitro from the bacterial SP6 promoter. The 3 'untranslated region (3'-UT) of the APP is shown in black.

Example 2: In Vitro Transcription and Translation of pGK003 The plasmid pGK003 was linearized with EcoRV and the complete digestion was confirmed by electrophoresis on agarose gel. The sample was extracted twice with phenol / chloroform, followed by two extractions with chloroform and ethanol precipitation. The resulting pellet was washed once in 70% ethanol, partially dried under vacuum and resuspended in TE at a concentration of 1 μg / μl. In vitro transcripts using linearized patterns at 30 μg / ml were prepared in 80 mM HEPES-KOH buffer (pH 7.5) with a content of 10 mM MgCl 2, 2 mM spermidine; 40 mM DTT, 3 mM ATP / CTP / GTP / UTP, 800 units / ml of RNAin Ribonuclease Inhibitor (Promega Corp.), 5 units / ml of Inorganic Yeast Phosphatase (Sigma Corp.), and 1800 units / ml of RNA polymerase SP6 (Promega Corp.). The reaction mixture was maintained at 37 ° C for 4 hours. The resulting transcript was verified by electrophoresis through 1.2% agarose / TBE / EtBr gel with denatured samples (65 ° C x 10 min). The transcripts were translated using wheat germ extract (Sigma Corp.). Briefly, transcripts were heated (65 ° C x 10 min), mixed with wheat germ extracts containing KAC, RNAsin, and a mixture of amino acids minus methionine, and were translated at 25 ° C for 1 hour in presence of methionine labeled with 35S (Amersham). Translation of a 4 kD ßA4 tip (kilo daltons) was verified by SD-PAGE using 16% Tris / Tricine Gel (Novex). The gels were fixed and the proteins were visualized fluorographically using a commercial system, "Amplify" (Amersham).

The incorporation of the tag into the translated ßA4 in vitro, which contains a methionine residue per molecule, was determined by dividing in gel. 2 mm slices were solubilized in 1 ml of 30% hydrogen peroxide, 0.75M NH40H overnight at 37 ° C. A volume of 10 ml of "Raedy Val é" flashing cocktail (Beckman) was then added and the DPM (Decay per minute) was determined using a Beckman LS6000IC flashing counter in the automatic DPM mode. Typical reactions produce ~ 250 ng of ßA4 / ml, or -56 nM. In vitro transcription followed by translation of the ßA4 clone, pGK003, in a wheat germ system resulted in a unique 4 kD protein product when visualized by fluorography on a 16% Tris-Polyacrylamide gel with tricine SDS (Figure 4A). Figure 4A shows the results of the SDS-PAGE on 16% Tris / Tricine Gel. Lane 1: Markers of high PM.

Lane 2: Markers of low PM. Lane 3: A ßA4 translated in vitro into a wheat germ system. The identity of this 4 kD product was confirmed by immunoprecipitation with antibodies specific for ßA4 (Figure 4B). Figure 4B shows the results of SDS-PAGE on 16% Tris / Tricine Gel. Lane 1: Markers of high PM. Lane 2: Markers of low PM. Lane 3: ßA4 translated in vitro from wheat germ system immunoprecipitated with MAb 286.8A. The transcription and translation of this as well as of other ßA4 clones in a combined reticulocytic lysate (TnT) system does not result in the same yield or purity of radiolabelled ßA4 (data not shown). This could be due to the short transcript or to the peculiar nature of the ßA4 peptide itself. The monoclonal antibody 286.8A, which was raised against crude peptide 1-42 and traces the 3-8 region of ßA4, was able to precipitate this protein in a concentration-dependent manner (Figure 5). Figure 5 is a graph of this immunoprecipitation in translated ßA4 in vitro (IVT ßA4). Increasing amounts of IVT ßA4 were immunoprecipitated with a fixed amount of 286.8A (7.4 μg) in 100 μl in RIPA buffer.

