Pathology biomarker assay

1. A method of diagnosis or of quantitation of fibrosis comprising:

obtaining a patient biofluid sample,

conducting an immunoassay to measure an aggregate amount of N- or C-terminal neo-epitope containing protein fragments naturally present in said biofluid sample, and

associating an elevation of said measure in said patient above a normal level with the presence or extent of fibrosis, wherein said immunoassay is conducted by a method comprising;

contacting the N- or C-terminal neo-epitope containing protein fragments, formed by cleavage of an intact protein by a proteinase, that are naturally present in said sample with an immunological binding partner specifically reactive with the N- or C-terminal neo-epitope, but not reactive with the intact protein, and

measuring the extent of binding of the N- or C-terminal neo-epitope protein fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, wherein said immunological binding partner is raised against a synthetic peptide corresponding to an N- or C-terminal neo-epitope amino acid sequence formed by cleavage of an intact protein by a proteinase and specifically binds a neo-epitope constituted by said N- or C-terminal amino acid sequence, said N- or C-terminal amino acid being formed by cleavage of said intact protein at any one of the cleavage sites shown in the following table:

Collagen Type I

Cleavage sites marked ‘.’;

Protease

SEQ ID NO in ( )

FAP

Q.GA P GLQG M PG.E (1)

FAP

D.RGE P G P PGPAGFAGP P GAD.G (2)

FAP

D.GVRGLTGPIGPPG P AGA P GD.K (3)

FAP

A.GL P GA K GLTGSPGS P G P DGK.T (4)

FAP

D.AGPVGPPGP P G PP G P PGPPSAG.F (5)

FAP

D.GLNGLPGPIG PP G P RGRTGD.A (6)

FAP

S.PGKDGVRGLTGPIGP P GPAGA P .G (7)

FAP

L.PGP P G PP GPPGPPGLGGNFAPQ.L (8)

FAP

L. P G PP GPPGPPGPPGLGGNFA P Q.L (9)

FAP

P.GPPG PP G P PGLGGNFAPQLSY.G (10)

FAP

P.GPPG PP G PP GPPGLGGNFAPQL.S (11)

FAP

F.PGARGPSG P QG P GG P PGP K GNSG.E (12)

FAP

G.L P G PP GPPGPPGPPGLGGNFA P Q.L (13)

FAP

G.L P GPPGPPGPPG PP GLGGNFA P Q.L (14)

FAP

G.L P G PP GPPGPPGP P GLGGNFA P Q.L (15)

FAP

D.AGPVGPPGP P G PP G PP GPPSAGFD.F (16)

FAP

D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP G

PPSAG.F (17)

FAP

P.GPPG PP G PP GPPGLGGNFAPQLSYGYDE K STGGISVP

GP M GP.S (18)

FAP

D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PG

PPSAGFD.F(19)

MMP9 + FAP

D.RGE P G P PGPAGFAGP P GAD.G (20)

MMP9 + FAP

D.GVRGLTGPIGP P GPAGA P GD.K (21)

MMP9 + FAP

D.AGPVGPPG PP G PP GPPGPPSAG.F (22)

MMP9 + FAP

D.GLNGL P GPIG PP GPRGRTGD.A (23)

MMP9 + FAP

D.AGPVGPPGPPG PP G P PGPPSAGF.D (24)

MMP9 + FAP

L. P GPPGPPGPPG PP GLGGNFA P Q.L (25)

MMP9 + FAP

P.G PP GPPGP P GLGGNFA P QLSY.G (26)

MMP9 + FAP

P.GPPG PP G PP GPPGLGGNFAPQL.S (27)

MMP9 + FAP

G.L P G PP GPPGPPGPPGLGGNFA P Q.L (28)

MMP9 + FAP

L.PGP P G PP G PP GPPGLGGNFAPQL.S (29)

MMP9 + FAP

P.GPPG PP G PP GPPSAGFDFSFLPQPPQE K AH

DGGRYYR.A (30)

MMP9 + FAP

D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G

PP G P PGPPSAGFD.F (19)

MMP9

G.AAGPPGP.T (31)

MMP9

P.VGPVGARGP.A (32)

MMP9

P.AGPVGPVGARGP.A (33)

MMP9

P.RGL P GP P GA P GP.Q (34)

MMP9

G.EAGR P GEAGLPG.A (35)

