Note: Descriptions are shown in the official language in which they were submitted.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
1
DIAGNOSTIC METHOD FOR BRAIN DAMAGE-RELATED DISORDERS
BACKGROUND OF THE INVENTION
Field of the invention
This invention relates to a diagnostic method for brain damage-related
disorders.
No biological marker is currently available for the routine diagnosis of brain
damage-related disorders including cerebrovascular, dementia and
neurodegenerative diseases. This invention relates to the use of cerebrospinal
fluid
from deceased patients as a model for the discovery of brain damage-related
disorder markers, and to the use of such markers in diagnosis.
Description of the related art
Over the last two decades, a number of biological markers (biomarkers) have
been
studied in the cerebrospinal fluid (CSF) and serum of patients with brain
damage-
related disorders, including creatine ldnase-BB [1], lactate dehydrogenase
[2],
myelin basic protein [3], S100 protein [4], neuron-specific enolase (NSE) [5],
glial fibrillary acidic protein [6] and tau [7]. Most of them have not proved
useful
indicators of the extent of brain damage and accurate predictors of clinical
status
and functional outcome. In fact, the diagnostic value of biomarkers for brain
damage-related disorders has been hampered by their late appearance and a
delayed peak after the damage event, their poor sensitivity and specificity,
and the
limited understanding of the mechanisms governing the release of these
molecules
into the CSF and ultimately in the blood. As a result of these limitations,
the use
of brain damage-related disorder biomarkers is currently limited to research
settings and none has been recommended for routine assessment [8].
WO 01/42793 relates to a diagnostic assay for stroke in which the
concentration
of heart or brain fatty acid binding protein (H-FABP or B-FABP) is determined
in
a sample of body fluid.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
2
SUMMARY OF THE INVENTION
Ideally, a biomarker for the diagnosis, monitoring and prognosis of brain
damage-
related disorders should include at least the following characteristics: (1)
it should
be brain-specific; (2) because of obvious difficulties to obtain CSF samples
in
patients, detection in serum is highly desirable; (3) it should appear very
early;
(4) its peak level, alternatively the area under the curve of sequential
concentrations, should reflect the extent of brain damage; finally (5) it
should be
indicative of functional outcome. We demonstrate here new brain damage-related
disorder bioniarkers and provide a comparison with S100 and NSE, the two
molecules, which have been most extensively assessed for this purpose.
We describe how proteins have been identified as new diagnostic biomarkers for
brain damage-related disorders using a proteomics-based analysis of CSF from
deceased patients as a model of massive brain damage. And we report as an
example on results obtained after serum FABP levels have been sequentially
determined using an ELISA assay in patients with acute stroke, as compared to
S100 and NSE. A diagnostic assay for stroke using FABP has been described in
WO 01/42793. Use of the polypeptides according to the present invention can be
validated in a similar way.
According to a first object of the invention, compositions are provided which
comprise polypeptides for which the level was found increased in the
cerebrospinal fluid from deceased patients compared to cerebrospinal fluid
from
healthy donors. According to this same object, compositions are disclosed
which
comprise antibodies which are derived from the above polypeptides
According to a second object of the invention, methods are provided which
utilize
the inventive compositions in the diagnosis and prognosis of brain damage-
related
disorders including cerebrovascular, dementia and neurodegenerative diseases.
The present invention provides the following:
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
3
1 A method of diagnosis of a brain damage-related disorder or the
possibility
thereof in a subject suspected of suffering therefrom, which comprises
detecting at
least one polypeptide, or a variant or mutant thereof, selected from A-FABP, E-
FABP, H-FAl3P, B-FABP, PGP 9.5, GFAP, Prostaglandin D synthase,
Neuromodulin, Neurofilanaent L, Calcyphosine, RNA binding regulatory subunit,
Ubiquitin fusion degradation protein 1 hornolog, Nucleoside diphosphate ldnase
A,
Glutathione S tranferase P. Cathepsin D, DJ-1 protein, Peroxiredoxin 5 and
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A) in a sample of body
fluid
taken from the subject.
2 A method according to 1, in which the polypeptide is differentially
contained in the body fluid of brain damage-related disorder-affected subjects
and
non-brain damage-related disorder-affected subjects, and the method includes
determining whether the concentration of polypeptide in the sample is
consistent
with a diagnosis of brain damage-related disorder.
3 A method according to 1 or 2, in which an antibody to the
polypeptide is
used in the detection or the determination of the concentration.
4 A method according to any of 1 to 3, in which the body fluid is
cerebrospinal fluid, plasma, serum, blood, tears, urine or saliva.
5 A method according to any of 1 to 4, in which the polypeptide is
present in
the body fluid of brain damage-related disorder-affected subjects and not
present
in the body fluid of non-brain damage-related disorder-affected subjects,
whereby
the presence of the polypeptide in a body fluid sample is indicative of brain
damage-related disorder.
6 A method according to any of 1 to 4, in which the polypeptide is not
present in the body fluid of brain damage-related disorder-affected subjects
and
present in the body fluid of non-brain damage-related disorder-affected
subjects,
whereby the non-presence of the polypeptide in a body fluid sample is
indicative
of brain damage-related disorder.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
4
7 A method according to any of 1 to 6, in which a plurality of
peptides is
determined in the sample.
8 A method according to any of 1 to 7, in which the polypeptide is
differentially subject to post-translational modification in the body fluid of
brain
damage-related disorder-affected subjects and non-brain damage-related
disorder-
affected subjects, and the method includes detecting the post--translational
modification of the polypeptide in the sample and determining whether this is
consistent with a diagnosis of a brain damage-related disorder.
9 A method according to 8, in which the post-translational
modification
comprises N-glycosylation.
10 A method according to any of 1 to 9, in which the brain damage-
related
disorder is stroke and the polypeptide is Ubiquitin fusion degradation protein
1
homolog.
11 A method according to any of 1 to 9, in which the brain damage-
related
disorder is stroke and the polypeptide is RNA binding regulatory subunit.
12 A method according to any of 1 to 9, in which the brain damage-
related
disorder is stroke and the polypeptide is Nucleoside diphosphate lcinase A.
13 A method according to any of 10 to 12, in which two or more markers
selected from antibodies to Ubiquitin fusion degradation protein 1 homolog,
RNA
binding regulatory subunit, Nucleoside diphosphate lcinase A and H-FABP are
used in a single well of an ELISA microtiter plate.
14 A method according to 13, in which all four markers are used in a
single
well.
15 A method according to any of 10 to 12, in which two or more
polypeptides
selected from Ubiquitin fusion degradation protein 1 homolog, RNA binding
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
regulatory subunit, Nucleoside diphosphate kinase A and H-FABP are separately
assayed, and a predictive algorithm is used for diagnosis.
16 Use of a polypeptide, or a variant or mutant thereof, selected from A-
5 FABP, E-FABP, FI-FABP, B-FABP, PGP 9.5, GFAP, Prostaglandin D synthase,
Neuromodulin, Neurofilament L, Calcyphosine, RNA binding regulatory subunit
Ubiquitin fusion degradation protein 1 homolog, Nucleoside diphosphate kinase
A,
Glutathione S tranferase P, Cathepsin D, DJ-1 protein, Peroxiredoxin 5 and
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A), or a combination of
such
polypeptides, for diagnostic, prognostic and therapeutic applications relating
to
brain damage-related disorders.
17 Use according to 16, in which the polypeptide is differentially
contained in
a body fluid of brain damage-related disorder-affected subjects and non-brain
damage-related disorder-affected subjects.
18 Use for diagnostic, prognostic and therapeutic applications, relating
to
brain damage-related disorders, of a material which recognises, binds to or
has
affinity for a polypeptide, or a variant or mutant thereof, selected from A-
FABP,
E-FABP, H-FABP, B-FABP, PGP 9.5, GFAP, Prostaglandin D synthase,
Neuromodulin, Neurofilament L, Calcyphosine, RNA binding regulatory subunit,
Ubiquitin fusion degradation protein 1 homolog, Nucleoside diphosphate kinase
A,
Glutathione S tranferase P, Cathepsin D, DJ-1 protein, Peroxiredoxin 5 and
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A).
19 Use according to 18 of a combination of materials, each of which
respectively recognises, binds to or has affinity for a polypeptide, or a
variant or
mutant thereof, selected from A-FABP, E-FABP, H-FABP, B-FABP, PGP 9.5,
GFAP, Prostaglandin D synthase, Neuromodulin, Neurofilament L, Calcyphosine,
RNA binding regulatory subunit, Ubiquitin fusion degradation protein 1
homolog,
Nucleoside diphosphate kinase A, Glutathione S tranferase P, Cathepsin D, DJ-1
protein, Peroxiredoxin 5 and Peptidyl-prolyl cis-trans isomerase A
(Cyclophilin
A).
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
6
20 Use according to 18 or 19, in which the or each material is an
antibody or
antibody chip.
21 Use according to 20, in which the material is an antibody to A-FABP.
22 Use according to 20, in which the material is an antibody to E-FABP.
23 Use according to 20, in which the material is an antibody to PGP 9.5.
24 Use according to 20, in which the material is an antibody to GFAP.
25 Use according to 20, in which the material is an antibody to
Prostaglandin
D synthase.
26 Use according to 20, in which the material is an antibody to
Neuromodulin.
27 Use according to 20, in which the material is an antibody to
Neurofilament
L.
28 Use according to 20, in which the material is an antibody to
CalCyphosine.
29 Use according to 20, in which the material is an antibody to RNA
binding
regulatory subunit.
30 Use according to 20, in which the material is an antibody to Ubiquitin
fusion degradation protein 1 homolog.
31 Use according to 20, in which the material is an antibody to
Nucleoside
diphosphate kinase A.
32 Use according to 20, in which the material is an antibody to
Glutathione S
tranferase P.
33 Use according to 20, in which the material is an antibody to
Cathepsin D.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
7
34 Use according to 20, in which the material is an antibody to DJ-1
protein.
35 Use according to 20, in which the material is an antibody to
Peroxiredoxin
5.
36 Use according to 20, in which the material is an antibody to
Peptidyl-prolyl
cis-trans isomerase A (Cyclophilin A).
37 An assay device for use in the diagnosis of brain damage-related
disorders,
which comprises a solid substrate having a location containing a material
which
recognizes, binds to or has affinity for a polypeptide, or a variant or mutant
thereof, selected from A-FABP, E-FABP, H-FABP, B-FA13P, POP 9.5, GFAP,
Prostaglandin D synthase, Neuromodulin, Neurofdament L, Calcyphosine, RNA
binding regulatory subunit, Ubiquitin fusion degradation protein 1 homo log,
Nucleoside diphosphate kinase A, Glutathione S tranferase P, Cathepsin D, DJ-I
protein, Peroxiredoxin 5 and Peptidyl-prolyl cis-trans isomerase A
(Cyclophilin
A).
