CA2086165A1 - Diagnostic assay for alzheimer's disease based on the proteolysis of alzheimer's precursor protein - Google Patents

Abstract

ABSTRACT
A method useful in the diagnosis of Alzheimer's Disease in a patient in which an amyloid protein precursor (APP) substrate is combined with a sample of cerebrospinal fluid or blood obtained from the patient to be tested, and proteolytic cleavage of the APP substrate is detected. The absence of detectable proteolytic cleavage, or the detection of a substantially lesser degree of proteolytic cleavage, in the presence of the patient's sample compared to that detected when an APP substrate is combined with test samples from control individuals, indicates affliction with Alzheimer's Disease. Convenient test reagents and kits for aiding the diagnosis of Alzheimer's Disease are provided, such as comprising an APP substract and immunoreagents for detecting a fragment formed by proteolytic cleavage as well as chromogenic APP substrates.

Description

A DIAGNO.SIIC .~SSAY FOR ALZliEIMEX ' S DISEASE
BASED ON THE PROTEOLYSIS OF T~IE PMYLOID PRECUKSOR r~iO'l`EII`I

B~Cl~(~ROIJNO

Th~ inv~ntion relates to methods of diagnosis for Al~heimer's Di~sase in humans~ More particul~rly, th~ present invention i~ a diagnost~c a~s~y b~sed on the det~ctlon o~ proteolysi~ of the precursor to the Alzheimer's Disease beta-amyloid protein in the presence of a sa~ple of cerebrospinal fluid or blood obtained from a patient to be tested.
Alzhei~er's Disea~e (hereinafter "AD") is a progressive, degenerative disorder of the ~rain, characterized by progressive atrophy, usually in the frontal, parietal and occipital cortices:
The clinical ~anifestations of AD include progressive ~emory impairments, loss of lallguage and visuospatial skills, ~nd behavioral deficits (McKhan et al., 1986, Neurology 34:939-944).
Ov~rall cognitive i~pairment is attributed to de~eneration of neuronal cells located t2lroughout the cerebral hemisphere~ (Price, 1986, Annu. Rev. Neurosci. 9:489-5120).
Pathologically, the primary distinguishing features of the post-mortem brain of an AD patient are, 1) pathological lesions comprised of neuronal perikarya containing accu~ulations of neurofibrillary tangles; 2) cerebrovascular amyloid deposits; and 3) neuritic plaques. Both the cerebrovascular amyloid and the neuritic plaques contain a distinctive peptide simply designated, "A4" or "beta-amyloid".
Beta-amyloid is an insoluble, hi~hly aggragating, small po~ypeptide of relative molecular mass 4,500, and is composed of 39 to 42 amino acids. Kang et al., 1937, Nature 325:733-736, described the beta-amyloid protein as originatir.g from and as a part of a larger precursor protein. ~o identify this precursor, a full-length complementary DNA clone coding for the protein was 2 ~

isolated and sequenced, using oligonucleotide prob~s desi~n~
the known ~eta-amyloid sequence. Th~ predicted pr~cur~or contained 695 residues and is currently designated, ~A~P 6~5" (.~l~rlr,i precursor protein 695).
APP 69' is the most abundant form of APP ~ound in the hu~an brain, but three other for~s exist, APP 714, APP 751 and APP 770.
The ~ifferent length ~oforcs arise from alternative splicing from a single APP gene locatQd on human chromosome 21 (Goldga~er et al., 1987, Science 2~ 77-880; Tanzl et al., 1987, Science ~ 880-885).
Subssquent cloning of the gene encoding the APP proteins revealed that the A4 region ~as encoded on two adjacent exons, ruling out the possibility that A4 accumulation is the result of direct expression of an alternatively spliced ~RNA. This implied that A4 accumulation must result from abnormal proteolytic degradation of the APP at sites both N- and C-te~minal to the peptide region within the APP.
Recent studies have shown that APP fragments extending from t~e N-terminus of A4 to the C-terminus of the full lenqth APP
molecule (referred tc hereinafter as the "C-100 fragment", because it is comprised of a~proximately 100 amino a~ids) are also capable of aggregation (Wolf et al., 1990, EMBO 9:2079-2084). F~rthermore, overexpression of C-100 fragments in transgenic mice results in the accumulation of neurofibrillar~ tang}es and neuritic plaque co-incident with neuronal degeneration (Kawabata et al., 1991, Nature 32~:476-478). Collectively these data suggest that a single proteolytic cleavage of APP at the N-terminus of the A4 region is sufficient to initiate the pathophysiology associated with AD.
APP is also cleaved at a site within the A4 region in the physiological pathway for secretion of the APP extracqllular domain ~Esch et al., 1990, Science 248:1122-1124; Wang et al., ~991, J.
Biol. Chem. 266:16960-16964). This pathway is operative in several cell lines and necessarily results in the destr~ction of the A4, amyloidic region of the precursor. Evidence that such a pathway is ~ 0 8 6 1 ~ ~

also operative in the human brain ~as b~en obtcine(~ rt ~t ,11, 1989, 8iochem. F3iophys. Res. Comm. L6S:1~2-188).
The ~nzymes responsible for th~ nol~al, n~n-p~th~
processing of APP have been termed 'sccretases". C-ter~ln~l fragments resulting from secretase action ar~ smaller than th~ C-100 fr~gments (deined above) by 17 amino acids, and will hereina~ter bo referred to as the "phy~iological C-terminal ~r~ent. n It has been postulated that ~he ne~ pathologlcal accumulation o~ A4 i~ controll~d ~y tile relative activity of the pathologic and physiologic pathways of APP degradation. It is uncertain whether th~ i~balance resulting in t~e dramatic increase in accumulation of A4 in the brains of AD patients results from a decrease in the activity of tn* secretases or an increase in the pathologic protease activi~y, or a comblnaticn of both.
Sever~l studies have undertaken the purification and characterization of both the secretases and purported pathologic proteases. Initial studies utili~ed assays featuring synthetic peptide substrates that only mimicked the expec_ed cleavage sites within APP. These assay~ failed to provide thc necessary protease speci~icity, and the peptidase activities thus quantified were used without success to pursue the purification of candidate APP
processing enzyme activities from human brain tissue. To date, no credible candidate protease(s) for either process have emerged, the results of the variou~ studies have been conflicting.
For exam~le, the numerous available studies have proposed that the pathologic protease is: a lysosomal cathepsin, Cataldo,et al., 1990, Proc. Natl. Acad. Sci VSA 87:3861-3865; a calcium dependent serine protease, Abrahams et al., 1990, J. Neuropathol. Exp.
Neurol., 49:333 (abstract); Calpain I, Siman et al., 1990, J.
Neuroscience ~Q:2400-241l; a multicatalytic protease, Ishiura et al., 1989, FEBS. Lett. ~:38~3-392; or a chymotryptic-like serine protease, Nelson et al., 1990, J. Biol Chem. 265:3836-3843.
Similar inconsistencies have arisen in the efforts to identify 2 ~ 5 the secretase, which has been claimed to be: a metallo-p~ptid~-e, McDer~ott et al., 19~1, Biochem. Biophys. Res. Comm. 179~
1154: an ~cetylcholinesterase associated protease~ S~all et al., 1991, Biochemistry lQ:10795-10799; or Cathepsin B, Tagawa et al., Biochem. Biophys. Res~ Co~. 177:377-387.
The gen~ral lack o~ succe~s of past and current efforts to identi~y the nature of t~e APP processing enzy~es haYe stem~ed from po~r specif~city of t~e assays employed, and from the complex heterogeneity of proteases associated with the cerebral tissue.
The present lnvention arose fro~ efforts ai~ed at identifying the APP processing en2ymes using speGific assays ~ased on the proteolytic degradation of recombinant APP. It has been di~covered t~at such assays have utility in the diagnosis of AD by the u~expected findi~g o~ substantially different levels of APP
degrading enzyme activity in samples of cerebrospinal fluid (hereinafter ~CSF~) obtained from AD patients compared with healthy controls.
The present invention is an in vitro assay ~or detecting AD~
related di~ferences in the levels of proteolytic enzy~e activity sp~cific ~or ~PP 695 in a body fluid derived from a patient. It has been unexpe~tedly discovered that CSF derived from AD patients contain no detectable levels of protease activity or significantly and con~istently low~r }evels of protease activity than corresponding control samples of CSF derived from non~AD
individuals. Thus, this assay is suitable for use as a diagslostic for AD in humans, and would provide means of early detection as required for more effective early therapeutic intel~ention.
Based on available knowledge and data prior to the present disclosure, the logical expectation is for relatively increased protease activity resulting in C-100 fragments for fluids continuous with the CNS from AD patients. The results disclosed herein show the opposite, CSF from non-AD subjects show relatively and consistently higher enzyme activity resulting in the C-100 fragments.