MMP9

Q.DGR P G PP G PP GA.R (36)

MMP9

K.DGLNGL P GPIGP P GP.R (37)

MMP9

G.AKGE P G P VGVQGP P GP.A (38)

MMP9

P.AGARGNDGATGAAGP P GP.T (39)

MMP9

P.VGPPG PP G P PGPPGPPSAGF.D (40)

MMP9

A.GA P G K DGLNGLPGPIGPPGP.R (41)

MMP9

D.AGPVGPPGP P G PP G P PGPPSAG.F (42)

MMP9

S.AGAPGKDGLNGL P G P IG PP GP.R (43)

MMP9

G.PPGPPG PP GLGGNFA P QLSYG.Y (44)

MMP9

P.GP P G PP GPPGPPGLGGNFAPQL.S (45)

MMP9

G.L P G PP GPPGPPGPPGLGGNFA P Q.L (46)

MMP9

G.ERGPPGP M GPPGLAGPPGESGREGAPGAEG

SPG.R (47)

FAP

853‘.IGNVGAPGAK’862 (alpha 2 chain)

(48)

FAP

462‘.AGKEGPVGLP’471 (alpha 2 chain)

(49)

FAP

249‘.IGSAGPPGFP’258 (alpha 2 chain)

(50)

FAP

1153‘.DGLNGLPGPI’1162 (alpha 1 chain)

(51)

FAP

1153‘.D(β)GLNGLPGPI’1162 (alpha 1

chain) (52)

FAP

751‘.KGADGSPGKD’760 (alpha 1 chain)

(53)

FAP

171‘STGGISVPGP.’180 (alpha 1 chain)

(54)

FAP

167‘.YDEKSTGGIS’176 (alpha 1 chain)

(55)

Collagen Type III

Protease

Cleavage sites marked ‘.’

FAP

E.AGIPGVPGAK.G (56)

FAP

P.KGDPGPPGIP.G (57)

FAP

G.PQGPKGDPGPP.G (58)

FAP

G.PGMRGMPGSPGGP.G (59)

FAP

D.GPPGPAGNTGAPGSPGVSGPKGD.A (60)

FAP

H.AGAQGPPGPPGINGSPGGKGEMGPAGIP.G (61)

FAP

D.GPRGPTGPIGPPGPAGQPGD.K (62)

MMP9

D.GPPGPAGNTGAPGSPGVSGPKGD.A (63)

MMP9

P.GSPGPAGQQGAIGSPGPAGPRGP.V (64)

MMP9

G.ERGRPGLP.G (65)

MMP9

G.KGDRGENGSPG.A (66)

MMP9

E.PGKNGAKGEPGP.R (67)

MMP9

S.PGERGETGPPGP.A (68)

MMP9

P.GGPGADGVPGKDGP.R (69)

MMP9

G.PPGKDGTSGHPGP.I (70)

MMP9

R.GLPGPPGIKGPAGIPG.F (71)

MMP9

L.PGENGAPGPMGPRGAPGE.R (72)

MMP9

G.APGAPGGKGDAGAPGERGPP.G (73)

MMP9

A.PGLKGENGLPGENGAPGPMGPRGAPG.E (74)

MMP9

G.IAGITGARGLAGPPGMPGPRGSPGPQ.G (75)

MMP9

G.SPGAKGEVGPAGSPGSNGAPGQRGEPGP.Q (76)

MMP9

G.LKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAA.

G (77)

MMP9 + FAP

D.GPPGPAGNTGAPGSPGVSGPKGD.A (78)

ADAMTS-4

267‘.GFDGRNGEKG’276 (79)

FAP

533‘.PGMRGMPGSP’542 (alpha 1 chain)

(80)

Unknown

642‘.GLPGTGGPPG’651 (81)

Unknown

652‘ENGKPGEPGP.’661 (82)

Unknown

861‘.GERGSPGGPG’870 (83)

unknown

899‘PGKDGPPGPA.’908 (alpha 1 chain)

(84)

MMP9,

442‘.GLPGTGGPPG’451(85)

ADAMTS-4

Collagen Type IV

Protease

Cleavage sites marked ‘.’

MMP9

333‘.IVIGTGPLGE’342 (alpha 1 chain) (86)

MMP9

328‘PGPPGIVIGT.’337 (alpha 1 chain) (87)

Collagen Type V

Protease

Cleavage sites marked ‘.’