38 An assay device according to 37, in which the solid substrate has a
plurality
of locations each respectively containing a material which recognizes, binds
to or
has affinity for a polypeptide, or a variant or mutant thereof, selected from
A-
FABP, E-FABP, H-FABP, B-FABP, PGP 9.5, GFAP, Prostaglandin D synthase,
Neuromodulin, Neurofilament L, Calcyphosine, RNA binding regulatory subunit,
Ubiquitin fusion degradation protein 1 homolog, Nucleoside diphosphate kinase
A,
Glutathione S tranferase P, Cathepsin D, DJ-1 protein, Peroxiredoxin 5 and
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A).
39 An assay device according to 37 or 38, in which the material is an
antibody
or antibody chip.
An assay device according to 39, which has a unique addressable location
for each antibody, thereby to permit an assay readout for each individual
polypeptide or for any combination of polypeptides.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
8
41 An assay device according to any of 37 to 40, including an antibody
to A-
FABP.
42 An assay device according to any of 37 to 40, including an antibody to E-
FAI3P.
43 An assay device according to any of 37 to 40, including an antibody
to PGP
9.5.
44 An assay device according to any of 37 to 40, including an antibody
to
GFAP.
45 An assay device according to any of 37 to 40, including an antibody
to
Prostaglandin D synthase.
46 An assay device according to any of 37 to 40, including an antibody
to
Neuromodulin.
47 An assay device according to any of 37 to 40, including an antibody to
Neurofilament L.
48 An assay device according to any of 37 to 40, including an antibody
to
Calcyphosine.
49 An assay device according to any of 37 to 40, including an antibody
to
RNA binding regulatory subunit.
50 An assay device according to any of 37 to 40, including an antibody
to
Ubiquitin fusion degradation protein 1 homolog.
51 An assay device according to any of 37 to 40, including an antibody
to
Nucleoside diphosphate ldnase A.
CA 02 932 077 2 016-0 6-03
WO 2005/029088
PCT/OB2004/050012
9
52 An assay device according to any of 37 to 40, including an antibody
to
Glutathione S tranferase P.
53 An assay device according to any of 37 to 40, including an antibody
to
Cathepsin D.
54 An assay device according to any of 37 to 40, including an antibody
to DJ-
1 protein.
55 An assay device according to any of 37 to 40, including an antibody to
Peroxiredcodn 5.
56 An assay device according to any of 37 to 40, including an antibody
to
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A).
57 A kit for use in the diagnosis of brain damage-related disorders,
comprising
an assay device according to any of 37 to 56, and means for detecting the
amount
of one or more of the polypeptides in a sample of body fluid taken from a
subject.
The new markers used in the present invention are as follows:
A-FABP (P15090), which has the sequence (SEQ ID NO.1):
1CDAFVGTWKLVSSENFDDYMICEVGVGFATRKVAGMAIGNMIISVNGDVITIKSESTFICNTEISFILG
QEFDEVTADDRKVKSTITLDGGVLVHVQKWDGKSTTIKRKREDDICLVVECVMKGVTSTRVYERA
131
E-FABP (Q01469), which has the sequence (SEQ ID NO.2):
1MATVQQLEGRWRLVDSKGFDEYMECELGVGIALRICMGAMAKPDCLITCDGICNLITECTESTLICTIQF
SCTLGEKFEETTADGRICTQTVCNFTDGALVQHQEWDGICESTITRICLKDGKLVVECVMNNVTCTRIY
EKVE 135
PGP 9.5 (P09936), which has the sequence (SEQ ID NO.3):
I MQLKPMEMP EMLNKVLSRL GVAGQWRFVD VLGLEEESLG SVPAPACALL LLFPLTAQBE 60
NFRKKQIPET KGQEVSPKVY FMKQT1GNSC GTIGLEIAVA NNQDKLGFED GSVLKQFLSE 120
TEKMSPEDRA KCFEKNEAIQ AAHDAVAQEG QCRVDDICVNF IIFILFNNVDG FILYELDGRMP 180
FP'VNHGASSE DTLLICDAAKV CREFTEREQG EVRFSAVALC KAA 223
GFAP (P14136), which has the sequence (SEQ ID NO.4):
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
1 MERRRTTSAA RRSYVSSGEM MVGGLAPGRR LGPGTRLSLA RMPPPLPTRV DFSLAGALNA 60
GFICETRASER AEMMELNDRF ASYIEICVRFL EQQNKALAAE LNQLRAKEPT KLADVYQAEL 120
RELRLRLDQL TANSARLEVE RDNLAQDLAT 'VRQKLQDBIN LRLEAENNLA AYRQEADEAT 180
LARLDLERKI ESLEEEIRFL RICIBBEEVRE LQEQLARQQV HVELDVAKPD LTAALKEIRT 240
5 QYEAMASSNM HEABEWYRSK FADLTDAAAR NAELLRQAKH EANDYRRQLQ SLTCDLESLR 300
GTNESLERQMREQEERTIVRE AASYQEALAR LEEEGQSLKD EMARELQEYQ DLLNVKLALD 360
EIA'TYRICLL EGEENRTI1P VQTFSNLQ1R ETSLDIKSVS EGHLICRNIVV KTVEMRDGEV 420
IKESKQEHKD VM 432
10 Prostaglandin D synthase (P41222), which has the sequence (SEQ ID NO.5):
23 APEAQVSV QPNFQQDKFL GRWFSAGLAS NSSWLREKKA. 60
ALSMCKSVVA PATDGGLNLT SFELRICNQCB TRTMLLQPAG SLGSYSYRSP IIWGSTYSVSV 120
VETDYDQYAL LYSQGSKGPG EDFRMATLYS RTQTPRAELK EKFTAFCKAQ GFTEDT1VFL 180
PQTDKCMTEQ
Neuromodulin (P17677), which has the sequence (SEQ ID NO.6):
1 MLCCMRRTKQ VEECNDDDQKI EQDGIKPEDK AIIKAATKIQA SFRGBITRICK. LKGEK1CDDVQ 60
AABAEANKKD EAPVADGVEK KGEGTTTAEA APATGSKPDE PGKAGETPSE EICKGEGDAAT 120
EQAAPQAPAS SEEKAGSAET ESATKASTDN SPSSKAEDAP AKEEPKQADV PAAVTAAAAT 180
TPAAEDAAAK ATAQPPTETG ESSQAEENIE AVDET'KPKES ARQDEGKEEE PEADQEHA 238
Neurofilament L (P07196), which has the sequence (SEQ ID NO.7):
1 SSFSYEPYYS TSYICRRYVET PRVIIISVRSG YSTARSAYSS YSAPVSSSLS VRRSYSSSSG 60
SLMPSLENLD LSQVAAISND LKS1RTQEKA QLQDLNDRFA SFIERVITET E QQNKVLBAEL 120
LVLRQUISEP SRFRALYEQE IRDLRLAAED ATT'NEKQALR GEREEGLEET LRNLQARYEE 180
EVLSREDAEG RLMERRKGAD EAALARAELE KRIDSLIViDEI SELKKVIIEBE IAELQAQIQY 240
AQIS'VEMDVT KPDLSAALKD IRAQYEICLAA KNMQNAEEWF KSRFTVLTES AAKNTDAVRA 300
AKDEVSESRR LLKAICTLEI ACRGMNEALE KQLQELEDKQ NADISAMQDT INKLENELRT 360
TKSEMARYLK EYQDLLNVKM ALDIE1AAYR ICLLEGEETRL SFTSVGSITS GYSQSSQVFG 420
RSAYGGLQTS SYLMSTRSFP SYYTSITVQEE QTEVEETIEA SICAEEAKDEP PSEGEAEEEE 480
KDKEEAEEEE AAEEEEAAKE ESEEAKEEEE GGEGEEGEET KEABEEEKKV EGAGEEQAA_K 540
ICKD 543
Calcypbosine (Q13938), which has the sequence (SEQ ID NO.8):
1 MDAVDATMEK LRAQCLSRGA SGIQGLARFF RQLDRDGSRS LDADEFRQGL AICLGUVLDQA 60
BAEGVCRKWD RNGSGTLDLE EFLRALRPPM SQAREAVIAA AFAKLDRSGD GVVTVDDLRG 120
VYSGRAIIPKV RSGEWTEDEV LRRFLDNFDS SEICDGQVTLA EFQDYYSGVS ASMNTDEEFV 180
AMMTSAWQL 189
RNA binding regulatory subunit (014805), also referred to as RNA-BP, which
has the sequence (SEQ ID NO.9):
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
11
1 MASKRALVIL AKGAEEMETV IPVDVMRRAGIKVTVAGLAG KDPVQCSRDV VICPDASLED 60
AKKEGPYDVV VT2GGNLGAQ NLSESAAVKEILICEQENRKG LIAAICAGPT ALLAHEIGFG 120
SKVTTHPLAK DKMMNGGEITT YSENRVEKDG LILTSRGPGT SFEFALAIVE ALNGICEVAAQ 180
VICAPLVLKD 189
Ubiquitin fusion degradation protein 1 hornolog (Q92890), also referred to as
UFD1 or UFDP1, which has the sequence (SEQ ID NO.10):
MFSFNMFDHP 1PRVFQNRFS TQYRCFSVSM LAGPNDRSDV EKGGKIIMPP SALDQLSRLN 60
ITYPMLFKLT NKNSDRMTHC GVLEFVADEG ICYLPHWMMQ NLLLEEDGLV QLETVNLQVA 120
TYSKSTCFCYL PHWMMQNLLL EEGGLVQVES VNLQVATYSK FQPQSPDFLD ITNPKAVLEN 180
ALRNFACL'IT GDVIAINYNE ICIYELRVMET TCPDKAVSBE CDMNVDFDAP LGYKEPERQV 240
QHEESTEGEA DHSGYAGELG FRAFSGSGNR LDGKKKGVEP SPSPEPGDI ICRGIPNYEFK 300
LGKITFIRNS RPL'VKKVEED EAGGRFVAFS GEGQSLR1CKG RICP 343
Nucleoside diphosphate ldnase A (P15531), also referred to as NDK A, which has
the sequence (SEQ ID NO.11):
1 MANCERITIA1KPDGVQRGL VGEBRRFEQ KGFRLVGLICF MQASEDLLKE BYVDLKDRPF 60
FAGLVICYMHS GPVVAMVWEG LNVVICTGRVM LGETNPADSK PG IIRGDFCI QVGRNITTIGS 120
DSVESAEKEI GLWFHPEELV DYTSCAQNWI YE 152
Glutathione S tranferase P (P09211), which has the sequence (SEQ ID NO.12):
1 PPYTVVYFPV RGRCAALRML LADQGQSWKE EVVTVETWQE GSLKASCLYG QLPKFQDGDL 60
TLYQSNTILR BLGRTLGLYG KDQQBAALVD MVNDGVEDLR CKY1SLIYTN YEAGKDDYVK 120
ALPGQLKPFE TLLSQNQGGK TFTVGDQISF ADYNLLDLLL IHEVLAPGCL DAFPLLSAYV 180
GRLSARPICLK AFLASPEYVN LP1NGNGKQ 209
Cathepsin D (P07339), which has the sequence (SEQ ID NO.13):
65 GP1PEV LKNYMDAQYY GEIGIGTPPQ CFTVVFDTGS SNLW'VPSIFIC ICLLDIACW111 120
HICYNSDKSST YVKNGTSFDI HYGSGSLSGY LSQDTVSVPC QSASSASALG GVKVERQVFG 180
EATKQPGITF IAAKFDGTLG MAYPRISVNN VLPVFDNLMQ QKLVDQNIFS FYLSRDPDAQ 240
PGGELMLGGT DSICYYKGSLS YLNVTRICAYW QVIILDQVEVA SGLTLCKEGC EAIVDTGTSL 300
MVGPVDEVRE LQKAIGAVPL IQGEYMIPCE KVSTLPAITL KLGGKGYKLS PEDYTLKVSQ 360
AGKTLCLSGF MGMDIPPPSG PLWILGDVFI GRYYTVFDRD NNRVGFAEAA RL 412
DJ-1 protein (Q99497), which has the sequence (SEQ ID NO.14):
1 MASICRALVIL AKGAEEMETV IPVD'VMRRAG 1KVTVAGLAG ICDPVQCSRDV VICPDASLED 60
AICKEGPYDVV VLPGGNLGAQ NLSESAAVKE ILICEQENRKG LIAAICAGPT ALLALIEIGCG 120
SKVTITIPLAK DKMMNGGITYT YSENRVEKDG LTLTSRGPGT SFEFALAIVE ALNGKEVAAQ 180
VKAPLVLKD 189
Peroxiredoxin 5 (P30044), which has the sequence (SEQ ID NO.15):
CA 02 932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
12
1 MGLAGVCALR RSAGY1LVGG AGGQSAAAAA RRCSEGEWAS GGVRSFSRAA AAMAPIKVGD 60
AIPAVEVFEG EPGNKVNLAE LFKG1CKGVLF GVPGAFTPGC SKTELPGFVE QAEALKAKGV 120
QVVACLSVND AFVTGEWGRA HKAEGKVRLL ADPTGAFGKE TDLLLDDSLV SIFGNRRLKR
180FSMVVQDGIV KALNVEPDGT GLTCSLAPNI ISQL 214
Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A) (P05092), which has the
sequence (SEQ ID NO.16):
1 VIVPTVFFDIA VDGEPLGRVS FELFADKVPK TAENFRALST GEKGFGYKGS CHIREPGFM 60