2 ~

Presently, th~ only means f~r conclusive confirmation of clinic~l diagnosis is po~t mortem examin~tion of the brains of diagnosed patient~ for the presence of ce~ebrovascular amyloid deposits, neuritic plaques and neurofibrillary tangles. By contrast, the present assay can be perforMed on body fluid samples derived fro~ live p~tients, to quantify proteas2 activity.
Ot~er reports of bioche~ic~l differences between control ~nd AD patients which may be of potential diagnostic utility are known including, the detection of dermal amyloid deposits in AD using radioactive iodine substituted derivatives (U.S. Patent No.
5,039,Sll); elevated plas~a alpha-l-anti chymotrypsin levels in AD
(Matsubara et al., 1990, Ann. Neurol. X~:S61-567~; and the presence of i~munochemical markers such as AS8, a 68 kDa protein sxpressed by degenerating neurons and d~tectable with the monoclonal antibody ALZ-50 (Wolozin et al., 1986, Science, ~ 648-650).
U.S. Patent No. 4,874,694, describes a method of testlng CSF
for non-sp~cific peptides suscept~ble to protein kinase C -ph~sphorylation. Protein ~inase C activity deviating ~rom norm is said to be an indicator for a wide ~ariety of neurological or psychiatric disorders, without anv particular specificity.
Thus, there is a need in t.he art for a convenient diagnostic method which can con~irm clinical indications of AD prior to the death of the patient. The large qualitative differences in acti~ities ~btained betweer, control and AD patients in the present invention provides for a reliable clinical diagnostic method. ~he differences depend on enzy~atic activity which offers the potential for further signal amplification by increasing the time period of incubation of the enzyme with the APP substrate.
Additionally, the format of the presently disclosed assays afords the capacity to process reasonably large numbers of samples and yields good sensitivity due to the immunochemical method of detection. Furthermore, the simplicity of the assay allows for ready adaptation for routine use by lab technicians and yields consistent, reproducible results. These and other improvements are 2 ~ `3 described hereinbelow.

~XHARY OF INV2NTIO~

The prPsent invention provides a method for use in the diagnosis o~ Alzheimer's Disease in which a sample of cerebrospinal fluid or blood taken fro~ a patient (hereinafter referred to as the "test samplen) is comblned with an APP subs~rate and proteolytic cleavage of the APP s~bstrate in the presence of said sample is detected. It has been found tha~ test samples obtained from individuals known ~o be afflicted with ~lzheimer's ~isease lack a charac.teristic APP proteolytic activity exhibited by test samples obt~ined from control individuals.
The method can be per~ormed un~er conditions in which essentially no detectable proteolytic cleavage is produced in the presence of test samples from AD individuals, whereby the absence o~ detectable proteolytic cleavage in the presence of the patient sample indicated afliction wi~h Alzheimer's Disease.
Proteolytic cleavage of ~he APP substrate can be detected in ~ number of convenient manners, including the detection of polypeptide or pep~ide fragments produced by proteolys.is of the substrate. Such fragments can be detected by any convenient means, such as by antibody binding. Another convenient method for detecting proteolytic cleavage is through the use of a chromogenic APP substrate whereby cleavage of the substrate reagent releases a chromogen, e.g., a ~olored or ~luo:escent, product.
Accordingly, ~he present invention further provides a test kit for use in the testing o~ a cerebrospinal fluid or blood sample of a patient as an aid in the diagnosis of Alzheimer's Disease, comprising one or more containers holding ~a) an APP substrate, and (b) an antibody re~g~nt, preferably comprising a detectable label such as an enzyme, which binds with a polypeptide or peptide fragment which is characteristic of proteolytic cleavage which occur~ in the presence of samples from control individuals, but 2 ~

which ls su~stantially not present in, or w~ich is prl sf r.t at substantially lower l~vels in, test samples of individ afflicted with Alzheimer's Disease.
Alternatlvely, the present invention provides a chromogenic substrate for use in the testing of a tcst sample from a patient a~;
an nid in the diagnosis o~ Alzheimer's Disease. Such chromogenic ~ubstr~te i8 for.~ed of an APP substrate portion linked to ~
c~romogenic indicator portion through ~ ~eptide linkage t~at is cleavable by protease present in cer~brospinal fluid or blood of control individuals but which i~ sub~tantially not cleav~ble, or cleavable to a substantially les er degree, in the presence of cerebrospinal fluid or blood of individuals afflicted with Alzheimer's Disease. Pre~erably, cleavage of such chromogenic APP
substrat~ r~leA~es an colored ind.ic~tor moiety.

~RI~F D~8CRIP~ION OF TH~ FIG~RE

Fi~ure 1 show~ three gels which represent typical i~munoblot analysis results ~or the present invention. The results depict the relative capaci.ties o~ CSF, from contro] and Alzheimer's Disease patients, to catalyze the in vitro degradation of amyloid precursor protein to yield C-terminal APP fragnents (denoted by the lower arrows in gel~ a,b, and c). The proteolytic activities of nine A~
and five control patients are compared. The results sho~ that while C5F from four o~ the five control cases contained detectable proteolytic activity only one of nine AD CSF samples possessed the same activity.