MMP2

920‘ERGPRGITGK.’929 (alpha 1 chain) (88)

MMP2/9

1584‘.RRNIDASQLL’1593 (alpha 1 chain) (89)

MMP2/9

229‘.QQGGAGPTGP’238 (alpha 2 chain) (90)

MMP2/9

355‘.QRGAHGMPGK’364 (alpha 2 chain) (91)

MMP9

525‘.RVGKMGRPGA’534 (alpha 3 chain) (92)

MMP2/9

1317‘.HMGREGREGE’1326 (alpha 3 chain) (93)

Collagen Type VI

Protease

Cleavage sites marked ‘.’

MMP8

873‘.RIAVAQYSDD’883 (94)

MMP8

1192‘.QLGTVQQVIS’1202 (95)

MMP12

1231‘.RDVVFLIDGS’1241 (96)

ApoE

Protease

Cleavage sites marked ‘.’

MMP9

81‘.LMDETM’86 (97)

Cat K

246‘RLDEVK.’251 (98)

MMP9

101‘TPVAEE.’106 (99)

Cat K

126‘.MEEMGS’131 (100)

Biglycan

Protease

Cleavage sites marked ‘.’

MMP3

219‘.KLTGIPKDLP’228 (101)

MMP3

226‘DLPETLNELH.’235 (102)

MMP12

220‘LTGIPKDLPE.’229 (103)

MMP13

329‘.RAYYNGISLF’338 (104)

MMP13

334‘GISLFNNPVP.’343 (105)

MMP12

87‘.EISPDTTLLD’96 (106)

MMP12

97‘LQNNDISELR.’106 (107)

MMP9

188‘.IEMGGNPLENS’198 (108)

MMP9

200‘FEPGAFDGLK.’209 (109)

MMP9

148‘.NHLVEIPPNL’157 (110)

MMP9

151‘VEIPPNLPSS.’160 (111)

MMP9

232‘.NELHLDHNK’ 240 (112)

MMP9

344‘.YWEVQPATFR’ 353 (113)

MMP9, MMP12

241‘.IQAIELEDLL’ 250 (114)

Decorin

Protease

Cleavage sites marked ‘.’

ADAM-TS4

75‘.VPKDLPPDTT’84 (115)

Versican

Protease

Cleavage sites marked ‘.’

Cathepsin K

3247‘.YENWRPNQPD’3256 (116)

Cathepsin K

3255‘PDSFFSAGED.’3264 (117)

Cathepsin K

3221‘.HDYQWIGLN’3229 (118)

MMP8

3306‘.KTFGKMKPRY’3316 (119)

MMP8

486‘.SVTQIEQIEV’495 (120)

MMP8

491‘EQIEVGPLVT.’500 (121)

CRP

Protease

Cleavage sites marked ‘.’

Signal peptide

013‘LSHAFG.’018 (122)

MMP3/8/9,

011‘TSLSHA.’016 (123)

/Cat S/K

Elastin

Protease

Cleavage sites marked ‘.’

Signal peptide

21‘LHPSRP.’26 (124)

ADAMTS1/4/8

379‘KAAKYG.’384 (125)

MMP8/9

MMP9/12

547‘GIGPGG.’552 (126)

Lumican

Protease

Cleavage sites marked ‘.’

MMP9

75‘NNQIDHIDEK.’84 (127)

LAMC1

Protease

Cleavage sites marked ‘.’

MMP2/9

664‘SAGYLDDVTL.’673 (128)

MMP9

1232‘LNRKYEQAKN.’1241 (129)

LAMA2

Protease

Cleavage sites marked ‘.’

MMP2

1240‘.QFEGKKLMAY’1249 (130)

MMP2/9

2314‘.GLWNFREKEG’2323 (131)

LAMB1

Protease

Cleavage sites marked ‘.’

MMP9

209‘ALDPAFKIED.’218 (132)

MMP2/9

1629‘.SIESETAASE’1638 (133)

MMP2/9

1639‘ETLFNASQRI.’1648 (134)

LAMA5

Protease

Cleavage sites marked ‘.’

MMP9

2402‘.NSRNQERLEE’2411 (135)

MMP2/9

2901‘EMDTLNEEVV.’2910 (136)

MMP9

3056‘DLELADAYYL.’3065 (137)

wherein P indicates hydroxyproline, M indicates oxidised methionine, and K indicates hydroxylysine.