CQGGDFTRIN GTGGKSIYGE KFEDENFILK IITGPGILSMA NAGPNTNGSQ FFICTA1CTEW 120
LDGKI1VVFGK VKEGMNIVEA MERFGSRNGK TSKKITIADC GQLE 164
The polypeptides useful in the present invention are not restricted to the
above
sequences, and include variants and mutants thereof. A variant is defined as a
naturally ocurring variation in the sequence of a polypeptide which has a high
degree of homology with the given sequence, and which has substantially the
same functional and immunological properties. A mutant is defined as an
artificially created variant. A high degree of homology is defined as at least
90%,
preferably at least 95% and most preferably at least 99% homology. Variants
may
occur within a single species or between different species. The above
sequences
are of human origin, but the invention encompasses use of the corresponding
polypeptides from other mammalian species, e.g. bovine animals.
Brain damage-related disorders in the context of the present invention include
the
following: head trauma, ischemic stroke, hemorrhagic stroke, subarachnoid
hemorrhage, intra cranial hemorrhage, transient ischemic attack, vascular
dementia, corticobasal ganglionic degeneration, encephalitis, epilepsy, Landau-
Kleffner syndrome, hydrocephalus, pseudotumor cerebri, thalamic diseases,
meningitis, myelitis, movement disorders, essential tremor, spinal cord
diseases,
syringomyelia, Alzheimer's disease (early onset), Alzheimer's disease (late
onset), multi-infarct dementia, Pick's disease, Huntingdon's disease,
Parkinson,
Parkinson syndromes, frontotemporal dementia, corticobasal degeneration,
multiple system atrophy, progressive supranuclear palsy, Lewy body disease,
amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Dandy-Walker
syndrome, Friedreich ataxia, Machado-Joseph disease, migraine, schizophrenia,
mood disorders and depression. Corresponding disorders in non-human mammals
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
13
are also included, such as transmissible spongiform encephalopathies (TSEs),
e.g.
bovine spongifonn encephalopathy (BSE) in cattle or scrapie in sheep.
H-FABP (P05413) and B-FABP (015540) are also useful in the present invention
for diagnosis of brain damage-related disorders or the possibility thereof,
especially those other than stroke and CJD.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows results of an assay for H-FABP (measured in OD units on the
vertical axis) for three groups of patients: a control group, a group with
acute
myocardial infarction (AlvI), and a group with acute stroke;
Figure 2 shows the results of sequential determination of H-FABP levels
(measured in OD units on the vertical axis) for the stroke group of patients
at
different time intervals after stroke;
Figure 3 shows portions of 2-DE maps for healthy and post-mortem CSF, with
upward-directed arrows indicating spots corresponding to RNA binding
regulatory subunit or DJ-1 protein. Enlargements of healthy CSF and deceased
CSF 2-DE maps are shown. Forty five jig of protein was loaded on a rPG gel (pH
3.5-10 NL, 18cm). Second dimension was a vertical gradient slab gel (9-16%T).
Gel was silver stained. The spots corresponding to the RNA binding regulatory
subunit or to the DJ-1 protein are indicated by upward-directed (red) arrows;
Figure 4 shows portions of 2-DE maps for healthy and post-mortem CSF, with the
right-hand arrows indicating spots corresponding to peroxiredoxin 5.
Enlargements of healthy CSF and deceased CSF 2-DE maps are shown. Forty
five jig of protein was loaded on a IPG gel (pH 3.5-10 NL, 18cm). Second
dimension was a vertical gradient slab gel (9-16%T). Gel was silver stained.
The
spot corresponding to Peroxiredoxin 5 is indicated by the right-hand (red)
arrows;
Figure 5 shows portions of 2-DE maps for healthy and post-mortem CSF, with
the right-hand pair of arrows indicating spots corresponding to peptidyl-
prolyl cis-
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
14
trans isomerase A (cyclophylin A). Enlargements of healthy CSF and deceased
CSF 2-DE maps are shown. Forty five pg of protein was loaded on a EPG gel (pH
3.5-10 NL, 18cm). Second dimension was a vertical gradient slab gel (9-16%T).
Gel was silver stained. The spots corresponding to Cyclophylin A are indicated
by the right-hand pair of (red) arrows;
Figure 6 shows ELISA intensity values for marker polypeptides obtained in a
survey of stroke patients;
Figure 7 shows UFD1 detection in plasma samples from said survey;
Figure 8 is an ROC curve of UFD1 from the data in Figure 7;
Figure 9 shows UFD1 detection corresponding to Figure 7;
Figure 10 shows RNA-BP detection in plasma samples from said survey;
Figure 11 is an ROC curve of RNA-BP from the data in Figure 10;
Figure 12 shows RNA-BP detection corresponding to Figure 10;
Figure 13 shows NDK A detection in plasma samples from said survey;
Figure 14 is an ROC curve of NDK A from the data in Figure 13;
Figure 15 shows NDK A detection corresponding to Figure 13;
Figure 16 shows portions of 2-DE maps for healthy and post-mortem CSF
indicating prostaglandin D synthase levels;
Figure 17 shows prostaglandin D2 synthase spot intensities on mini-2-DE gels
prepared with CSF of a CJD patient and a healthy patient as a control;
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
Figure 18 shows ELISA intensity values for H-FABP obtained in a survey of
stroke patients and a control group;
Figure 19 shows UFDP-1 spot intensities on mini-2-DE-gels prepared with CSF
5 from a control and a deceased patient;
Figure 20 shows UFDP1 plasma concentration measured by ELISA for two
cohorts of stroke patients and controls from Geneva and from the USA;
10 Figure 21 shows RNA-BP spot intensities on mini-2-DE-gels prepared with
CSF
from a control and a deceased patient;
Figure 22 shows RNA-BP plasma concentration measured by ELISA for three
studies of controls and stroke patients;
Figure 23 shows NDKA spot intensities on mini-2-DE-gels prepared with CSF
from a control and a deceased patient;
Figure 24 shows NDKA plasma concentration measured by ELISA for two
cohorts of stroke patients and controls from Geneva and from the USA;
Figure 25a shows the time onset of symptoms, showing the stroke marker (SM)
concentration for UFDP1, RNA-BP and NDKA, in each case respectively for
controls, stroke patients at less than 3 hours from the time of
cerebrovascular
accident, and stroke patients at more than 3 hours from the time of
cerebrovascular accident;
Figure 25b shows data for type of stroke, showing the stroke marker
concentration.
for UFDP1, RNA-BP and NDKA, in each case respectively for controls,
hemorrhagic stroke patients, transient ischemic attack(TIA) patients and ische-
mic
stroke patients;
Figure 26 is a summary of information for a panel of early plasmatic markers
of
stroke;
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
16
Figure 27 shows ELISA intensity values for a mix of UFD1, RNA-BP, NDKA
and H-FABP in the same well;
Figure 28 is a graphic representation of combinations of two out of the four
biomarkers from Figure 27, showing selected cut-off values for diagnosis;
Figures 29A and 29B show information related to 37 stroke and 37 age/sex
matched control plasma samples in a further study. Diagnosis (Diag) is shown
as
I (ischemic stroke), H (hemorrhagic stroke), TIA (transient ischemic attack)
or ctrl
(control). The concentrations determined by ELISA of UFD1, RNA-BP and
NDK A are also shown. ELISA was performed as previously described;
Figure 30 shows the results from this further study for 37 stroke and 37
control
plasma samples tested in Geneva for UFD1. USA-1 (non age sex matched
controls) data for UFD1;
Figure 31 shows the results from this further study for 37 stroke and 37
control
plasma samples tested in Geneva for RNA-BP. USA-1 (non age sex matched
controls) and USA-2 (age sex matched controls) data for RNA-BP;
Figure 32 shows the results of a large scale study USA3 on 633 patients for
RNA-
BP;
Figure 33 shows a statistical analysis (Kruskal-Wallis) on USA-3 for RNA-BP;
Figure 34 shows results for 33 stroke and 33 control plasma samples tested in
Geneva for NDKA. USA-1 (non age sex matched controls) data for NDK A;
Figure 35 shows results of a large scale study USA3 on 622 patients for NDKA;
Figure 36 shows a statistical analysis (Kruskal-Wallis) on USA-3 for NDK A;
Figure 37 shows stroke marker concentration as a function of time onset of
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
17
symptoms (Geneva data, new 37 stroke and 37 control plasma samples);
Figure 38 shows stroke marker concentration as a function of type of stroke
(hemorrhagic, ischemic, TIA) using USA-1 data.