DE8CRIPTION OF T~ I~V~NTION

The present invention is based on the unexpected finding that cerebrospinal fluid of individuals afflicted with AD contain measurably lower levels of, or under particular conditions no detectable levels of, APP protease (or proteases with APP protease-2 ~ o ~

li~e activity) relative to the level of APP prot~o1ytie activity ir,cerebrospinal fluid of healthy or con~rol i~dividuals. rOr purposes hereof, control individuals are thos,e who do not manifest clinical or pathological indications of A~. The phenomenon responslble for such decrease in APP proteolytic activit~ in AD
test samples is not clear.
It ~ po~tulated that Alzhei~er'~ Di~ea~e 1s due to a dQ~l~iency in th~ levels of the secretase en~yne which serves to cleave APP in such a fash$on as to render it innacessible to the pathway of amyloido~is. In this regard it is possible that the enzy~ic actlvity measured in the present invention corresponds to the for~ation of the physiological C-ter~inal fragment by the secretase. This conclusion however is mzde less 1ikely by the fact t~at the physiologic ~ra~ent would be appro~imately 17 amino acids ~mall~r than C-~OO and would not be expected to co-migrate with reco~bin~nt C~100, as has been obse~ved in the present invention.
In any event, the unexpected observation provides the basis for a c~nvenien~ and ~ersatile method useful as an aid in the di~gnosis of Alzheimer's Di~ease. Moreover, the finding is extendable to the testing o~ blood samples, particularly samples of peripheral blood, including plasma and serum, since various isoforms of APP are found in a variety of blood components, most notably platelets. Although obtainable by more invasive ~eac;ures, cerebrospinal fluid may be the preferred test sample due to the presence of higher level~ of APP proteolytic activity in normal CSF, as well as having to deal with protease inhibitors appearing in blood.
The APP substrate used in the present method comprises a site for the differential proteolytic cleavage observed between control individuals and AD patients. Accordingly, such APP substrate can be provided as a test reagent in a variety of forms. Although preferably derived from, or corresponding at least in part with the amino acid sequence of, APP 695, derivatives or analogs o~ other APP iosoforms (supra) are contemplated for use in the present 2 ~

method as well. APP 695 can be obtzlLned by biochen~ic~ olat~n or pu~ification from natural sources such as described in Schu!~ rt et al., 1989, Proc. Natl. Acad. sci. ~6:2066; or by exprcssion of:
reco~binant DNA clones encoding the protein or a functional portion thereof (Rnops et al., 1991, J. Bio. Che~. 2S6:7285; Bhasin et al., 1991, Proc. Natl. Acad. Sci. 88:10307~.
As used herein, "APP substrate" ~h~ll mean ~ull length APP, whether derived by isc)lation or purification from a biological source or by expression of a cloned gene encoding APP or its analogs, and fragments of any such protein, including fragments obtained by digestion of the protein or a portion thereof, fragoents obtained by expression of a gene coding for a portion of the APP protein, and synthetic peptides ha~ing amino acid se~uences corresponding to a portion o~ the ~PP pro~ein. The aforesaid frag~ents of the APP protein will comprise a secluence of amino acids sufficient for recognition and cleavage by the pertinent proteolytic test sample activity (supra).
Isolation of APP from biological material usually will involve puri~ication by conventional ~echnic~es such as chromatography, partie~larly affinity chromatogsaph~. Purified APP can be used to prepare monoclonal or polyclonal antibodies which can then be used in a~finity purification aecording to conventional procedures.
Resulting purified APP material can be fur~her processed, e.g., fragmented, by chemi~al or en~ymatic digestion. Useful fragments will be identifi~d by screening for desired susceptibility to the pertinent proteolytic test sample activity ~supra)~
As previously stated, the APP substrate can also be prepared by expression of recombinant ~NA clones coding for APP or a portion thereof. The cloned APP gene may itself be natural or synthetic, with the natural gene obtainable for cDNA or genomic libraries using degenerate probes based on known amino acid sequences (Kang et al~, 1987, Nature 325:733). Other techniques for obtaining suitable recombinant DNA clones, as ~ell as methods for expressing the cloned gene, will be evident to the worker in the field.

2 ~1 8 ~ 3 Furthermore, the APP substrate can be prepared to a con~eni~rl size by conventional peptide synthesis in correspondence ~ith the deduced amino acid sequence of the desired AP~ i50tor'm. ] n particular, based on amino acid analysis, overlapping peptides carl be synthesized and tested for the pertinent proteolytic susceptibility. As useful peptides are found, smaller peptides can be prepared in order to ~ap small~r reacting uni~s, if deYirable.
A vari~ty o~ convenient methods are applicable to the detection o~ proteolytic cleavage of the APP s~ostrate in the presence of the test sample~ Several of the presently ~ore preferred methods are described below, however, it will be reco~nlzed by the skilled worker in the field that many other methods can be applied to this step without departing from the inventiv~ features hereof. In general, any method can be used for tbis purp~se which is capable of detecting the occurrsnce of proteolytic cleavage of the APP substrate. Such can be afforded by appropriate design of the APP substrate such that cleavage produce~
a signal produeing species, e.g., an optically responsive product sueh as a eolored or fluorescent dye. Another prineipal approaeh involves the sensitive detection of one or more cleavage products such as by immunoassay. Such eleavage product or products to be detected is that whieh is eharacteristically produced by reaction of the APP subs~rate with test samples from control individuals eo~pared to those with AD~ Presently, such cleavage product is preferentially a C-terminal ~rag~ent of the APP substrate: however, any fra~ment which appears upon incubation with control samples b~t which is absent from, or appears in substantially lesser amounts in, as assay mi~ture comprising an AD test sample can be the object of detection.
The detection of one or more cleavage products characteristic of the differential proteolytic activity observed in control samples compared to AD samples, can be accomplished in many ways.
One such method, which is further exemplified in the examples which follow, involves the procedure commonly known as Western blot.

2~8~

Typically, after the incubation of ~>P wi~h te t 5a electrophoresis is performed to separ-~te the componerlts re-;ultin~
in the reaction ~ixture. The separated protein components a~-~ th~n transferred to a solid matrix 5UCh as a nitrocellulos~ ~t~brarle.
An antibody specific to a fraqment characteristic of control versus AD individuals is then reacted with the components fixed to the membr~ne and detected by ad~ition of a secondary enzy~e-labeled antibody coniugate. ~he location o~ the resulting bound conjùgate is developed with a chromogenic substrate ~or the enzyme label.
A variety of immunoassay formats which are a~enable to currently availablo diagnostic test systems can also be applied to the detection of APP fragments. Typically, the APP substrate will be incubated with the test sa~ple and resulting intact APP rendered i~obilized (suc~ as by capture onto a solid phase), or alternatively, the test sample is incubated with an immobil:ized form of the APP substrate. Proteolytic cleavage characteristic of the control population compared to AD individuals is then detected by reacting the im~obilized APP substrate with an antibody reagent directed to a portion o~ the APP substrate which is cleaved from the APP substrate or which defines the cleavage site. The antibody reagent can compr.isc whol~ antibody or an antibody fra~nent co~prising an antigen combining site such as Fab or Fab', and can be of the monoclonal or polyclonal type. The detec~ion of antibody reagent bound to the immo~ilized phase is indicative of the absence o~ the characteristic proteolytic cleavage and thus indicates affliction with AD. The detection of binding of the antibody reagent will generally involve use of a labeled for~ of such antibody reayent or use of a second, or anti-~antibody), antibody which is labeled.
Capture or immobilization of APP can be accomplished in many ways. An antibody can be generated specific to an epitope of APP
which is not on the cleavable fraqment. Such an antibody can be immobilized and used to capture or immobilize intact APP.
Alternatively, a ligand or hapten can be covalently attached to APP