Example 3: Immunogen and Selection of Peptide Preparation The peptides were prepared by standard Fmoc solid phase procedure (see for example Gras-Masse et al., 1985). Peptide # 959, is a 14-residue synthetic peptide having an N-terminal cysteine attached to a hydrophilic separator DGDGD and residues 35-42 of human ßA4 (which results in the complete sequence: CDGDGDMVGGWIA (SEQ ID NO: 1 )), was coupled to a carrier of KLH (Keyhole Eyelid Hemocyanin) activated with maleimide using the commercially available Imjet Activated Immunogen conjugation kit (Pierce). Briefly, 2 mg of peptide and 200 μl of conjugation buffer were dissolved and allowed to react at room temperature for 2 hours with 2 mg of reconstituted maleimide-activated KLH. The conjugate was purified by gel filtration and used as an immunogen for the production of monoclonal antibody using the standard protocols as described in Example 4. Peptide # 958, a 14 residue synthetic peptide having an N-terminal cysteine attached to a GGGGG separator and residues 35-42 of human ßA4 (resulting in the complete sequence: CGGGGGMVGGWIA (SEQ ID NO: 2)), was coupled to ovalbumin by dissolving 2 mg of peptide in 200 μl of 6M guanidine, 0.01M phosphate pH 7.0 and conjugated as above 2 mg of an ovalbumin activated with reconstituted maleimide. The purified conjugate was used in the selection by ELISA of the monoclonal fusion products. The antibodies selected in this manner are specific for the "35-42" determinant rather than the separator, cysteine bridge or portions of the immunogen carrier. Figure 6: Illustrates the peptide used to generate the immune response (the immunogen) and the peptide used to select sera from mice, and also shows the fusions in the enzyme immunoassay plate (EIA). Sequence 35-42 of ßA4 was synthesized together with a separator and a C-terminal Cysteine, which was then used to couple this covalently via a bridge of the maleimide to a large carrier molecule. Both the separator and the carrier molecule in the immunogen and in the selection peptide are different to select antibodies specific for the ßA4 signaling.

ELISA (Immunosorbent Assay Linked to the Enzyme) Biotinylation of the MAb N-hydroxysuccinimide ester was used to biotinylate the monoclonal antibody 286.8A. The integrity of the reagent was checked first by observing its spontaneous hydrolysis in the presence of primary amines: a 0.2 mg / ml solution of NHS-LC-Biotin (Vector Labs, Burlingame, CA) in PBS was verified at 260 nm over time. An OD260 of 1.0 after about 2 hours (rinse from initial DO260 ~ 0.55) indicates the expected hydrolysis. In the biotinylation reaction it was found that a molar ratio of 66: 1 Biotin to monoclonal 286.8A at neutral pH has optimal results when the biotinylated 286.8A was tested in an Elisa format. 0.6 mg of NHS-LC-Biotin in H0 were added at a concentration of 0.1 mg / ml (with 5 minutes of dissolution) to 1 ml (2 mg) of 286.8A in PBS. The nucleophilic attack of the NHS ester was allowed to occur at 25 ° C for 2 hours after which 10 mg of glycine in 50 μl of H20 was added to stop the reaction. The reaction was then placed on a desalted column with 10 ml crosslinked dextran equilibrated with PBS and aliquots of 0.5 ml were collected. The first peak representing the IgG peak was collected and stored at 4 ° C until it was used.

Elisa procedures Corning 25801 96-well ELISA plates were coated overnight at 4 ° C with 100 μl of monoclonal 4G8 or other capture antibody at 5 μg / ml typically in H20 or buffer. The plate was then washed with PBS containing 1% Triton X-100 in a Dynatech Ultrawash plus. The wells were then blocked for 90 minutes with 300 μl of PBS containing 1% Triton X-100 and 1% BSA Elisa grade (Blocking Buffer). After washing the antigen or unknown diluted in blocking buffer was added to the wells in triplicate and incubated at room temperature for 2 hours. The plate was washed 2 times and another 400 ng of biotinylated 286.8A or other detection antibody was added. 30 minutes later the plate was l- ^ or more extensively (washed 2 times, rinsed 2 minutes, washed 2 times) and 100 μl of preformed Avidin-Biotin-Alkaline Phosphatase Complexes (Vector Labs). The plate was washed (washed 2 times, rinsed 2 minutes, washed 2 times, rinsed 5 minutes, 4 washes) and MUP substrate was added at 0.06 mg / ml lx of diethanolamine buffer. The plates were read on a Millipore Cytoflour 15 minutes later using a 360 nm excitation filter and a 460 nm emission filter.