DESCRIPTION OF PREFERRED EMBODIMENTS
The invention presented here is directed towards compositions and methods for
detecting increasing or reducing polypeptides levels in body fluids including
blood components (e.g. plasma or serum) or cerebrospinal fluid from patients
affected by a brain damage-related disorder including cerebrovascular,
dementia
and neurodegenerative diseases. For this purpose, use can be made of
antibodies
or any specific polypeptide detection method.
Antibodies against brain damage protein markers, in particular their protein-
binding domains, are suitable as detection tools. Molecular biological and
biotechnological methods can be used to alter and optimize the antibody
properties of the said molecules in a specific manner. In addition to this,
the
antibodies can be modified chemically, for example by means of acetylation,
carbamoylation, fonnylation, biotinylation, acylation, or derivatization with
polyethylene glycol or hydrophilic polymers, in order to increase their
stability.
A specific polypeptide marker selected from A-FABP, E-FABP and any other
FABP, i.e. H-FABP or B-FABP, PUP 9.5, GFAP, Prostaglandin D synthase,
Neurornodulin, Neurofilament L, Calcyphosine, RNA binding regulatory subunit,
Ubiquitin fusion degradation protein 1 homolog, Nucleoside diphosphate kinase
A, Glutathione S tranferase P, Cathepsin D, DJ-1 protein, Percairedo)dn 5 and
Peptidyl-proly1 cis-trans isomerase A (Cyclophilin A) is determined in a body
fluid sample, for example by using an antibody thereto. The marker is
preferably
measured by an immunoassay, using a specific antibody to the polypeptide and
measuring the extent of the antigen (polypeptide)/antibody interaction. The
antibody may be a monoclonal antibody or an engineered (chimeric) antibody.
Antibodies to the polypeptides are known and are commercially available. Also,
the usual Kohler-Milstein method may be used to raise antibodies. Less
CA 02932077 2016-06-03
WO 2005/029088
PCT/G132004/050012
18
preferably, the antibody may be polyclonal. In the context of the present
invention, the term "antibodies" includes binding fragments of antibodies,
such as
single chain or Fab fragments.
Any known method of immunoassay may be used. In a sandwich assay an
antibody (e.g. polyclonal) to the polypeptide is bound to the solid phase such
as a
well of a plastics microtitre plate, and incubated with the sample and with a
labelled second antibody specific to the polypeptide to be detected.
Alternatively,
an antibody capture assay (also called "indirect immunoassay") can be used.
Here, the test sample is allowed to bind to a solid phase, and the anti-
polypeptide
antibody (polyclonal or monoclonal) is then added and allowed to bind. If a
polyclonal antibody is used in this context, it should desirably be one which
exhibits a low cross-reactivity with other forms of polypeptide. After washing
away unbound material, the amount of antibody bound to the solid phase is
determined using a labelled second antibody, anti- to the first.
A direct assay can be performed by using a labelled anti-polypeptide antibody.
The test sample is allowed to bind to the solid phase and the anti-polypeptide
antibody is added. After washing away unbound material, the amount of antibody
bound to the solid phase is determined. The antibody can be labelled directly
rather than via a second antibody.
In another embodiment, a competition assay can be performed between the sample
and a labelled polypeptide or a peptide derived therefrom, these two antigens
being
in competition for a limited amount of anti-polypeptide antibody bound to a
solid
support. The labelled polypeptide or peptide can be pre-incubated with the
antibody on the solid phase, whereby the polypeptide in the sample displaces
part
of the polypeptide or peptide thereof bound to the antibody.
In yet another embodiment, the two antigens are allowed to compete in a single
co-
incubation with the antibody. After removal of unbound antigen from the
support
by washing, the amount of label attached to the support is determined and the
amount of protein in the sample is measured by reference to standard titration
curves established previously.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
19
Throughout, the label is preferably an enzyme. The substrate for the enzyme
may
be colour-forming, fluorescent, chemiluminescent or electrochemical, and can
be
soluble or precipitating. Alternatively, the label may be a radioisotope or
fluorescent, e.g. using conjugated fluorescein.
The enzyme may, for example, be alkaline phosphatase or horseradish percaidase
and can conveniently be used colorimetrically, e.g. using p-nitrophenyl
phosphate
as a yellow-forming substrate with alkaline phosphatase.
For a cherniluminescent assay, the antibody can be labelled with an
acridinitun
ester or horseradish permddase. The latter is used in enhanced
chemihuninescent
(ECL) assay. Here, the antibody, labelled with horseradish peroxidase,
participates in a chemiluminescent reaction with luminol, a peroxide substrate
and
a compound, which enhances the intensity and duration of the emitted light,
typically, 4-iodophenol or 4-hydroxycinnamic acid.
An amplified immunoassay such as immuno-PCR can be used. In this technique,
the antibody is covalently linked to a molecule of arbitrary DNA comprising
PCR
primers, whereby the DNA with the antibody attached to it is amplified by the
polymerase chain reaction. See E. R. Hendrickson et al., Nucleic Acids
Research
1995; 23, 522-529 (1995) or T. Sano et al., in "Molecular Biology and
Biotechnology" ed. Robert A. Meyers, VCH Publishers, Inc. (1995), pages 458 -
460. The signal is read out as before.
In one procedure, an enzyme-linked immunosorbent assay (ELISA) can be used to
detect the polypeptide.
The use of a rapid microparticle-enhanced turbidimetric immunoassay, developed
for H-FABP in the case of AMI, M.Robers et al., "Development of a rapid
microparticle-enhanced turbidimetric immunoassay for plasma fatty acid-binding
protein, an early marker of acute myocardial infarction", Clin. Chem.
1998;44:1564-1567, significantly decreases the time of the assay. Thus, the
full
automation in a widely used clinical chemistry analyser such as the COBASTM
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
MIRA. Plus system from Hoffiaann-La Roche, described by M.Robers et al. supra,
or the AxSY11/Irm system from Abbott Laboratories, should be possible and
applied
for routine clinical diagnosis of brain damage-related disorders.
5 The polypeptide concentrations can be measured by other means than
immunoassay. For example, the sample can be subjected to 2D-gel
electrophoresis
and the amount of the polypeptide estimated by densitometric scanning of the
gel
or of a blot therefrom. However, it is desirable to carry out the assay in a
rapid
manner, so that the patient can be treated promptly.
In principle, any body fluid can be used to provide a sample for diagnosis,
but
preferably the body fluid is cerebrospinal fluid (CSF), plasma, serum, blood,
urine,
tears or saliva.
According to the invention, a diagnosis of brain damage-related disorders may
be
made from determination of a single polypeptide or any combination of two or
more of the polypeptides.
The invention also relates to the use of one or more of the specified
polypeptides
which is differentially contained in a body fluid of brain damage-affected
subjects
and non-brain damage-affected subjects, for diagnostic, prognostic and
therapeutic
applications. This may involve the preparation and/or use of a material which
recognizes, binds to or has some affmity to the above-mentioned polypeptide.
Examples of such materials are antibodies and antibody chips. The term
"antibody" as used herein includes polyclonal antiserum, monoclonal
antibodies,
fragments of antibodies such as Fab, and genetically engineered antibodies.
The
antibodies may be chimeric or of a single species. The above reference to
"prognostic" applications includes making a determination of the likely course
of a
brain damage-related disorder by, for example, measuring the amount of the
above-mentioned polypeptide in a sample of body fluid. The above reference to
"therapeutic follow-up" applications includes making a determination of the
likely
course of a brain damage-related disorder by, for example, measuring the
amount
of the above-mentioned polypeptide in a sample of body fluid (and evaluating
its
level as a function of the treatment, the disability recovery or not, the size
of the
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
21
lesions etc.). The above reference to "therapeutic" applications includes, for
example, preparing materials which recognize, bind to or have affinity to the
above-mentioned polypeptides, and using such materials in therapy. The
materials
may in this case be modified, for example by combining an antibody with a
drug,
thereby to target the drug to a specific region of the patient.
The above reference to "presence or absence" of a polypeptide should be
understood to mean simply that there is a significant difference in the amount
of a
polypeptide which is detected in the affected and non-affected sample. Thus,
the
"absence" of a polypeptide in a test sample may include the possibility that
the
polypeptide is actually present, but in a significantly lower amount than in a
comparative test sample. According to the invention, a diagnosis can be made
on
the basis of the presence or absence of a polypeptide, and this includes the
presence of a polypeptide in a significantly lower or significantly higher
amount
with reference to a comparative test sample.
The above references to "detecting" a polypeptide should be understood to
include a reference to compositions and methods for detecting post-
translational
modifications of the polypeptides in addition to quantitative variations.
As an example, we detected differences in the post-translational modifications
pattern of prostaglandin D synthase between post-mortem and control CSF.
Similar differences were also detected between CSF from a patient suffering
from
Creutzfeldt-Jakob disease and control CSF. This is described in Example 5
below. The invention therefore encompasses the detection of post-translational
modifications in general, and determining whether such modifications of a
polypeptide are consistent with a diagnosis of a brain damage-related
disorder.
Kits and assay devices for use in diagnosis of brain damage-related disorders
are
also within the scope of the invention. These may include one or more
antibodies
to a polypeptide selected from A-FABP, E-FABP and any other FABP, i.e. H-
FABP or B-FABP, POP 9.5, GFAP, Prostaglandin D synthase, Neuromodulin,
Neurofilament L, Calcyphosine, RNA binding regulatory subunit, Ubiquitin
fusion
degradation protein 1 homolog, Nucleoside diphosphate kinase A, Glutathione S
tranferase P, Cathepsin D, DJ-1 protein, Peroxiredoxin 5 and Peptidyl-prolyl
cis-
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
22
trans isomerase A (Cyclophilin A). The antibodies will bind to the appropriate
polypeptides in a fluid sample taken from a patient. The antibodies may be
immobilised on a solid support. Preferably, each antibody is placed in a
unique
addressable location, thereby to permit separated assay readout for each
individual
polypeptide in the sample, as well as readouts for any selected combination of
polypeptides.
The following Examples illustrate the invention.
EXAMPLE 1
Using two-dimensional gel electrophoresis (2-DE) separation of cerebrospinal
fluid (CSF) proteins and mass spectrometry techniques, 15 polypeptides named
in
Table 1 were found elevated or decreased in the CSF of deceased patients, used
as
a model of massive brain damage.
Study population and samples handling
Eight CSF samples were used for the proteomics-based approach aiming at
discovering brain damage-related disorder markers. Four of these samples were
obtained at autopsy from deceased patients 6 hours after death with no
pathology
of the central nervous system. Four others were collected by lumbar puncture
from living patients who had a neurological workup for benign conditions
unrelated to brain damage (atypical headache and idiopathic peripheral facial
nerve palsy). CSF samples were centrifuged immediately after collection,
aliquoted, frozen at -80 C and stored until analysis.