~ a2 -2 ~ 3 and ~ corresponding immobilized receptor or ~ntihody c/~lt~ u ~I-J~
capture or immobilize APP. A typical lig~nd:receptor pair u~cf~l]
for thi~ purpose is biotin:avidin. Examples of haptens ~s~ful for thi~ purpo~e are fluorescein and digitoxigenin.
The solid pha~e on which the APP substrate i~ immobilized or captured can be compo~ed of a variety of materials including ~icrotiter plate wells, te~t tubes, strips, beads, particles, and thQ lik~. A particularly useful ~olid ph~e i8 magnetic or paramagnetic particles. Such particle6 can be derivatized to contain chemically active groups that ca~ be coupled to a variety o~ co~pounds by si~ple chemical reactions. ~he particles can be cleared from suspension by bringing a magnet close to a vessel cont~ining the particles. Thus, the particles can be washed rep~tedly without cu~bersom~ centrifugation or filtrat.ion, providing the basis for ~ully auto~atin~ the assay procedure.
Labels ~or the primary or secondary antibody reagent can be sel~ctad from those well known in the art. So~e su_h l~bsls are fluorescent or chemiluminescent label6, radloisotopes, and, ~ore preferably, en~ymes for this purpose are alkaline phosphata~e, peroxidase, and B-galactosidase. These enzymes are stable under a variety of conditions, havs a high catalytic turnover rate, and can be detected using simple chromogenic substrates.
Proteolytic cleavage of the APP substrate can also be detected by chromatographic techniques which ~ill separate and then detect the APP fragments. High pressure liquid chromatography (HPLC) is particularly useful in this regard. In applying this technique, a ~luorescently tagged APP substrate is prepared. After incubation with the test sample, the reaction mixture is applied to the chromatographic column and the differential rate of migration of fluorescent fragments versus intact APP is observe~.

2 ~

~xam~l~ 1. Dav~lopm~ne o Chl~D ~u~t~r Ovary ~c~lol CHll lln~
e~proDo~nq ~eco~b~Dant ~PP ~5.

Y0ctor ooust~uatlo~. A known 2.36 Kb NruI/Spel fragment of APP
695 cDNA from FC-4 (Kang et al., sUPr~) was filled in by the large fragment of E. coli DNA polymerase I and blunt end inserted into the S~aI cloning sit~ o~ KS Bluescript M13+ ~Stratagene Cloning Systems, La Jolla, CA.) creating pMTI-5 (App 695 ~nder the T3 pro~oter), I new optlmal ~oz~k consensus DNA s~quence was then created u~ing site-specific mutagene~i~ tRunkel et al., 1987, ~L~ L~L~y~Q~ 367-3~2) with the oligo: s'-CTC~AGAACTAGTGGGTCGACACGATGCTGCCCGGTTTG-3' (SE~ ID NO: 1) to create PMTI-39. This plas~id was next altered by sit~ specific mutagenesis (Kunkel et al., ~.) to change the valine at position 614 to a glutamate (open reading frame numbering according to Xan~
et al., I~
The full l~ngth APP cDNA containlng the opti~al Kozak consensus sequence and Val to Glu mutation was then cut out of PMTI-39 with NotI and a HindIII partial digest. The 2.36 Kb APP
695 ~rag~ent was then gel purified and ligated into NotI/HindIII
cut pcDNAINeo (Invitrogen Corp. San Diego, CA.) to create PMTI 90 in which the APP 695 expression is placed under th~ control of the CMN promoter. ~he Val to Glu mutation was sequence confirmed and th~ vector used to stably transform CHO c~lls.

b) G~or~io~ of ~tnbl~ ~HO c~ hns ~pro3s~ng ~PP 5~5 ~utenes.
Chinese Hamster Ovary ~-1 cells (~TCC CCL 61) ~ere used for transfection with the APP 695 construct. Twenty micrograms of an expressiori plasmid containing APP 695 and a neomycin drug resistance marker was transfected into lx107 C~O cells in 0.5 ml PBS by electroporation using a ~io Rad Gene Apparatus (Bio-Rad L~boratories, Richmond, CA.). A single pulse of 1200 V at 25 yf capac.itance was administered to the cells.

Following electroporation, cells were incubdt~d in icc f~r minutes and collected by centrifugation. The cell pell~t ~
res~spended in Alpha MEM, 10~ fetal calf serum at a density of SxlO~ cells/ml, and 1 ml aliquots were distributed into e.~ch w~
of ~iva 24-well tissue culture cluster plates. After 48 hours incubation, c~lls containing the neomycin drug resistance marker were select~d by addition o~ 1 ml of media containing 1 mg/ml G~netic1J~ ~GI~CO-BRL, Grand X~land, NY.) and incubation wa3 continued ~nd bi-w~ekly changes o~ drug containing media.
Drug r~sistant cells were tested for APP 695 expression by Western blotting. Cells positive for APP 695 expr~ssion were cloned by llmiting dilution, and individual clones were isolated and tested ~or APP 695 expression. A clone positive for APP 695 expres~ion was subcultured and expanded into roller bottles for large scale pr~duction of APP 69S expressing cells an~ subsequent isolation of r~co~binan~ protein.

~a~pl~ 2. ~ovalop~nt ~ ~pr0s~1~D ~ector~ ~or tho pro~uc~on o~
~o~bl~nt ~ 100 st~ndRr~ by tran~l~nt i~gect~on o~ ~a~llan c~
The C-100 peptide fragment contains the C-terminal portion of APP which spans from t~le ~-teroinus of the A4 peptide to the C-terminus of full leng~h APP ~see above, BAC~GRO~ND section)~ The C-100 frag~ent is the purported initial degradation product leading to the ultimate formation of the A4 peptide.
In one embodi~ent of the present invention, cell lysates from }lela S3 cells ~ATCC CCL 2.2) expressing reco~`oinant C-100 are analyzed in the immunoblot assay in parallel with the recombinant APP samples that have been incubated with CSF (see Figure 1). The migration and detection of the C-100 fras~ents serves both as a size marker for the genesis of products formed by pathologic proteases as well as a positive control for the immunodetection of C-terminal APP fragments in general.
Comparison of the size of enzymically senerated products with ~ 9~ ~

the size o~ the c-loo fragment c~n provide in~hts into wheth~r not the en~ymically generated frdgments resu~t fro~l cl~avd~
to the N-terminus of the A4 peptide or alternative~y within the ~4 segment as would be c~talyzed by secr~t~se.