2. A method as claimed in claim 1 , wherein said immunological binding partner is specifically reactive with any of the following sequences at the N terminal of the protein fragment:

Collagen Type I

Cleavage sites marked ‘.’/SEQ ID NO ( )

.AAGPPG

(138)

.AGPVGP

(139)

.DGR P G P

(140)

.EAGR P G

(141)

.GA P GLQ

(142)

.GLNGLP

(143)

.GLNGL P

(144)

.GL P GA K

(145)

.GPPG PP

(146)

.G PP GPP

(147)

.GVRGLT

(148)

.L P G PP G

(149)

.L P GPPG

(150)

.PGARGP

(151)

.PGKDGV

(152)

.PGP P G P

(153)

. P G PP GP

(154)

. P GPPGP

(155)

.RGE P G P

(156)

.RGL P GP

(157)

.VGPVGA

(158)

.DGLNGL

(159)

.AKGE P G

(160)

.AGARGN

(161)

.VGPPG P

(162)

.GA P G K D

(163)

.IGSAGP

(164)

.AGAPGK

(165)

.PPGPPG

(166)

.GP P G PP

(167)

.YDEKST

(168)

.ERGPPG

(169)

.IGNVGA

(170)

.AGKEGP

(171)

.KGADGS

(172)

.D(β)GLNGLP

(173)

Collagen Type III

Cleavage sites marked ‘.’/SEQ ID NO ( )

.AGAQGP

(204)

.AGIPGV

(205)

.APGAPG

(206)

.ERGRPG

(207)

.GERGSP

(208)

.GFDGRN

(209)

.GGPGAD

(210)

.GLPGPP

(211)

.GLPGTG

(212)

.GPPGPA

(213)

.GPRGPT

(214)

.GSPGPA

(215)

.IAGITG

(216)

.KGDPGP

(217)

.KGDRGE

(218)

.LKGENG

(219)

.PGENGA

(220)

.PGERGE

(221)

.PGKNGA

(222)

.PGLKGE

(223)

.PGMRGM

(224)

.PPGKDG

(225)

.PQGPKG

(226)

.SPGAKG

(227)

Collagen Type IV

Cleavage site marked ‘.’/SEQ ID NO ( )

.IVIGTG

(251)

Collagen Type V

Cleavage sites marked ‘.’/SEQ ID NO ( )

.RRNIDA

(253)

.QQGGAG

(254)

.QRGAHG

(255)

.RVGKMG

(256)

.HMGREG

(257)

Collagen Type VI

Cleavage sites marked ‘.’/SEQ ID NO ( )

.RIAVAQ

(259)

.QLGTVQ

(260)

.RDVVFL

(261)

Biglycan

Cleavage sites marked ‘.’/SEQ ID NO ( )

.KLTGIP

(262)

.RAYYNG

(263)

.EISPDT

(264)

.IEMGGN

(265)

.NHLVEI

(266)

.NELHLD

(267)

.YWEVQP

(268)

.IQAIEL

(269)

Decorin

Cleavage sites marked ‘.’/SEQ ID NO ( )

.VPKDLP

(276)

Versican

Cleavage sites marked ‘.’/SEQ ID NO ( )

.YENWRP

(277)

.HDYQWI

(278)

.KTFGKM

(279)

SVTQIE

(304)

ApoE

Cleavage sites marked ‘.’/SEQ ID NO ( )

.LMDETM

(288)

.MEEMGS

(289)

LAMA2

Cleavage sites marked ‘.’/SEQ ID NO ( )

.QFEGKK

(294)

.GLWNFR

(295)

LAMB1

Cleavage site marked ‘.’/SEQ ID NO ( )

.SIESET

(296)

LAMA5

Cleavage sites marked ‘.’/SEQ ID NO ( )

.NSRNQE

 (299).

3. A method as claimed in claim 1 , wherein said immunological binding partner is specifically reactive with any of the following sequences at the C terminal of the protein fragment:

Collagen Type I

Cleavage sites marked ‘.’/SEQ ID NO ( )

AEGSPG.

(174)

AGA P GD.

(175)

AGPPGP.

(176)

AGP P GP.

(177)

APQLSY.

(178)

A P QLSY.

(179)

EAGLPG.

(180)

GGRYYR.

(181)

GNFAPQ.

(182)

GNFA P Q.