CSF 2-DE
All reagents and apparatus used have been described in detail elsewhere [9].
250 1 of CSF were mixed with 500111 of ice-cold acetone (-20 C) and
centrifuged
at 10000g at 4 C for 10 minutes. The pellet was mixed with 10111 of a solution
containing 10% SDS (w/v) and 2.3% DTE (w/v). The sample was heated to 95 C
for 5 minutes and then diluted to 60111 with a solution containing 8M urea, 4%
CHAPS (w/v), 40mM Tris, 65mM DTE and a trace of bromophenol blue. The
whole fmal diluted CSF sample corresponding to 45pg was loaded in a cup at the
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
23
cathodic end of the 1PG strips. 2-DE was performed as described previously
[10].
In brief, the first dimensional protein separation was performed using a
commercial 18cm non-linear LPG going from pH 3.5 to 10 from Amersham
Biosciences (Uppsala, Sweden). The second dimensional separation was
performed onto in-house manufactured vertical gradient slab gels (9-16% T,
2.6%
C). Analytical gels were then stained with ammoniacal silver staining [11].
Gels
were scanned using a laser densitometer (Amersharn Biosciences, Uppsala,
Sweden). 2-DE computer image analysis was carried out with the MELANIE 3
software package [12].
Mass spectrometry identification
Differentially expressed spots were found through the comparison of analytical
gels of deceased vs. healthy CSF (n=4). Spots of interest were analysed by
peptide mass fingerprinting using a matrix-assisted laser
desorption/ionization
time-of-flight mass spectrometer (PerSeptive Biosystems Voyager STR MALDI-
TOF-MS, Framingham, MA, USA) [10] and identified through database using the
PeptIdent tool (http://www.expasy.ch/sprot/peptident.html).
Table 1:
A-FABP P15090
E-FABP Q01469
PGP 9.5 P09936
GFAP P14136
Prostaglandin D synthase P41222
Neuromodulin P17677
Neurofilament L P07196
Calcyphosine Q13938
RNA binding regulatory subunit 014805
Ubiquitin fusion degradation protein 1
homolog Q92890
Nucleoside diphosphate lcinase A P15531
Glutathione S tranferase P P09211
Cathepsin D P07339
H-FABP P05413
B-FABP 015540
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
24
EXAMPLE 2
Using two-dimensional gel electrophoresis (2-DE) separation of cerebrospinal
fluid (CSF) proteins and mass spectrometry techniques, FABP was found elevated
in the CSF of deceased patients, used as a model of massive brain damage.
Since
H-FABP, a FABP form present in many organs, is also localised in the brain, an
enzyme-linked immunosorbant assay (ELISA) was developed to detect H-FABP
in stroke vs. control plasma samples. However, H-FABP being also a marker of
acute myocardial infarction (AMA Troponin-I and creatine kinase-MB (CK-MB)
levels were assayed at the same time in order to exclude any concomitant heart
damage. NSE and S1 00B levels were assayed simultaneously.
Study population and samples handling
The population used for the assessment in plasma of the various markers
detailed
below included a total of 64 prospectively studied patients (Table 2) equally
distributed into three groups: (1) a Control group including 14 men and 8
women
aged 65 years (ranges: 34-86 years) with no known peripheral or central
nervous
system condition; (2) a group of patients with acute myocardial infarction
(AMI
group) including 14 men and 6 women aged 65 years (ranges: 29 to 90 years);
the
diagnosis of AMI was established in all cases by typical electrocardiography
modifications and elevated levels of CK-M13 (above a cut-off value of 9.3
ng/ml)
and of Troponin-I (above a cut-off value of 2 ng/ml) ; (3) a group of patients
with
acute stroke (Stroke group) including 14 men and 8 women aged 65 years
(ranges:
to 87 years); the diagnosis of stroke was established by a trained neurologist
25 and was based on the sudden appearance of a focal neurological deficit
and the
subsequent delineation of a lesion consistent with the symptoms on brain CT or
MRI images, with the exception of transient ischemic attacks (TIAs) where a
visible lesion was not required for the diagnosis. The Stroke group was
separated
according to the type of stroke (ischemia or haemorrhage), the location of the
30 lesion (brainstem or hemisphere) and the clinical evolution over time
(TIA when
complete recovery occurred within 24 hours, or established stroke when the
neurological deficit was still present after 24 hours).
CA 02932077 2016-06-03
WO 2005/029088 PCT/GB2004/050012
Table 2
Group Control AM! Stroke Stroke
Diagnosis Location TA=
lschemia Haemorrhage
Brainstem Hemisphere TIA CVA
Diagnosis
Number 22 20 22
Stroke 22
H-FABP
OD>0.531 0 20 15 11 4 3 12 3 12
OD<0.531 22 0 7 5 2 1 6 2 5
Troponin-1
>2ng/m1 0 20 1
<2ng/m1 22 0 21
For each patient of the three groups, a blood sample was collected at the time
of
5 admission in dry heparin-containing tubes. After centrifugation at 1500g
for
15min at 4 C, plasma samples were aliquoted and stored at ¨20 C until
analysis.
For the Stroke group, three additional blood samples were collected after the
neurological event: <24 hours; <48 hours; and >48 hours. In this group, the
time
interval between the neurological event and the first blood draw was 185
minutes
10 (ranging from 40 minutes to 3 days). This parameter was taken into
account in
the data analysis. Each patient or patient's relatives gave informed consent
prior
to enrolment.
FABP measurement
15 H-FABP levels were measured in plasma by a sandwich ELISA. A 96-well
polystyrene microtitre plate (NUNC, Polylabo, CH) was coated with 100111/well
polyclonal goat anti human muscle FABP (Spectral Diagnosis HC, Ontario,
USA), 20.41.1g/m1 in carbonate buffer 0.1M pH 9.6, overnight at 4 C. The plate
was automatically washed with PBS (15naM Na2PO4-120mM NaC1-2.7naM KC1
20 pH 7.4, Sigma) on a BioRad NOVAPATHTm WASHER (Hercules, CA). Every
washing step was performed with fresh PBS. Non-specific binding sites were
blocked with 200111/well 2% casein (w/v) in carbonate buffer for 2h at 37 C.
After the washing step, the samples were pipefted in duplicate at 100 1/well.
The
plate was incubated 2h at 37 C. After the washing step, 100 1/well of mouse
25 anti-human Heart FABP (clone 66E2, HyCult biotechnology b.v, Uden,
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
26
Netherlands), 0.3ug/m1 in PBS-1% BSA (w/v), were incubated for lh at room
temperature (RN) with shaking. After the washing step, 100 1/well of
phosphatase labelled anti-mouse immunoglobulins (Dako, Denmark), 15 g/m1 in
PBS, were incubated 1h30 at R.T. with shaking. After the washing step,
50ml/well of phosphatase substrate, 1.5mg/m1paranitrophenylphosphate in
diethanolamine, were incubated 30min. Reaction was stopped with 10041/well
NaOH 1M. Colour development was assessed with a tnicroplate reader,
MilenkzTM kinetic analyzer (DPC, LA, USA), at a wavelength of 405ntn.
CK-M33 and Troponin-I measurement
Plasma samples were centrifuged at 1500g for 15min, and aliquots were stored
at
¨20 C. Serum CK-MB and Troponin-I levels were determined using a
fluorescent microparticle enzyme immunoassay (META) with an automated
chemical analyser AxSYlVFm system (ABBOTT Laboratories, Abbott Park, IL).
The formation rate of fluorescent products was directly proportional to the
amount
of Troponin-I in the sample. The detection limit for Troponin-I was 0.3 g/l.
CK-
MB measurement is proportional to the amount of fluorescent probes and the
detection limit was 0.7141.
NSE and S100 measurement
Similar to H-FABP measurements, NSE and SlOOB were assayed in the four
serial plasma samples of the Stroke group. The SMART S-100 and SMART-NSE
ELISA kits were used. Both have been commercialised by Skye PharmaTech Inc.
(Ontario, CA). The detection limits for NSE and SlOOB were 1 g/1 and 0.01 g/1
respectively.
Statistical analysis
H-FA13P levels were expressed in optical density (OD) values as mean + SD.
Because recombinant H-FABP was not available, external calibration could not
be
performed to express results as concentration units (ng/ml). Troponin-I and CK-
MB levels, were expressed in ng/ml. Because plasma H-FABP, troponin-I and
CK-MB concentrations did not fulfill the criteria for a gaussian distribution
in
neither of the normal, stroke and AMI populations according to the Kolmogorov-
Smimov test, comparisons between the three groups was carried out using the
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
27
non-parametric Kruskall-Wallis test with post hoc Dtum's procedure.
Comparisons between the stroke subgroups defined above were made by means of
the Mann-Whitney Utest and longitudinal assessment of H-FABP concentrations
over time were analyzed using the repeated measures analysis of variance
(ANOVA). Reference limits for H-FABP aiming at distinguishing stroke versus
normal patients were delineated using receiver operating characteristic (ROC)
curves (AnalyseItTM software for Microsoft ExcelTM) and, subsequently,
sensitivity, specificity, positive and negative predictive values were
calculated at
each time point. Statistical significance was set at p<0.05.
Results
Individual results of the H-FABP assay in the three populations, expressed in
OD
units, are graphically shown in Figure 1 and detailed in Table 3. Mean plasma
H-
FABP concentration was 0.221 + 0.134 OD in the Control group, 1.079 + 0.838
OD in the Stroke group and 2.340 + 0.763 OD in the AMI group. The coefficient
of variation found for this ELISA was 5.8% 3.8. Using the Kruskall-Wallis
test,
all three concentrations were found significantly different (p<0.001) from
each
other. The best cat-off value to discriminate between the Control and the
Stroke
groups was set at OD > 0.531 as determined by the ROC curves for II-FABP level
(data not shown). Using this cut-off value, validity measures of H-FABP for
the
diagnosis of stroke were as follows: sensitivity was 68.2 % with 15 out of 22
stroke patients above the cut-off, specificity was 100 % with all of the 22
control
subjects below the cut-off, positive predictive value was 100 % and negative
predictive value was 75.9 %.