Example 4: Generation of Monoclonal Antibodies Balb / c mice were immunized with inoculations I.P. multiple of peptide # 959 conjugated to KLH. Splenocytes from immunized animals were fused with AG8 mouse myeloma using standard protocols (Wunderlich et al., 1992, J. Immunol, Methods 147: 1-11). The supernatants of the resulting hybridomas were selected for the immunoreactivity of peptide # 958 coupled to the ovalbumin using standard Elisa protocols as described above. Hybridomas positive for the expression of the immunoreactive AMb were cloned at least twice by limiting dilution and the analysis of the AMb isotype was performed. Purified AMb IgG was prepared from ascitic fluid using affinity chromatography with protein A. Subsequently, fusion selection showed that immunization of mice with peptide # 959 conjugated to KLH and selection in a solid-phase ELISA format with the peptide # 958 coupled to the ovalbumin results in six signals of positive origin (identified as 369.1 to 369.6). Both peptides have amino acids 35-42 from the ßA4 region, different N-terminal separators, and a cysteine for covalent coupling to the carrier proteins (Figure 6). The free C terminal with the charged carboxy group and a limited length of only 8 amino acids favors the generation of antibodies directed specifically against larger forms of the ßA4 peptide; the shorter ßA4 peptides that terminate before amino acid 42 could not be recognized in this way.

Figure 6 illustrates the diagrams of the structure of the immunogen (carrier-peptide) and the selection peptide (Carrier-selection peptide) used. Two to six signals of origin were finally not cloned. Of the remaining four, two gave immunoprecipitation / flare signals only one percent higher than normal non-immune controls; the other two (identified as 369.6 and 369.2) showed the signals of 18% and 19% respectively. The production of monoclonal antibodies of ascitic fluid and the subsequent in unpurification of these clones was carried out. Table 1 compares the data obtained with IPSA for hybridoma supernatants and purified antibodies.

Table I Cell Line Isotype I PSA (supernatant) IPSA (purified) 369. 1 IgGl / IgG2b 3% N. D. 369.2 IgGl 19% 25% (with 5μg) 369. 3 IgGl 2% N. D. 369.6 IgG2b 18% 7% (with 10 μg) Table 1. Comparison of antibody activities in hybridoma cell lineages. The IPSA data represent the percent of ßA4 translated into a vitro that could be immunoprecipitated by supernatants of hybridoma or purified antibody.

Example 5: Immunoprecipitation / Flashing Test for the Selection of the Hybridoma To develop and select the monoclonal antibodies that recognize the ßA4 peptide in solution instead of when bound to a solid phase, an assay was performed in which the immunoprecipitation of translated ßA4 in vitro marked with 35S-methionine (IVT ßA4) was measured . A standard amount of translated ßA4 in vitro was allowed to form antibody / antigen complexes in a solution that could be optimized for ionic strength, pH and detergent composition. After the immune complexes are precipitated with Protein G (Omnisorb cells) and washed extensively, the bound radioactivity was counted in a liquid flashing counter, the background noise was extracted and the efficiency of the precipitation was calculated. This immunoprecipitation / flashes assay (IPSA) allows rapid identification and characterization of antibodies, and has been used to test a variety of ßA4 antibodies. The assay can be applied in general to monoclonal hybridoma supernatants as well as to polyclonal sera to identify antibodies that can be used for immunoprecipitations. Typically, 18 IPSAs can be performed in one day. This is therefore a rapid and informative secondary hybridoma selection method. In summary, approximately 1.5 x 105 were added DPM of ßA4 translated in vitro labeled with 35S-methionine (IVT ßA4) at 10 μl of lOx of immunoprecipitation buffer (150mM NaCl, 10% NP-40, 5% deoxycholic acid, 1% SDS, 500mM Tris, pH 8). To this, 90 μl of monoclonal cell supernatant of the monoclonal fusion of interest (our designation # 369) was added and allowed to react for 2 hours at 4 ° C. The background noise was determined using 90 μl of supernatant of a non-reactive clone; the positive control was 90 μl of supernatant containing monoclonal antibody 286.8A which was previously smoothed against a crude synthetic ßA4 (1-42) preparation. 2 hours later, 40 μl of a 10% solution of Omnisorb (Calbiochem) cells equilibrated in lx of immunoprecipitation buffer (RIPA buffer, 150mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, Tris 50mM, pH 8) and allowed to react for an additional 2 hours at 4 ° C with stirring. The cells were pelleted by centrifugation for 5 minutes at 4500 g and 4 ° C, and washed 3x with 800 μl lx of cold immunoprecipitation buffer. The pellets were quantitatively transferred to flask flasks and counted in a Beckman LS6000 flashing counter in the automatic DPI mode. The percentage of immunoprecipitated ßA4 was calculated. The immunoprecipitation / flashes assays were performed with 1 μg of purified monoclonal antibody 369.2B in a total volume of 100 μl of immunoprecipitation buffer to which 5 μg of competitive peptide was added. The incubations and precipitations were as described above. Figure 7 describes the percentage of immunoprecipitated ßA4 IVT as a function of antibody concentration for MAbs 369.2, 369.6 and MAb 286.8A. Under the conditions of this assay, 369.6 (and the additional subclone 369.2B) is approximately four times better than 369.6 in the immunoprecipitation of soluble ßA4 IVT, but precipitates a little less than half that of 286.8A. Figure 7 shows the results of the immunoprecipitation of ßA4 translated in vitro vs. antibody concentration (μg antibody / 100 μl RIPA buffer) where; Hor cent of immunoprecipitated ßA4 = (dpms with MAb) - (dpms without or with preimmune control) (dpms of total ßA4 / reaction) Percents with a given MAb concentration varied only a few percentage points between and within the experiments .