~) ~lh~1~ con~tru~tlon Two ~ethods wer~ u~ed to make plasmids for C-loO expression.
~ach pla~mi ~hall bc identified separately as either PMTI 73 or PMTI 100.

P~ 7~ Co~tructl~ns The co~ercially avail~ble plasmid PUC-~l9 was dige3ted with Eco~I to eliminate its polylinkers. Co~ercially available PWE16 was then inserted into the digested PUC-l9 to create PMTI 23Q0. PMTI 2301 was derived from PMTI 2300 following Ba~HI~E~ind III diges~ion using an oligonucleotide adapter. The EcoRI pro~oter fragment of APP was lnserted into th~ HindIII site Gf pMTI 2301 by blunt end ligation to produce PMTI 2307.
PMTI 2311 was g~nerated by ligating the BamHI fragment Prom FC-4 (Xang et al., supra) into the ~amHI site of PMTI 2307. The XhoI fragment erom FC-4 was inserted into the XhoI ~ite of PMTI
2311 to generate PMTI 2312. PMTI 2323 was generated by insertion o~ the 2.2 kb BglII/EcoRI fragoent from the EcoRI genonic clone of the ~ouse ~etallothionein-I gene into the ClaI site of PMTI 2312.
To generate minigene PMTI 233~, the sequences between the ECpnI and BglII sites of P~TI 2~23 were delet~d and the clone was ligated using synthetic oligonucleotide adaptor, sp-spacer-A4.
PMTI 2337 was cut with 8am Hl/SpeI and the fragment ligated into the B~m Hl/Xbal restriction sites of bluescript ECS (+) (Stratagene) to create PMTI 2371. PMTI 2371 was cut Hind III/NotI
to release a 0.7 kb fragment coding for the terminal 100 a~ino acids of APP 695. Also encoded ~as the sequence for signal peptide.
This insert was ligated into the Hind III/NotI site of pcDNAINE0 (Invitrcgen Corp.) to create the plasmid PMTI 73.

- 16 ~

2 ~ 8 ~

PYTI 100 C~n~tructl~s P~TI 90 (see Example I) wa~ cut XbaI/;~indIII
to rel~ase a 0.6 kb frag~ent again coding for the terminal loO
amino acids o~ APP 6gs ~nd this was ligated to the XbaI/HindIII
site of pcDNAINE0 to create PMTI 100. In each case vectors,inserts and pl~smid~ wQre purified by ~ethods know~ to those ~killed in the art.

b) Tra~ t~o~ ~nd ~pro~slo~ o~ C-100 ~rag~s~t~ Preparation for ~mall scal~ expr~ssion o~ C-100 ~tandarcl was initla~ed by seeding 5x105 cQll Hela Sl cell~ in each well of a 6 well costar cluster (3.5cm diamet~r) 24 hours before use.
Suf~icient v~ccinia virus vTF7-3 was trypsin treated to infect ~t a multlplicity o~ 20 plague forming units per cell, ~ixing an equ~l vDlume of crude virus stock and 0. 2S mg/ml trypsin, then vor~exed vigorously. The trypsin tr~ated virus was i~cubated at 37-~ for 30 minutes, witb vortexing a~ 10 ~inute in~ervals. Where clumps persis~ed, th~ in~uba~ion ~ixture was chilled to O'C and sonicated for 30 seconds in a sonicating water bat~. The chilled sonication was repeated until no more clumps were detected.
The trypsin treated virus was the~ diluted with suf~icient seru~ free DMEM for each well with Hela Sl cells to have 0.5 ~1 of virus. Medium was aspirated way, then the cells were infected with virus for 30 minutes, wi~h rocking a~ 10 ~inutes interYals to distribute the ~irus.
Approximately S ~inutes before in~ection was ceased, fresh transfection mixture was prepared as follows: To each well was added 0.015 ~1 lipofection reagent (Betheseda Research Labs, Gathersburg, MD.) to 1 ml OPTIMEM (Betheseda Research l,abs, Gathersburg, MD.) in a polystyrene tube, ~nixing gently. Vortex was avoided. T~en, 3 ~9 CsCl purified DN~ was added and mixed gently.
Virus ~ixture was aspirated from cells, then the transfection solution was introduced. The resulting mixture was incubated for three hours at 37-C. Each well was then overlaicl with 1 ml of 2 ~ g ~ ;3 OPTIMUM and incubated at 37 c ln a co2 incubator o~rniq~lt Cells were harvested at 20 hours post transfecti~n by centrifugation, and lys~tes were prepared on ice with the addition of 0.2ml of a lysis buffer which contained lS Triton X-lOo, lO
~/~1 BPTI, 10 ~g/ml Leupeptin, 200 mM Nacl, lO mM H~PES, 1 mM
CaCl2, 1 ~M MgCl2, 1 mM EDTA, ad~usted to pH 7.5. Complete lysis was monitored by ligbt microscopy, and harvested ~mmediately.
Lysis took less than l minute to complete, with delay at this step causing lysis of nuclei resulting in a gelatinous mess.
Reco~binant lysates were stored at -20-C for later use.
Prefera~ly, reco~binant lysates should be diluted 1:50, in (3X) ~S
PAGE sample buffer which is devoid of 2-mercapto ethanol prior to freezi~g.
A comparison o~ the size of the proteins produced by expression with either PMIT 73 or PMTI 100 using SDS--PAGE/
im~unoblot with the APP C-terminal antibody was performed. The study showed that PMTI 100 directed the expression of a single imnunoreactive band, wherease, PMTI directed the expression of three band~ of similar molecular si~e. The band of intermediate size was less intense when compared to the upper and lower bands, and co-migrated wit~ the single product band observed when using PMT~ 100 to direct the expression.
The largest of the three bands produced by PMTI 73 was slightly larger than the single band observed with PMTI lO0. Amino ~cid sequence analysis of the largest band from PMTI 73 expression showed that the signal peptide sequence was cleaved from the initial translation product to yield a C-lOo fragment containing 5 extra amino acids at the N-terminus. Although our initial results in example S showed the result of a study in which the protein product of PMTI 73 was used as a migration marker, use of the protein encoded by PMTI lO0 is preferred.

pl~ 3. Pr~p~ration o~ R~bbit polyclonal ~n~lser~.
Antisera were elicited to the C-terminal domain ot- ~u~an ~
69S, ~nd were prepared in accordance with the method as described in Buxbaum et al., 1990, Proc. Nat'l. Acad. Sci. 87:6003-6006. A
~ynthQtic peptide (hereina~ter ~B APP 645-694n) corresponding to ~ho CVOH-termin~l reqion ~f APP 695 was obtained fro~ the Yale Unlversity, Protein and Nucleic Acid Chemi~t~y Facillty, New H~ven, CT .
n APP 645-69~ was used to immunize rabbits to elicit polyclonal antibodies. Sera were ~creened by immunoblot analysis of lysates of E. coli that expressed a fusion protein including the amino acids l9 through 695 of human AP~ 695. Sera which were immunoreactive against the recombinant fusion protein were further ~creened for immunoprecipitating activity against [~S] methionine-labeled APP 695, which was produced from B APP 695 cDNA by successive in vitro transcription tkit purchased from Stratagene, La Jolla, CA~ and translation treticulocyte lysate ki~ purchased fro~ Promega Corp., Madison, WI).