Table 3
Group Control AMI Stroke
H-FABP mean 0.221 2.434 1.079
SD 0.134 0.638 0.838
Significance <0.001 <0.001
Troponin-I mean 0.0 164.6 0.5
SD 0.1 205.6 1.3
Significance <0.001 as
CK-MB mean 1.3 63.8 7.9
SD 0.9 51.5 21.3
Significance <0.001 ns
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
28
To discriminate, at the biological level, between patients from the AMI and
the
Stroke groups, Troponin-I and CK-MB were further assayed in each group with
cut-off values set at 2 ng/m1 for the AxSYM Troponin-I assay and 3.8 ng/ml for
the AxSYM CK-MB assay (Table 3). As expected, the concentrations of these
AMI markers were significantly higher (p<0.01) in the AMI group as compared to
both the Control and the Stroke groups. No difference was found between the
last
two groups, thus confirming that Troponin-I and CK-MB do not increase as a
result of a brain insult and that stroke patients did not sustain a
concomitant AMI
at the time of their stroke. Taken together, H-FABP, Troponin-I and CK-MB
concentrations allowed a correct discrimination between AMI (increase of all
three markers) and stroke (increase of H-FABP with normal Troponin-I and CK-
MB) in all the 20 AMI patients and in 15 stroke patients, with the exception
of
one stroke patient showing, along with increased H-FABP levels, moderately
elevated levels of Troponin-I and CK-MB in the absence of EKG modifications,
all of which being consistent with a concomitant non-AMI heart damage.
In the Stroke group, seven false negative results were found with H-FABP
levels
below the cut-off value of OD 0.531 at any time point following the
neurological
event Of these seven patients, two had a rapid and complete recovery of their
neurological deficits within 24 hours consistent with a transient ischernic
attack
(TIA), and two have had a lacunar stroke on MAI images, one located in the
brainstena. While TIA and lacunar stroke may explain false negative results in
a
majority of patients, no explanation was consistently found for the three
remaining stroke patients with low TI-FAD? levels.
Sequential determinations of H-FABP level after stroke showed that 10 out of
15
(67 %) H-FABP positive stroke patients had a very early increase of H-FABP
levels (<12 hours). Moreover, as shown in Figure 2, when all stroke patients
were
considered, the mean H-FABP concentrations decreased steadily after the
insult,
the highest value being found before 12 hours. The differences between the
initial
measurement and the less than 48 hours and afterwards measurements were
significant (ANOVA, p<0.05). When H-FABP levels were compared between the
different subgroups of the Stroke group, no statistically significant
differences
were found. H-FABP levels were similar for ischemia (0.955 + 0.668, N=15)
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
29
versus haemorrhage (1.346 + 1.139, N=7) strokes, and for hemispheric (0.987 +
0.783, N=18) versus brainstem (1.493 + 1.080) strokes, but the statistical
power of
the analyses was limited by the small size of the samples to be compared.
However, when comparing established strokes versus TIAs, the former (1.2002 +
0.892) showed nearly twice as high II-FABP levels as the latter (0.652 +
0.499),
although this difference failed to reach significance (Mann-Whitney U test,
p=0.24).
NSE and S1 00B were assayed in the Control and the Stroke groups, and
the results were compared with the H-FABP assay. The cut-off values using the
SMART-NSE and SMART SlOOB protein ELISA tests for the diagnosis of stroke
were 10 ng/ml for NSE and 0.02 ng/rril for S1 00B. NSE and SlOOB levels were
slightly increased in the Stroke groups (14.12 ng/ml and 0.010 ng/ml,
respectively) as compared to the Control group (15.88 rig/m1 and 0.004 ng/ml,
respectively). As shown on Table 4, specificity, sensitivity, PPV and NPV for
the
diagnosis of stroke were found much lower for NSE and SlOOB than for H-FABP.
These differences are relevant since the three markers have been tested in the
same samples.
Table 4
H-FABP NSE S10013
Sensitivity 68.2 55 15
Specificity 100 36.4 95.5
Positive 100 44 75
predictive
Negative 75.9 47.1 55.3
predictive
EXAMPLE 3
Three new proteins have been identified on 2-DE gels prepared with CSF samples
from deceased patients. These proteins correspond to spots that have been
previously shown increased in CSF samples from deceased patients relative to
healthy controls. However, previous attempts to identify these proteins using
MALIN-TOP mass spectrometry were unsuccessful. The current experiments
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
were performed. by LC-MS-MS using ESI-Ion Trap device (DecaLCQ XP,
ThennoFinnigan). Furthermore, the increasing amount of data in databases could
lead to the successful identification of previously uncharacterized spots.
5 (1) RNA-binding protein regulatory subunit (014805) DJ-1 protein
(Q99497):
RNA-binding protein regulatory subunit has been previously described in
deceased CSF samples (see Example 1 above). Here, we have obtained the same
identification with an adjacent spot (Figure 3). We also confirmed the
previous
10 identifications. Figure 1 shows enlargements of healthy CSF and deceased
CSF
2-DE maps. 270 pg of protein was loaded on a TPG gel (pH 3.5 ¨ lONL, 18cna).
The second dimension was a vertical slab gel ( 12 %T). The gel was stained
with
a fluorescent dye. The upward-pointing arrows indicate the spots corresponding
to the RNA binding regulatory subunit or to the DJ-1 protein.
Furthermore, our results indicate that these spots could correspond to another
homologous protein called DJ-1. The RNA-binding protein regulatory subunit
and DJ-1 sequences differ from one another only by one amino acid. The single
peptide detected during the current experiments did not contain this specific
residue.
DJ-1 gene mutations are associated with autosomal recessive early-onset
parldnsonism (Bonifati et al., Science, 2003). Different results suggest that
the
DJ-1 protein could be involved in cellular oxidative stress response and
neurodegenerative pathologies (Bonifati et al., Science, 2003; Wilson et al.,
PNAS, 2003).
(2) Peroxiredoxin 5 (P30044):
The 2-DE spot corresponding to Peroxiredoxin 5 is shown in Figure 4. This is
an
enlargement of healthy CSF and deceased CSF 2-DE maps prepared in the same
way as for Figure 3. The spot corresponding to Peroxiredoxin 5 is shown by the
arrows on the right-hand side.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
31
Peroxiredoxin 5 is an antioxidant enzyme that could have a neuroprotective
effect
(Plaisant et al., Free Radic. Biol. Med., 2003). Aberrant expression pattern
of
proteins belonging to the Peroxiredoxin family was also described in brains of
patients with different neurodegenerative diseases (Krapfenbauer et al.,
Electrophoresis, 2002; Krapfenbauer et al., Brain Res., 2003).
(3) Peptidyl-prolyl cis-trans isomerase A or Cyclophilin A (P05092)
Two spots were identified as being the Peptidyl-prolyl cis-trans isomerase A
(Figure 5). This is an enlargement of healthy CSF and deceased CSF 2-DE maps
prepared in the same way as for Figure 4. The basic spot corresponding to
Cyclophilin A is just adjacent to the spot corresponding to the Peroxiredoxin
5.
Cyclophilin A was described as a protective factor against cellular oxidative
stress
(Doyle et al., Biochem J., 1999). rt binds to Peroxiredoxin enzymes and could
be
involved in the peroxidase activity (Lee et al., JBiol. Chem., 2001).
Furthermore,
a publication suggests that Cyclophilin A is secreted by vascular smooth
muscle
cells (VSMC) in response to oxidative stress and stimulate VSMC growth (Jin et
al., Circ. Res., 2000). These results suggest the implication of Cyclophilin A
in
vascular diseases processes. A link was also described with a familial form of
amyotrophic lateral sclerosis (a neurodegenerative pathology) associated with
a
mutation in the antioxidant enzyme Cu/Zn Superoxide Dismutase-1 (Lee at al.,
PNA.S, 1999). Cyclophilin A seems to have a protective effect against the
mutant
SOD-induced apoptosis.
EXAMPLE 4
Introduction
A survey of stroke patients was carried out and the results are shown in
Figures 6
to 15. An ELISA intensity signal was obtained for Ubiquitin fusion degradation
protein 1 homo log (UFD1), RNA binding regulatory subunit (RNA-BP) and
nucleotide diphosphate lcinase A (NDK A) in plasma samples of the patients and
of negative control patients. Plasma samples were taken from patients between
0-
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
32
24 hours and/or after 72 hours of arrival at emergency hospital, and were
matched
for age/sex with samples from control patients.
Protocol
ELISA was performed. using 96-well Reacti-Bindm NeutrAvidinTM coated Black
Plates (Pierce, Rockford, IL). Plates were first rinsed in Borate Buffer
Saline pH
8.4 (BBS) (100 mM II3B03, 25 mM Na2B407 (Sigma, St Louis, MO, USA), 75
mM NaC1 (Merck, Darmastadt, Germany)) on a NOVAPATHTm washer (Bio-
Rad, Hercules, CA). Then, 50 1 of antibody-biotin conjugated (2 gg/mL)
prepared in the dilution buffer A at p117 (DB, Polyvinyl Alcohol, 80%
hydrolyzed, Mol. Wt. 9000-10,000 (Aldrich, Milwaukee, WI, USA), MOPS (3-
[N-Morpholino] propane sulfonic acid) (Sigma), NaC1, MgC12 (Sigma), ZnCl2
(Aldrich), p116.90, BSA 30% Solution, Manufacturing Grade (Serological
Proteins Inc., Kankakee, IL)), were added and incubated for one hour at 37 C.
Plates were then washed 3 times in BBS in the plate washer. 50 p..1 of antigen
was
then added and incubated for one hour at 37 C. Recombinant proteins were
diluted at 100, 50, 25, 12.5, 6.25 ng/m1 in the dilution buffer A to establish
a
calibration curve. Plasma samples were diluted at the appropriate dilution in
the
dilution buffer A. After the washing step, 501.d of alkaline phosphatase
conjugated antibodies were added at the appropriate dilution in the dilution
buffer
A and incubated for one hour at 37 C. The 96-well plate was then washed 3
times
with BBS in the plate washer and 50 !IL of fluorescence Attophos AP
Fluorescent substrate (Promega, Madison, WI) were added. Plates were read
immediately on a SpectraMax GEMINI-XS, (Molecular Devices Corporation,
Sunnyvale, CA, U.S.A.) fluorometer rnicrotiter plate reader relative
fluorescence
units (RFU) (Xacitation'444 nnl and Xtinission=555 urn).
We read plates in fluorescence using a SpectraMax GEMINI-XS (Molecular
Devices) fluorometer microplate reader (Xexcitation-411 nm and Xemission=555
nm). Results are expressed in RFU and can be obtained in endpoint mode (only
one reading) or in kinetic mode on 10 minutes. In kinetic mode, for each well
we
used 6 flashes (per well) which are integrated into an average and read each
well 6
times using minimal interval time between each reading. This ends up being 2
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
33
minutes between readings. We determined a slope and this is what we used for
our valuations. The best cut-off value to discriminate between the Control and
the
Stroke (Ischemic plus hemorrhagic or Ischemic vs. Hemorrhagic) groups was
determined by the ROC curves using GraphPad Prism 4 software.
Conclusion
We can clearly see from Figures 7, 10 and 13 that UFD1, RNA-BP and NDK A
respectively are overexpressed in stroke patients compared to control
patients.