IPSA for Monoclonal Characterization Approximately 1.5 x 10 5 DMS of in vitro translated βS4 labeled with 35S-methionine was added to various amounts of purified mnonoclonal antibody, either 369.2B, 369.6 or 286.8A, in a total volume of 100 μl of ex immunoprecipitation buffer, and they were allowed to react as described above. Immune complexes were precipitated with Omnisorb, washed, and counted as described above.

Example 6: Characterization of MAb 369.2B To better characterize the cell lineage, 369.2B, a competitive immunoprecipitation / flashing test was performed (Competitive IPSA). In this variation the 369.2B was added in a molar excess of approximately 200 times of unlabeled competitor peptide at the same time as the ßA4 1-42 translated in vitro labeled. As expected, the peptides for the human ßA4 region, 1-40, 1-11, 1-28, 12-28, as well as the inverted 40-1 peptide do not compete with the in vitro translated ßA4 labeled with 35S-methionine by immunoprecipitation, while peptide 1-42 complete yes (Figure 8). These results were corroborated in a format of Solid phase ELISA: the absorbed ovalbumin-coupled selection peptide containing the ßA4 region 35-42, as well as the peptide 1-42, compete while the 1-40 does not (Figure 9). The decreased competitiveness of peptide 1-42 compared to 35-42 coupled to ovalbumin may be due to conformational and / or solubility factors involving the antigenic determinant, or perhaps more simply to the pcular stoichiometry of conjugation (ovalbumin, a carrier with molecular weight of 45 kD compared to 4 kD for peptide 1-42, and having somewhere between 5-15 maleimide groups conjugable by the carrier). Figure 8 shows the Competition Immunoprecipitation / Flashing peptide for the determination of the epitope of MAb 369.2. The peptide competitor (5 μg) was mixed with translated Aβ4 in vitro (~ 1.5 x 105 DPM or ~ 200 pg) and then immunoprecipitated with 2 μg of 369.2, where; For i-ipnto e ßA4 i nmunoprecipitated = (dpms with MAb) - (dpms without or with preimmune control) (dpms of total ßA4 / reaction) Percents with a given MAb concentration would typically vary only a few percentage points between and inside the experiments. Figure 9 shows the tracing of the epitope of MAb 369.2 by competitive assay. C369.2 (50ng IgG / 100 μl) was preincubated with or without synthetic competing peptides (22 ° C, 1 hour), then subjected to Elisa against 35-42 coupled to Ovalbumin bound to the plate (200ng / well) . The percent of competence was calculated in relation to the binding of MAb in the absence of the competitor, that is to say where; * of competitor = (signal with / without competitor) - [(signal with competitor) - (background noise) | (signal with / without competitor) The solid frame is the peptide conjugate 35-42 (OVA); the open box is peptide 1-42, and the solid diamond is peptide 1-40. From this data it is concluded} That the monoclonal 369.2B is specific for the C-terminal end of the full-length ßA4 (1-42) peptide. Although the exact antigenic determinant has not been mapped finely, it clearly involves residues beyond position 40 and, since the antibody was made for a short synthetic peptide the determinant probably does not involve other ßA4 residues that can be juxtaposed conformationally. Specifically, 369.2B is a very important tool for recognizing ßA4 species that end at position 42. An additional and interesting observation of the peptide competence assay is the increased immunoprecipitability of ßA4 translated in vitro by decapeptide 25-34. This phenomenon has also been observed in assays using another monoclonal antibody (i.e., 286.8A) as well as a polyclonal rabbit antiserum (data not shown). It is also known from other experiments using various amounts of detergent, specifically SDS, in IPSA assays with MAb 286.8A, which can immunoprecipitate more ßA4 by increasing the amount of detergent (data not shown). The SDS, interestingly, has shown not to be effective to solubilize the aggregates of ßA4, at least according to what is shown by the SDS-PAGE (Burdick et al., 1992). However, it is not immediately clear why SDS could increase the immunoprecipitability of ßA.