(183)

GPAGA P .

(184)

GP P GAD.

(185)

GPPSAG.

(186)

IGP P GP.

(187)

IGPPGP.

(188)

IG PP GP.

(189)

ISVPGP.

(190)

LQG M PG.

(191)

NFAPQL.

(192)

P GA P GP.

(193)

P G P DGK.

(194)

PGP M GP.

(195)

P G PP GA.

(196)

P K GNSG.

(197)

PPSAGF.

(198)

P QLSYG.

(199)

PSAGFD.

(200)

QGP P GP.

(201)

RGRTGD.

(202)

VGARGP.

(203)

Collagen Type III

Cleavage sites marked ‘.’/SEQ ID NO ( )

AGQPGD.

(228)

ENGSPG.

(229)

GDPGPP.

(230)

GERGPP.

(231)

GLPGAA.

(232)

GPAGIP.

(233)

GPPGIP.

(234)

GPPGPA.

(235)

GRPGLP.

(236)

GSPGGP.

(237)

GSPGPQ.

(238)

GVPGAK.

(239)

KGEPGP.

(240)

PAGIPG.

(241)

AGPRGP.

(242)

PGEPGP.

(243)

PGKDGP.

(244)

PRGAPG.

(245)

RGAPGE.

(246)

RGEPGP.

(247)

SGHPGP.

(248)

SGPKGD.

(249)

TGPPGP.

(250)

Collagen Type IV

Cleavage site marked ‘.’/SEQ ID NO ( )

GIVIGT.

(252)

Collagen Type V

Cleavage sites marked ‘.’/SEQ ID NO ( )

RGITGK.

(258)

Biglycan

Cleavage sites marked ‘.’/SEQ ID NO ( )

TLNELH.

(270)

PKDLPE.

(271)

FNNPVP.

(272)

DISELR.

(273)

AFDGLK.

(274)

PNLPSS.

(275)

Versican

Cleavage sites marked ‘.’/SEQ ID NO ( )

FSAGED.

(280)

VGPLVT.

(281)

Lumican

Cleavage sites marked ‘.’/SEQ ID NO ( )

DHIDEK.

(282)

CRP

Cleavage sites marked ‘.’/SEQ ID NO ( )

LSHAFG.

(283)

TSLSHA.

(284)

Elastin

Cleavage sites marked ‘.’/SEQ ID NO ( )

LHPSRP.

(285)

KAAKYG.

(286)

GIGPGG.

(287)

ApoE

Cleavage sites marked ‘.’/SEQ ID NO ( )

RLDEVK.

(290)

TPVAEE.

(291)

LAMC1

Cleavage sites marked ‘.’/SEQ ID NO ( )

LDDVTL.

(292)

YEQAKN.

(293)

LAMB1

Cleavage sites marked ‘.’/SEQ ID NO ( )

AFKIED.

(297)

NASQRI.

(298)

LAMA5

Cleavage sites marked ‘.’/SEQ ID NO ( )

LNEEVV.

(300)

ADAYYL.

 (301).

4. A method as claimed in claim 1 , wherein said detection of binding is quantitative.

5. A method as claimed in claim 1 , wherein the amount of binding is compared to control values established for populations of healthy individuals and of individuals characterised by a fibrotic disease or by an inflammatory condition.

6. A method as claimed in claim 1 , conducted as a competition assay such that protein fragments in said sample compete for binding to the immunological binding partner with a known concentration of a binding agent which binds said immunological binding partner.

7. A method of diagnosis of quantitation of fibrosis comprising:

obtaining a patient biofluid sample,

conducting an immunoassay to measure an aggregate amount of N- or C-terminal neo-epitope containing protein fragments naturally present in a biofluid sample,

associating an elevation of said measure in said patient above a normal level with the presence or extent of fibrosis, wherein said immunoassay is conducted by a method comprising:

contacting protein fragments naturally present in said sample with an immunological binding partner reactive with a neo-epitope formed by cleavage of a protein by a proteinase; and

measuring the extent of binding of protein fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, wherein said neo-epitope is formed by cleavage of collagen type V at the site 1317′.HMGREGREGE shown in SEQ ID NO: 93 and the neo-epitope is contained in the sequence HMGREG and wherein said immunological binding partner is raised against a synthetic peptide having an N-terminal sequence HMGREG shown in SEQ ID NO: 257.