Statistical analysis for each of the biomarker was performed and ROC curves
(GraphPad Prism 4 software) indicating sensitivity of the test as a function
of 1-
specificity (Figures 8, 11 and 14 for UFD1, RNA-BP and NDK A respectively)
were drawn. Best cutoff values to distinguish between stroke and control
patients
were deduced from these ROC curves. We obtained 94.4%, 94.4% and 100%
sensitivity for UFD1, RNA-BP and NDK A respectively and 77.8%, 72.2% and
83.3% specificity for UFD1, RNA-BP and NDK A respectively. For each marker,
a non parametric Mann Whitney test was performed to compare stroke and control
groups. For the 3 biomarkers, we obtained very low p values (<0.0001 for UFD1
and NDK A and p=0.0003 for RNA-BP) meaning that differences between stroke
and controls are very significant.
In Figure 6, we can also notice that RNA-BP and NDK A can detect a stroke only
minutes after symptoms onset, meaning that these are very early markers of
stroke. This result is confirmed by the decreasing signal observed between
arrival
at the hospital and after 72 hours. Patients 202 and 239 were tested at the
arrival
25 (between 0 and 24 hours) and after 72 hours and we can see that for all
the
markers, the signal significantly decreases.
These results demonstrate that Ubiquitin fusion degradation protein 1 homolog
(UFD1), RNA binding regulatory subunit (RNA-BP) and nucleotide diphosphate
30 ldnase A (NDK A) are useful markers for early diagnosis of stroke,
alone, in
combination, or combined with other biomarkers.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
34
EXAMPLE 5
This Example is concerned with post-translational modifications that can be
induced in neurodegenerative disorders. The study population and samples
handling, and the CSF 2-DE were as described in Example I.
2-DE imnaunoblotting assays
Proteins separated by 2-DE were electroblotted onto PVDF membranes
essentially as described by Towbin et al. [22]. Membranes were stained with
Amido Black, destained with water and dried. Proteins of interest were
detected
as previously described [29] using specific antibodies and BCLTM western
blotting
detection reagents (Amersham Biosciences, Uppsala, Sweden). We used the
following antibody: anti-human Prostaglandin D synthase (lipocalin type)
rabbit
polyclonal antibody (Cayman chemical, Ann Arbor, MI) diluted 1/250.
Figure 16(A) shows a comparison of PGHD spot intensities on 2-DE gels
prepared with CSF of deceased or control patients. Forty-five jig of protein
was
loaded on an 1PG strip (pH 3.5-10 NL, 18 cm). The second dimension was
performed on a vertical gradient slab gel (9-16 % T), stained with ammoniacal
silver. Apolipoprotein Al labelled in italic showed similar levels in the two
samples. PG1ID spot locations in control gel were deduced from previous
identifications [31]. In the gel from deceased patients, putative PGHD spot
locations are given. Figure 16(B) shows immunodetection of PGHD in 2-DE gels
prepared with CSF from deceased and control patients. 2-DE was performed as
indicated in A. Immunodetection was performed as previously described [29]
using an anti-human Prostaglandin D synthase (lipocalin type) rabbit
polyclonal
antibody and ECLTM western blotting detection reagents.
Results
Prostaglandin D synthase (PG111)) is a basic protein (p1= 8.37) known to be
post-
translationaly modified by N-glycosylation (Hoffmann A. et aL, J. Neurochem.
1994, 63, 2185-2196). On CSF 2-D gels from healthy living patients, five spots
were detected. On 2-D gels prepared with post-mortem CSF, the three acidic
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
spots are strongly decreased with a concomitant increase of the two basic
spots
(Figure 16A).
In order to confirm that these different spots correspond to PGHD, we
performed
5 immunoblot assays using a specific antibody (Figure 16B). The results
obtained
confirmed that the acidic PGPID spots were not present in the CSF from
deceased
patients while the basic spots were still present. Furthermore, the
measurement of
the total PGHD spot volume in the two gels using the Melanie 3 software
indicated that the PGHD level is similar in the two samples. This suggests,
10 therefore, that there was a deglycosylation of PGHD in the CSF of
deceased
patients but the total PGHD amount remained constant.
Data from the literature:
PGHD was found to be decreased in the CSF of patients suffering from AD
15 (Puchades M. et al., Brain Res. Mol. Brain Res. 2003, 118, 140-146).
However,
the study was performed using 2-DE gels and only the acidic spots were
analyzed.
As shown by our results on CSF from deceased patients, it is possible that PUT-
ID
was deglycosylated in the samples analyzed, resulting in the disappearance of
acidic spots but no decrease in the total protein level.
Using capillary isoelectrie focusing, Hiraoka and colleagues have identified
changes in the charge microheterogeneity of CSF PGIID associated with various
neurological disorders (Hiraoka A. at al., Electrophoresis 2001, 22, 3433-
3437).
The ratio of basic forms/acidic forms was found to increase in AD, in PD with
pathological brain atrophy, and in multiple sclerosis. It was speculated that
these
post-translational modifications were linked to changes in the N-glycosylation
pattern.
PGIID post-translational modifications (PTM) pattern in CSF of a
Creutzfeldt-Jakob (CJD) disease patient:
We compared the PTM pattern of PGHD in CSF samples collected from a CJD
patient and a healthy control. The proteins were separated by 2-DE,
electroblofted
on a PVDF membrane and PGHD was detected using a specific antibody, as
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
36
previously described. The CSF samples were collected by lumbar puncture. The
control patient had a neurological workup for benign conditions unrelated to
brain
damage. CSF samples were centrifuged immediately after collection, aliquoted,
frozen at -20 C and stored until analysis.
The results are shown in Figure 17 which is a comparison of prostaglandin D2
synthase spot intensities on mini-2-DE gels prepared either with CSF of a
patient
suffering from the Creutzfeldt-Jakob disease or with a control CSF from a
healthy
patient. Forty-five g of protein were loaded on a 1PG gel (p113-10 NL, 7 cm).
Second dimension was a vertical gradient slab gel (12% T). Immunodetection
was performed using an anti-human PGED (lipocalin type) rabbit polyclonal
antibody (Cayman chemical, Ann Arbor, MI) and ECLTM western blotting
detection reagents (Amersham Biosciences, Uppsala, Sweden).
The results showed that the PTM pattern of PGHD in the CSF from the CJD
patient is clearly different from the control, with a strong decrease of the 4
most
acidic spots (Figure 17). The pattern of the CJD patient is similar to the one
observed in post-mortem CSF. These data support the interest of changes in the
PTM pattern of PGIID as marker of neurological disorders.
EXAMPLE 6
This Example provides additional data showing plasma levels of UFDP1 in stroke
and control patients. Figure 19 shows the levels of UFDP1 in CSF of a control
and a deceased patient Additional data has been obtained from two cohorts of
patients and controls, the smaller from Geneva, and a more comprehensive panel
from the US. The methodology for this Example and following Examples 7 and 8
is the same, save that the antibodies being used have different specificities
for the
protein in question. The method in each of the studies is similar to that
given as
Example 4:
ELISA was performed using 96-well ReactiBindTM NeutrAvidinTm coated
Black Plates (Pierce, Rockford, IL). Plates were first rinsed in Borate Buffer
Saline pH 8.4 (BBS) (100 mM 113B03, 25 mM Na2B407 (Sigma, St Louis,
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
37
MO, USA), 75 mM NaC1 (Merck, Darmastadt, Germany)) on a
NOVAPATHI'm washer (Bio-Rad, Hercules, CA). Then, 50111 of relevant
biomarker specific antibody-biotin conjugate (2 i_tg/mL) prepared in the
dilution buffer A at p117 (DB, Polyvinyl Alcohol, 80% hydrolyzed, Mol. Wt.
9000-10,000 (Aldrich, Milwaukee, WI, USA), MOPS (3-[N-Morpholino]
propane sulfonic acid) (Sigma), NaC1, MgC12 (Sigma), ZnC12 (Aldrich),
p116.90, BSA 30% Solution, Manufacturing Grade (Serological Proteins Inc.,
Kankakee, IL), were added and incubated for one hour at 37 C. Plates were
then washed 3 times in BBS in the plate washer. 50 Al of antigen or plasma
was then added and incubated for one hour at 37 C. Recombinant protein
antigens were diluted at 100, 50, 25, 12.5, 6.25, 3.125, 1.56 ngiml in the
dilution buffer A to establish a calibration curve. Plasma samples were
diluted
at the appropriate dilution in the dilution buffer A. After a further washing
step, 50111 of relevant biomarker specific alkaline phosphatase conjugated
antibodies were added at the appropriate dilution in the dilution buffer A and
incubated for one hour at 37 C. The 96-well plate was then washed 3 times
with BBS in the plate washer and 50 IA of fluorescence Attophos AP
Fluorescent substrate (Promega, Madison, WI) were added. Plates were read
immediately on a SpectraMax GEMINI-XS, (Molecular Devices Corporation,
Sunnyvale, CA, U.S.A.) fluorometer microtiter plate reader
We read plates in fluorescence using a SpectraMax GEMINI-XS (Molecular
Devices) fluorometer microplate reader (?excitation- =444 tun and 2t-
-emission'=555 am).
Results are expressed in RFU and can be obtained in endpoint mode (only one
reading) or in kinetic mode on 10 minutes. In kinetic mode, for each well we
used
6 flashes (per well) which are integrated into an average and read each well 6
times using minimal interval time between each reading. This ends up being 2
minutes between readings. We determined a slope and this is what we used for
our valuations. The best cut-off value to discriminate between the Control and
the
Stroke groups was determined by the ROC curves using GraphPad Prism 4
software.
The results are shown in Figure 20.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
38
EXAMPLE 7
This corresponds to Example 6, except that the polypeptide is RNA-BP. Figure
21 shows the levels of RNA-BP in CSF of a control and a deceased patient.
Figure 22 shows RNA-BP plasma concentration by ELISA for three studies, each
comprising stroke patients and controls.
EXAMPLE 8
This corresponds to Example 6, except that the polypeptide is ND1CA. Figure 23
shows the levels of NDKA in CSF of a control and a deceased patient. Figure 24
shows NDICA plasma concentration by ELISA for the Geneva and US cohorts of
stroke patients and controls as in Example 6.
EXAMPLE 9
In addition to simple discrimination between stroke and control patients, the
data
from each of Examples 6, 7 and 8 can be analysed in relation to the time
between
cerebrovascular accident and sample collection, or alternatively in relation
to the
type of stroke ¨ ischaemic, haemorrhagic or transient ischaemic attack (TIA).
These separate analyses are shown in Figure 25a and Figure 25b and demonstrate
the utility of deceased CSF markers in the diagnosis of stroke. This is
particularly
relevant to clinical practice as it is essential to diagnose stroke within
three hours
of the event to allow administration of clot busting drugs such as TPA. It is
also
essential that tests can differentiate haemorrhagic stroke from ischaemic
attack as
TPA is only suitable for the treatment of ischaemia and can have catastrophic
effects in patients with an haemorrhagic stroke.