Example 7: Immunohistochemical studies We included immunohistochemical studies with 369.2B. The staining pattern of 369.2B (1 / 10,000 dilution of purified ascitic antibody solution of 22 mg / ml) when compared to the monoclonal antibody 286.8A which has been shown to recognize the 3-8 epitope of ßA4 and is specific to the human (data not shown) showed interesting differences. The results obtained by immunohistochemistry show that 369.2B is an excellent antibody (at a dilution of 1 / 10,000) to specifically mark the nuclei of amyloid plaques, diffuse amyloid deposits as well as fibrillary and vascular amyloid deposits (Figure 10). Figure 19 is a photomicrograph showing the ß-amyloids, blood vessels and perivascular deposits of ßA4 in a 10 μm thick slice submerged in paraffin from the brain of a 76-year-old female patient with Alzheimer's disease. The tissue sections were pretreated with 88% formic acid (30 minutes), then labeled using an avidin-biotin-peroxidase kit (Bector Laboratories, Burlington, CA) with nickel diaminobenzidine as the chromagen. The monoclonal antibody 369.2B marks plaques with a variety of morphologies, including nucleated, perivascular and diffuse (non-amyloidotic) plaques. It also marks a set of amyloidotic blood vessels. The studies also showed that ßA4 1- 43 peptide was not able to compete for staining (an excess of peptide more than 1000 times), while ßA4 1-42 completely abolished the signal (Table 2). Again as expected, the 1-40 or 40-1 does not compete for staining. From these studies it can be easily concluded that this antibody is an excellent tool for immunohistochemistry. As suggested by in vitro studies which show physicochemical differences between 1-40 and 1-42, it is possible that these two species of ßA4 show different patterns in the brains with Alzheimer's disease. With the monoclonal antibody of the present invention, we are now able to address this issue. Thus, the monoclonal antibody and the method of the present invention are useful for diagnostic and therapeutic uses in all immunological and related methodologies that can be applied to the detection, verification, extraction, inhibition and modification of the unique ßA4 species, in the diagnosis and treatment of AD.

Table 2 Monoclonal Antibody Used for Staining Peptide competent MAb 286.8AN MAb 369.2BC terminates 1 terminal None / buffer +++ +++ None / DMSO +++ +++ "40-1" Human +++ +++ "1- 16"Human - +++" 1-16"Mouse +++ +++" 1-40"Human _ +++" 1-42"Human" 1-43"Human +++" 35-42"Human with separator +++ Table 2. Results of competitive binding and stain inhibition experiments, +++ indicate strong staining, - indicates detectable staining. It should be understood that the foregoing description emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Konig, Gerhard Graham, Paul (ii) TITLE OF THE INVENTION: Monoclonal Antibody Specific for the Peptide ßA4 (iii) SEQUENCE NUMBER: 3 (iv) ADDRESS FOR CORRESPONDENCE: (A) ADDRESS: Allegretti & Witcoff, Ltd. (B) STREET: 10 South Wacker Drive Suite 3000 (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States (F) ZIP CODE: 60606 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION FROM THE MANDATORY / AGENT: (A) NAME: McDonnell, John J (B) REGISTRATION NUMBER: 26,949 (c) REFERENCE / DOCUMENT NUMBER: 95,216 [ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 312-715-1000 (B) TELEFAX: 312-715-1234 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l Cys Asp Gly Asp Gly Asp Met Val Gly Gly Val Val He Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Cys Gly Gly Gly Gly Gly Met Val Gly Gly Val Val He Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: excision site (B) LOCATION: 4..5 (D) OTHER INFORMATION: / level = Beta / note = "Beta splice site in APP" (ix) CHARACTERISTICS: (A) NAME / KEY: cleavage site (B) LOCATION: 20..21 (D) OTHER INFORMATION: / level = Alpha / note = "Alpha cleavage site in APP, waste 16/17 of ßA4". (ix) CHARACTERISTICS: (A) NAME / KEY: cleavage site (B) LOCATION: 46..47 (D) OTHER INFORMATION: / level = Gamma / note = "Gamma cleavage site in APP" (ix) CHARACTERISTICS: (A) NAME / KEY: Peptide (B) LOCATION: 5..47 (D) OTHER INFORMATION: / level = ßA4 / note = "ßA4 peptide" (ix) CHARACTERISTICS: (A) NAME / KEY: Region (B) LOCATION: 33..56 (D) OTHER INFORMATION: / level = Tm / note = "Transmembrane region of APP" (ix) CHARACTERISTICS: (A) NAME / KEY: Region (B) LOCATION: 1..32 (D) OTHER INFORMATION: / level = Ex / note = "N-terminal extracellular part of APP" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3; Glu Val Lys Met Asp Wing Glu Phe Arg His Asp Ser Gly Tyr Glu Val 1 5 10 15 His His Gln Lys Leu Val Phe Phe Wing Glu Asp Val Gly Ser Asn Lys 20 25 30 Gly Wing He He Gly Leu Met Val Gly Gly Val Val He Ala Thr Val 40 45 He Val He Thr Leu Val Met Leu Lys Lys Lys It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (20)