~a~plo ~. Pur~f~catlo~ o~ APP 695 from ~oco~blnant C~O ~
All steps ~ere per~or~ed at o to 4-C unless indicated otherwise. Holo-~PP 6ss was detected by immunoblot analysis using an anti human APP 695 C-terminal antibody essentially as described in Example 5, below.

ol~tio~ of pl~a ~e~bra~e~. ~hole cell pellets (179 g) from continuous culture of CHO cells in roller bottles were collected ~y centrifugation (lSOOg X 5 min), and resuspended to a total volume of 600 ml in 50 ~M tris-HCl buffer pH 8.0 containing sodium chloride (30 mM), magnesium chloride (l mM), EDr~ (lO mM), PMSF (200 ~g/ml), E-64 (42 ~g/ml) and pepstatin (3.8 ~g~ml). The cells were homogenized using ~ te~lon potter (lO return strokes), then layered (25 ml per centrifuge tube) onto lO ml of ~$61 3:-~

homogeni~ation bu~fer containing 41~ sucros~ and devoid of th~
protease inhibitors EDrA, PMSF, E-64 And pepstatin. ~oll~bir;~;
centrifugation (26"300 ~PM X 60 min, in a Beckman SW--28 rotor, t.le interfacial layer was carefully removed (approximately 150 ml in combined volume), diluted with an equal volume of homogenization buffer ~minus protease inhibitor~ esuspended with a teflon potter (3 return strokes), and recentrifuged as described above to yield a tightly packed pellet. The supernatant was deeanted and the pellet resuspended in 100 ml total volu~e with 50 mM tris HCl pH 8.0 (teflon potter 3 return strokes). Recentrifugation (50,000 RPM X 60 mln in a Beckoan 70 Ti rotor), yielded a pellet which was resuspended to a total volume of 57 ml in 50 mM tris HCl, pH 8~0.

b) ~olubili~atio~ o~ Pla~ o~br~no3. Thirty seven milliliters of the above resuspended CHO plasma me~brane preparation were added ~equentially to a cocktail of protease inhibitors and stock 20%
(v/v) triton X-100 to ac~i~ve the ~ollowing component concentrations: E~TA (1 ~M), E-64 (24 ~g/ml), PMSF (53 ~g/ml), pepstatin A (11 ~g~ml), an~ triton X-100 (2.~% v/v, ~inal), in the homogenization buffer (total solubilization volume of 45 ml) described above. A~ter gently rocking of the mixture at 4 C for 30 min, the non-solubilized material was removed by centrifugation (50,000 RPM X 40 ~in in a Beckman 70 Ti rotor). The supernatant containing solubilized holo-APP was filtered through a 0 45 ~M disc filt~r.

a) Purification o~ aolubili~d holo-APP 695 by stro~g anion o~cha~g~ ahromatography. The above supernatant containiny holo-APP
695 was diluted with an equal volume of distilled water and applied to a mono-Q ~H 10/10 column previously equilibrated with 20 mM
tris-HCl buffer pH 8.0 containing 0.1~ triton X-100. Once loaded the column was eluted in a linear gradient of 0 to lM NaCl contained within a to,al volume of 210 ml of equilibration buffer.
The flow rate was maintained at 3 ml/min throughout. Proteins 2 ~

eluting between a conductivity range of 17 to 22 m~ o (4 C) contained the majority of immunoreactive ~PP 6'l5, and were co".l.ined and dialyzed for 4 hours versus 2L oF 5 mM tris-~Cl ~ P . ~) containing 0.025~ triton X-loO, and clarified to remove slight turbidity by cent~ifugation (26,800 x 60 min in a Beckman SW 28 rotor).

~) ~op~rl~ ag~ro~0 ahro~togr~phy. The clarified ~ample was ~pplied to a colu~n of heparin agarose (15 x 1.6 cm) previously equ~llbrated wi~h dialysi~ buffer. Upon loading a light brown band formed within the top 1~3 of the colu~n. Once loaded, S ~in fr~ctions were collected (a 1OW rate of 1 ml/~in was used throughout~. The colu~n was then eluted stepwis~ with 85 ml of equilibration in whic~ the sodium chloride was successively ~djusted to the following ~inal conc~ntrations: 0, 150, 300, 600, and 2000 ~M. The ~ajority of the immunodetectable holo-APP eluted at 600 mM NaCl, with the next quantitative fraction being recovered at 300 ~M. The APP recovered at 300 ~M and 600 mM NaCl were collected separately ~nd stored in dllquots at -80 C, The APP used in the following studies ~ere from the 300 mM fraction. The yield of partially pur~ ~PP from the 300 ~M h~parin agarose eluent was 5.5 ~q ~Bradford assay) per gram of w~t CH0 cell pellet.

~mpl- S. Tho i~munobl~t a39~y ~or the dotectic~ of dif~0rcnc~
~n th~ ra~at~ OD o~ ~PP 695 cataly~e~ by control an~ AD C8~.

~) I~cu~tlo~ o~ C8F Mlth APP:
i) CSF (5 ~l) can be incubated with substrate APP 695 for between 24 to 48 hours. For this example, the 48 hour incubation period was chosen, and the other incubation parameters were: at 37 C with 10.75 ~l of recombinant human APP 695, which was adjusted to 140 mM
inal in MES buffer pH 6.5 by the addition of the required amount of 2M stock buffer. The final buffer concentration in the incubation being 95 mM, pH 6.S. The final concentration of ~ 21 ~ 3 partially pure APP preparation and MES buffer a~ter mi~in~ with CS~
were 37 ~g/ml and 95 mM, respectively, in a total reaction volume of 15.75 ~ uring the incuhation time, proteolytic deqr~dation of some of the APP 695 occurs to yield lower ~olecular weight fragments.

ii) The proteolytic reaction was ~ermlnated by addition of 7.5 ~l, o~ tbe following 3X L~emlie SDS-PAGE sample bufPer: 1.5 M Tris HCl, pH 8.45, conta~ning 36~ (v/v) glycerol and 12% (v/v) SDS, lOS (v/v~
2-~rc~ptoethanol, and trace bromophenal blue tracXing dye.
Samples were heated (lOO-C X 8 min), and then cooled.

b) ~D~3 Pl~a~ nn~ly~
The reaction ~i~ture~ (15 ~l) were app}ied to the wells of a 10 to 20t acryla~ide gradient Tricine gel (routinely a 1.0 ~m ~bick, 15 well Novex pr~cast gel, No~ex Experimental Technology, San Diego, CA). The gel was run under constant voltage conditions, and at 50 V until the sample enters the gel whereupon the voltag~
was raised to 100 V. Electrophosesis was discontinued when the tracking dye reaches ~o within 0.5 cm of the gel bottom. The gels were calibrated using prestained Mr markers r~nging in Mr from 3 to 195 kDa ~Bethesda Research Laboratories, Gaithersburq, MD.). Ten microlitres each of a kit containing high and low mole~ular weight ~arkers were mixed with 10 ~l oP 3X sample buffer, and treated as de~cribed in section (~