EXAMPLE 10
Whilst each of the deceased CSF markers have good individual performance for
the diagnosis of stroke, it is likely that a commercial product will require
the
measurement of levels of several proteins. This 'panel' approach can be
achieved
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
39
in two ways. In the simpler approach the antibodies for each separate marker
are
pooled and used to coat microtitre wells. The intensity of the signal will be
the
sum of that for each independent marker, though in this case it will be
impossible
to determine the individual levels of each of the markers. This may create
challenges in setting meaningful cut-off values, however, this presents the
most
user friendly commercial product.
Figure 26 summarises the markers which are used in this Example. Experimental
results are shown in Figure 27, in which antibodies against the deceased CSF
proteins UFD1, RNA-BP, NDKA and H-FABP were used at the same
concentrations as in Example 4. However, these antibody solutions were mixed
in
equal volumes, reducing the concentration of each antibody species to one
quarter
of the original level in the single analyte examples described above. The
protocol
used is as follows:
To overcome the problem of panel algorithm, we tested the four antibodies
directly in mixture in each well. The protocol is exactly the same as
previously described for separated antibodies (above), save that each of the
biomarker specific biotin-antibody conjugates were used at 12.5 1., per well
during the first antibody coating step. The standard curve was similarly
constructed by using 12.5 12L per well of each of the four recombinant protein
antigens UFDP1, RNA-BP, NDKA and H-FABP each prepared separately at
initial concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56 ng/ml in the
dilution buffer A to establish a calibration curve on the same plate. Plasma
samples were used at the same dilution and volume (50 III, per well) as for
the
individual biomarker assays. Detection of captured antigens was performed
using the same biomarker specific antibody-alkaline phosphatases conjugates
as the individual assays, with equal volumes (12.5 !IL) of the four biomarker
specific antibody-alkaline phosphatases conjugates being added to each well
for the standard curve and plasma samples. Measurement of fluorescence was
performed as described for the single biomarker assays as described in the
example above.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
Ten stroke and ten control (non age/sex matched) plasma samples 2-fold
diluted were tested (Figure 27). This experiment led to 100% sensitivity and
80% specificity. The two false positives samples correspond to patient's
control 368 and 450 that display prostate cancer and probable head trauma.
5
In this specific example the fluorescence signal obtained corresponds to the
sum of the signal generated by each biomarker specific antibody sandwich and
it is impossible to determine the relative contribution of each single
biomarker
to the whole when using alkaline-phosphatase conjugated antibodies for the
10 detection side of the assay. It is also an aspect of the invention that
each
biomarker specific antibody can be labelled with a different fluorophore with
sufficient difference between their excitation and emission wavelengths that
the level of each antibody can be determined without interference. In this
case
it is possible to accurately quantify the levels of up to four different
15 biomarkers in a sample in a single assay, providing benefits in reduced
sample
requirement and increased throughput.
EXAMPLE 11
20 In some circumstances it may not be desirable to measure levels of
multiple
analytes in a single well. For example the absolute levels of individual
proteins,
or the ratio between levels of multiple proteins may be necessary to make a
specific diagnosis. In this situation it may be desirable to measure the
levels of
each analyte in a separate assay. A predictive algorithm is then used to
interpret
25 these multiparametic datasets to provide a unique diagnosis for each
patient. In
this Example we have used a statistical algorithm to predict the theoretical
performance of different multi-analyte biomarker panels.
The datasets of individual biomarker levels generated in the various examples
30 above were analysed using a proprietary algorithm to determine the true
positive and true negative rates for each combination of the deceased CSF
proteins UFDP1, RNA-BP, NDKA and IT-FABP for the diagnosis of stroke.
For the analysis a Sample set (18 controls and 18 stroke for UFD1, RNA-BP,
NDK A and H-FABP) was divided into 2 random populations.
CA 02932077 2016-06-03
WO 2005/029088 PCT/GB2004/050012
41
80% of the total samples for training of the thresholds was performed by the
technique of naive bayes, and the remaining 20 % of the total samples were
then used to evaluate the thresholds (sensitivity and specificity) for each
marker, or combination of markers made 1000 fold.
Where the algorithm was applied to single proteins it was possible to compare
sensitivities and specificities values with those observed. The sensitivity
and
specificity for these data sets (figures in parentheses) were calculated based
on
the optimum cut-off determined from the ROC curve as described in the
examples above. In the following data, the first value in parenthesis
corresponds to standard deviation (e.g. 0.93 + 0.15). The second value in
parenthesis for the "1 protein" data corresponds to sensitivity (SE) and
specificity (SP) obtained without using the algorithm, but using simple ROC
curve (GraphPad Prism). The SE and SP values are indicated just to compare
the results with and without the algorithm.
The output of this algorithm analysis was as follows:
1 protein
True positive rate of UFD1 on training set: 0.93 (0.15) (SE 94%)
True negative rate of UFD1 on training set: 0.74 (0.24) (SP 78%)
True positive rate of RNA-BP on training set: 0.85 (0.21) (SE 94%)
True negative rate of RNA-BP on training set: 0.73 (0.23) (SP 72%)
True positive rate of H-FAI3P on training set: 0.47 (0.29) (SE 39%)
True negative rate of IT-FABP on training set: 0.80 (0.23) (SP 100%)
True positive rate of NDK A on training set: 0.79 (0.24) (SE 100%)
True negative rate of NDK A on training set: 0.89 (0.16) (SP 83%)
2 proteins
True positive rate of UFD1/RNA-BP on training set: 0.90 (0.17)
True negative rate of UFD1/RNA-BP on training set: 0.69 (0.25)
True positive rate of UFD1/H-FA_BP on training set: 0.82 (0.22)
True negative rate of UPT)1/H-FABP on training set: 0.83 (0.20)
True positive rate of UFD1/NDK A on training set: 0.92 (0.16)
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
42
True negative rate of UFD1/NDK A on training set: 0.79 (0.21)
True positive rate of RNA-BP/H-FABP on training set: 0.81 (0.24)
True negative rate of RNA-BP/H-FABP on training set: 0.73 (0.24)
True positive rate of RNA-BP/NDK A on training set: 0.91 (0.16)
True negative rate of RNA-BP/NDK A on training set: 0.83 (0.21)
True positive rate of H-FABP/NDK A on training set: 0.77 (0.27)
True negative rate of II-FAl3P/NDK A on training set: 0.84 (0.20)
3 proteins
True positive rate of RNA-BP/NDK A/H-FABP on training set: 0.96
(0.11)
True negative rate of RNA-BP/NDK A/11-FABP on training set: 0.83
(0.20)
True positive rate of UFD1/NDK A/II-FABP on training set: 0.92 (0.17)
True negative rate of IIFD1/NDK .AJH-FABP on training set: 0.83 (0.21)
True positive rate of UFD1/RNA-BP/NDKA on training set: 0.95 (0.14)
True negative rate of UFD1/RNA-BP/NDKA on training set: 0.82 (0.20)
True positive rate of UFD1/RNA-BP/H-FABP on training set: 0.93
(0.15)
True negative rate of UFD1/RNA-BP/HFABP on training set: 0.75
(0.23)
The 4 proteins
True positive rate of UFD1/RNA-BP/H-FABP/NDK A on training set
0.93 (0.13)
True negative rate of UFD1/RNA-BP/H-FABP/NDK A on training set:
0.73 (0.23)
Figure 28 is a graphical representation of combinations of two out of the four
biomarkers used in this Example. It shows the cut-off points (horizontal and
vertical lines) which we have determined for diagnosis.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
43
EXAMPLE 12
Further large scale studies were performed in Geneva and USA on UFD1, RNA-
BP and NDK A post naortem CSF markers. ELISA was carried out on samples as
described in the previous Examples (both for the Geneva as well as the USA
experiments). The results are shown in Figures 29-38.
References
[1] Vaagenes P, Unial P, Melvoll R, Valnes K: Enzyme level changes in the
cerebrospinal fluid of patients with acute stroke. Arch Neurol 1986;43:357-
362.
[2] Lampl Y, Paniri Y, Ethel Y, Sarova-Pinhas I: Cerebrospinal fluid lactate
dehydrogenase levels in early stroke and transient ischemic attacks. Stroke
1990;21:854-857.
[3] Matias-Guiu J, Martinez-Vazquez J, Ruibal A, Colomer R, Boada M, Codina
A: Myelin basic protein and creatine lcinase BB isoenzyme as CSF markers of
intracranial tumors and stroke. Acta Neurol Scand 1986;73:461-465.
[4] Persson L, Hardernark HG, Gustafsson 3, Rundstrom G, Mendel-Hartvig I,
Esscher T, Pahlman S: S-100 protein and neuron-specific enolase in
cerebrospinal
fluid and serum: markers of cell damage in human central nervous system.
Stroke
1987;18:911-918.
[5] Cunningham RT, Young IS, Winder 3, O'Kane MJ, MclCinstry S, Johnston CF,
Dolan OM, Hawkins SA, Buchanan KD: Serum neurone specific enolase (NSE)
levels as an indicator of neuronal damage in patients with cerebral
infarction. Eur
I Clin Invest 1991;21 :497-500.
[6] Herrmann M, Vos P, Wunderlich MT, de Bruijn CH, Lamers ICJ: Release of
glial tissue-specific proteins after acute stroke: A comparative analysis of
serum
concentrations of protein S-1 00B and glial fibrillary acidic protein. Stroke
2000;31:2670-2677.
CA 02932077 2016-06-03
WO 2005/029088
PCT/GB2004/050012
44
[7] Bitsch A, Horn C, Kemmling Y, Seipelt M, Hellenbrand U, Stiefel M,
Ciesielczyk B, Cepek L, Bahn E, Ratzka P, Prange H, Otto M: Serum tau protein
level as a marker of axonal damage in acute ischemic stroke. Fur Neurol
2002;47:45-51.
[8] Watson MAScott MG: Clinical utility of biochemical analysis of
cerebrospinal
fluid. Clin Chem 1995;41:343-360.
[9] Hochstrasser DF, Frutiger S, Paquet N, Bairoch A, Ravier F, Pasquali C,
Sanchez JC, Tissot JD, Bjellqvist B, Vargas R, et at: Human liver protein map:
a
reference database established by rnicrosequencing and gel comparison.
Electrophoresis 1992;13:992-1001.
[10] Sanchez J-C, Chiappe D, Converset V, Hoagland C, Binz P-A, Paesano S,
Appel RD, Wang S, Sennitt M, Nolan A, Cawthome MA, Hochstrasser DF: The
mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and
obesity. Proteomics 2001;1:136-163.
[11] Hochstrasser DFMerril CR: 'Catalysts' for polyacrylasnide gel
polymerization
and detection of proteins by silver staining. Appl Theor Electrophor 1988;1:35-
40.
[12] Appel RD, Palagi PM, Walther D, Vargas .TR, Sanchez JC, Ravier F,
Pasquali
C, Hochstrasser DF: Melanie II--a third-generation software package for
analysis
of two- dimensional electrophoresis images: I. Features and user interface.
Mectrophoresis 1997;18:2724-2734.