1. A monoclonal antibody that binds to the ßA4 peptide associated with diffuse amyloid, fibrillar amyloid, neurofibrillary tangles, and vascular amyloid, characterized in that the ßA4 peptide contains a free carboxy terminal amino acid corresponding to amino acid 42 of the amino acid sequence of the ßA4 peptide.

2. The monoclonal antibody according to claim 1, characterized in that it recognizes the C-terminal amino acid 42 of the βA4 peptide (SEQ ID NO: 3).

3. The monoclonal antibody according to claim 1, characterized in that it is an antibody of the IgG class.

4. The monoclonal antibody according to claim 1, characterized in that it is an antibody of subclass IgGl or IgG2b.

5. A monoclonal antibody, characterized in that it is identified as 369.2B, and is produced by the cell lineage deposited with the American Type Culture Collection (ATCC) with the accession number HB 11829.

6. An inmonulogically active fragment, characterized in that it is a fragment of the monoclonal antibody according to claim 1.

7. An immunologically active fragment, characterized in that it is a fragment of the monoclonal antibody according to claim 5.

8. A cell lineage, characterized in that it is identified by the access number ATCC HB 11829.

9. A method for detecting the presence of the ßA4 peptide in tissue, characterized in that it comprises contacting a tissue sample with the monoclonal antibody according to claim 1, and detecting the presence of the monoclonal antibody.

10. A method for selectively purifying the ßA4 peptide, characterized in that it comprises contacting a sample to be purified with the monoclonal antibody according to claim 1, separating the ßA4 peptide from the sample to be purified, and isolating the ßA4 peptide.

11. A method for detecting the ßA4 peptide associated with Alzheimer's disease, characterized in that it comprises contacting a sample to be tested with the monoclonal antibody according to claim 1 and detecting the presence of the ßA4 peptide.

12. A method for detecting the presence of the ßA4 peptide in tissue, characterized in that it comprises contacting a monoclonal tissue sample according to claim 5 and detecting the presence of the monoclonal antibody.

13. A method for selectively purifying the ßA4 peptide, characterized in that it comprises contacting a sample to be purified with the monoclonal antibody according to claim 5, separating the ßA4 peptide from the sample to be purified, and isolating the ßA4 peptide.

14. A method for detecting the ßA4 peptide detected with Alzheimer's disease, characterized in that it comprises contacting a sample to be tested with the monoclonal antibody according to claim 5, and detecting the presence of the ßA4 peptide.

15. A method for preventing aggregation of the ßA4 peptide, characterized in that it comprises administering the monoclonal antibody according to claim 1.

16. A method for preventing aggregation of the ßA4 peptide, characterized in that it comprises administering the monoclonal antibody according to claim 5.

17. Means for detecting the presence of the ßA4 peptide, characterized in that they comprise an immunologically reactive fragment of the monoclonal antibody according to claim 1.

18. Means for detecting the presence of the ßA4 peptide, characterized in that they comprise an immunologically reactive fragment of the monoclonal antibody according to claim 5.

19. Means for preventing the aggregation of the ßA4 peptide, characterized in that they comprise an immunologically reactive fragment of the monoclonal antibody according to claim 5.

20. A method for generating the antibody according to claim 1, characterized in that it comprises immunizing a mammal with the peptide CDGDGDMVGGWIA (SEQ ID NO: 1) conjugated with an immunologically suitable carrier.