~) ~ounoblotting:
i) The gel was then transferred to a mini trans--blot electrophoresis cell (Biorad Labs, Richmond, CA.). Proteins were electro-blotted onto a ProBlott (TM) membrane (Applied 8iosystems, Foster City, CA.), for 1 hour at loO v (constant), using the following transfer buffer maintained at 4 C:~0 mM Tris HCl buffer pH 8.5 containing 150 mM glycine and 20% (v/v) methanol.

il) The ProBlott membrane ~as removed an~ placed in lS mA o~
blockinq buffer of ~h~ following composition for 1 ho~r at r~o~i temperature: s~ (w/v) norl-fat dried milk in lo mM Tris HCl ~u!f~r pH 8.0 containing 150 mM NaCl.

a) ~mo~nod-tocti~ of a~P 8D~ C-t~ al degr~tlou pro~uct~:
The m~mbrane w~s transgerred to 15 ~l o~ blocking buffer containing ~ 1:1000 dllution of rabbit polyclonal anti~eruc ellcited to ~ Rynthetlc human APP 695 C-ter~inal peptide immunogen and incubated at 4-C over night.
T~e mQmAbrane was rinsed with three successive 15 ml volumes of blocking buffer with gentle shaking for S minutes. The me~brane was then tran~ferred to 15 ml of blocking buffer containing a l:1000 dilution of alkalinc phosphatase-coupled Goat anti-~abbit IgG (Fisher Scientific, Pittsburgh, PA.), and incubated at room temperature for 90 ~inutesO The ~embrane was ~hen rinsed with three successive 15 ~l volumes of blocking buffer with ~entle shaking ~or ~0 ~inutes.
The membrane was next washed with three consecutive 15 ml volumes of alkaline phosphatase buffer for 5 minutes each, comprising: iOO -~' Tris HC1 pH 9.5, containing 100 mM NaC' and S mM
MgClz. The gel was next incubated in the dark with 15 ml of lO0 rn~
Tris HCl pH 9.5, containing 100 r~M NaCl, 5 mM MgCl2 and 50 ~l of BCIP subctrate (50 mg~ml, Promega, Madison, WI.) and 99 ~l of NBT
substrate (50 mg/ml, Promega~. Incubation was continued until there was no apparent further intensification of low Mr immunoreactive bands ~typically 3 hours at room temperature).
The gel was then rinsed with deionized water and driecl. A
typical end result of such analysis is shown in Figure l, which is further tabulated in Tables 1, 2 and 3, and explained hereinbelow.

R~8ULT8 of ~ typical n~lysi~ co~paring ~ctivity in control ~ncl AD
C8F:
To examine potential differences between the levels of APP

2 ~

deqrading proteases in the CSF of oontrol and AD p~tl~nts, t~
following experiment was perforrned:
CSF from a total of nine t9) person~ diaqnosed with AD ~t post mortem (average age at death was ~7 years, range 69 to ~ years, average autolysis time is 4.38 hours, n=9), and five (5) persons dlagnosed as not havlng AD based on clinical assess~ent and post ~ortem ~x~minatlon (average age was 72 years, range 57 to ~5 years, n~4, average autolysis time is 3~5 hours, n~5) were individually testsd ~or APP d~grading activity using the assay conditions described in Example 5, above.
The ollowing controls were perfor~ed:
i) APP was incubated in the presenc~ of reaction buft`er and the absence o~ CSF to assess the e~tent of APP degradation arising ~rom trace proteases within the ~PP preparation itself ~Figure 1), lane 1 for each o~ gels a, b and c, see also Tables 1-3, below);
ii) cell lysate containing authentic reco~binant C-100 frag~ent derived by Example 2 was applied to each gel (Figure 1, lane 2 in each o~ gels a, b ~nd c, in an amount equivalent to that present in one microliter of origin~l Hela S3 cell culture;
iii) ~or each incubation containing CSF and ~PP, a corresponding incubation in which the APP was omitted was per~ormed to determine if the CSF contained endogenous immunoreactive bands which would complicate the interpretation of products derived fro~ recombinant ~PP proteolysis. This APP control was analyzed in an adjacent electrophoretic lane to the incubation containing the complete system.

.

sa~L~ ~ g~nd ~or ~g~ in ~lq~lr~ l) SAMPI~ AU~OI.Y',I
CASE CODE APP ~;95DIAGrlOSIS SEX AGE TIMr YRS ~ ~ !~S ) 1 - APP only 3 89-35 ~ APP AD ~78 9 89-34 ~ APP AD M80 4 7 89-29 ~ APP AD MA0 4 ~ 89-29 - ~PP
9 89 - 26 + APP AD M76 3 ll 89-2S * APP AD F78 5.5 13 89-08 -~ APP AD M75 3.5 Mr ~rkers*
*Mr ~arkers are: ~yosin H-chain ~196 XDa), phosphorylase B (106 kDa), bovine serum albu~in (71 kDa), Ovalbu~in (44 kDa), Carbonic anhydrase (28 kDa), be~alactoglobulin (18 kDa), lysozyme ~15 kDa) bovine trypsin inhibitor ~5.~ kDa) and Insulin A and B chains (3 kDa ) .
Note: The upper arrows in Figure 1 show the position o~
unco~verted APP 695 and t~e lower arrows s~ow ~e position o~
migration o~ the enzymically ~enerated band. Notice the absence oP
the lower Mr band in the AD c~ses, but its presence in 4 of 5 controls.

_ ;~5 _ TABL$ ~ (legend for "q~l b~ in Fl~ura 1) SAMPLE ~ ro~ Is CASE CODE APP 695 DIAGNOSIS SEX AGE TIM~
~ (YRS~ ~S~
1 - APP only 2 ~ C~100 3 90-05 + APP AD F 77 2.5 90-01 + APP AD F ~4 2.5 ~j 9~)-01 - ~PP
7 89-41 + APP AD M 69 5.5 8 Mr Mark~r~s*
9 89~41 - ~PP

~BL~ 3 (leg~nd for "g~l ~" in Figur~ 1) SAMPLE AUTOLYS~S
CASE COD~ APP 695 DIAGNOSIS SEX AGE TIME

1 - APP only 2 - C-l~O
3 89-43 * ~PP Control ~ 85 2 89-37 ~ APP Control M X 6 7 89-24 + APP Control M 71 2.5 8 89~24 - APP
g 89-22 + APP Control F 73 4 89-22 - ~PP
11 Mr Markers~
12 89-11 + APP Control F S7 3 13 8~ PP
X ~ The precise ago at death could not he deter~ined.
Mr Markers are the same as for I'able 1, above.

. - ~6 -2~$~

The results of tho experimental comparison ~ri? .~o~n in )~
1. The legends in Tables 1, 2 anl 3 provide, in dddition to the contents of each immunoblot lane, a detailed description of t~-patlent diagnosis, sex, a~e at death, and time after death whichelapsed before sa~pling of CSF (autolysis time). ~11 sc~mples were frozen either on dry ice or at -80 C as ~on as possible after r~oval and ctored fiC ~uch until ~naly~ed.
Thc arrow~ on Figuro 1 de~ignate the regions Or interest in the a~sessment o~ degr~dation of APP to low molecular cize C-terminal fragments. The upper arrow denotes the migration of the undegraded reco~oinant APP, which co-migrates with the phosphorylase ~ marXer suggesting an apparent size of around llo kDa. The lower arrow marks the position of migration of C-loO.
This fragment migrated slightly ahead of lyso~yme, suggesting an ~pparent molecular ~ass of around 14 kDa. In four out of five c~scs using CSF from di~f~rent control cases, incubation of the CSF
with APP caused the forDation of an immunoreactive band which co-migrated within the si~e range corresponding to recomhinant C-100 (Figure 1, gel c, lanes S, 7, 9 and 12).
By sharp contrast, this hand was found to be completely absent in eight of nine incubations using AD CSF (Figure 1, gel a, lanes 3, 5, 7, 9 and 11: and gel b, lanes 3, 5 and 7) and only faintly visible in the incubation of CSF from the ninth AD patient ~gel a, lane 13). The band is not present in any of the control incubations containing APP without CSF (lane 1, gels a, b and c) showing that it is not derived from proteolytic activities found within the APP preparation itself. Furthermore, none of the CSF
samples when incubated without recombinant APP showed the presence of the fragment (gel a, lanes 4, 6, 8, 10, 12 and 14; gel b, lanes 4, 6 and 9; gel c, lanes 4, 6, B, 10 and 13), showing that the fragment had to be derived from the breakdown of the recomhinant APP.
Thus, the data shows a significant difference between control ~ ~ g ~

and AD CSF in th~ levRla oP thla C-termlnal APP ~roduct fo~ned ~paci~ically by lncubatlon o~ CS~ wltn t~e APP su~str.

~ Q ~

8EQU~NC~ LI~TIN~
(1) GENERAL INFORMATION:
(i) APPLICANT: P~l P. Ta~burini, Robert N. ~reyer, and Kathryn M. B~usch (ii) TITLE OF INVENTION: A Diagnostic Ass~y For Alzhei~er'~ Dis.lce Based On The Proteolysls Of The A~yloid Precursor Pr~tein (lii) NU~BER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Miles Inc.
(B) ST~EET: 400 Morgan Lane ~C) CITY: We~.t ~aven (D) S~TE: Connecticu~
(E) COUNTRY: US~
(F) ZIP: 0~516 ~v~ COMPUTR~ READABL~ FORM:
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(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Displaywrite ~.0 (vi) CURRENT APPLICATION DATA:
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~B) FILING DATE:
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(A) NAME: Jae H. Kim, Esq.
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(viii) TELECOMMUNICATION INFORMATION:
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(A) LENGTH: 39 nucleotides ~B) TYPE: nucleic acids (C) STR~N~EDNESS: 6ingle strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
cDNA to ~RNA
~iii) PUBLICATION INFO~MATION:
(A) AUTHORS: Kang et al.
(~) JOURNAL: Nature (C) ~OLUME: 325 (D) PAGE: 733 lE) DATE: 1987 (iv) SEQUENCE DESCRIP~ION: SEQ ID NO:l

Claims (19)

1. A method for use in the diagnosis of Alzheimer's Disease in a patient, comprising the steps of:
(a) combining an APP substrate with a sample of body fluid selected from cerebrospinal fluid and blood obtained from said patient, and (b) detecting proteolytic cleavage of said APP
substrate in the presence of said sample, the degree to which such proteolytic cleavage of said APP substrate occurs in the presence of cerebrospinal fluid or blood samples from individuals known to be afflicted with Alzheimer's Disease being substantially different from that which occurs in the presence of cerebrospinal fluid or blood samples from control individuals.

2. The method of claim 1 wherein steps (a) and (b) are performed under conditions in which essentially no detectable proteolytic cleavage is produced in the presence of cerebrospinal fluid or blood samples from individuals known to be afflicted with Alzheimer's Disease, whereby the absence of detectable proteolytic cleavage in the presence of the patient sample indicates affliction with Alzheimer's Disease.

3. The method of claim 1 wherein said body fluid sample is cerebrospinal fluid.

4. The method of claim 1 wherein said APP substrate comprises a recombinantly expressed polypeptide.

5. The method of claim 1 wherein said APP substrate comprises a synthetic peptide.

6. The method of claim 1 wherein proteolytic cleavage is detected by detecting the production of a polypeptide or peptide fragment of said APP substrate which is characteristic of proteolytic cleavage which occurs in the presence of cerebrospinal fluid or blood samples from control individuals compared to that which occurs in the presence of cerebrospinal fluid or blood samples from individuals known to be afflicted with Alzheimer's Disease.

7. The method of claim 6 wherein said polypeptide or peptide fragment is a C terminal fragment.

8. The method of claim 6 wherein said polypeptide or peptide fragment is detected by antibody binding.

9. The method of claim 1 wherein said APP substrate is a chromogenic substrate and wherein proteolytic cleavage is detected by measuring the chromogenic response thereof.

10. A test kit for use in the testing of a cerebrospinal fluid or blood sample of a patient as an aid in the diagnosis of Alzheimer's Disease, comprising one or more containers holding:
(a) an APP substrate, and (b) an antibody reagent which binds with a polypeptide or peptide fragment which is eharaeteristie of proteolytic cleavage which occurs in the presence of cerebrospinal fluid or blood samples from control individuals compared to that which occurs in the presence of eerebrospinal fluid or blood samples from individuals known to be afflicted with Alzheimer's Disease.

11. The test kit of claim 10 wherein said APP substrate comprises a recombinantly expressed polypeptide.

12. The test kit of claim 10 wherein said APP substract comprises a synthetic peptide.

13. The test kit of claim 10 wherein said antibody reagent comprises a detectable label.

14. The test kit of claim 13 wherein said detectable label is an enzyme.

15. The test kit of claim 10 wherein said polypeptide or peptide fragment is a C-terminal fragment.

16. A chromogenic substrate for use in the testing of a cerebrospinal fluid or blood sample of a patient as an aid in the diagnosis of Alzheimer's Disease, comprising:
(a) an APP substrate portion, and (b) a chromogenic indicator portion that is coupled to said APP substrate portion through a peptide linkage that is cleavable by protease present in cerebrospinal fluid or blood of control individuals but which is substantially not cleavable, or cleavable to a substantially lesser degree, in the presence of cerebrospinal fluid or blood of individuals afflicted with Alzheimer's Disease.

17. The chromogenic substrate of claim 16 wherein said APP
substrate comprises a recombinantly expressed polypeptide.

18. The chromogenic substrate of claim 16 wherein said APP
substrate comprises a synthetic peptide.

19. The chromogenic substrate of claim 16 wherein cleavage by said protease releases an colored indicator moiety.