IP Library › Granted Patent US 9,606,130
Granted Patent B2
US 9,606,130 · App. 14/960,713 · Granted Mar 28, 2017

Fibrosis biomarker assay

Inventors: Sanne S. Veidal (Olstykke, DK); Morten A. Karsdal (Kobenhavn O, DK); Diana J. Oersnes-Leeming (Klampenborg, DK); Natasha Barascuk Michaelsen (Gentofte, DK); Helene Skjot-Arkil (Kolding, DK); Antonio Segovia-Silvestre (Copenhagen, DK); Efstathios Vassiliadis (Hammel, DK)
Assignee: Nordic Bioscience A/S
G01N33/6893C12Q1/37G01N2333/4737G01N2333/78G01N2800/085G01N2800/32G01N2800/50
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Quick Facts
Patent No.
US 9,606,130
App. No.
14/960,713
Granted
Mar 28, 2017
Kind
B2
Abstract

Provided herein are methods of diagnosis or of quantitation of fibrosis. An immunoassay is conducted to measure neo-epitope containing protein fragments of collagen type III, collagen type I, collagen type IV, collagen type V, or collagen type VI, elastin, biglycan, decorin, lumican, versican, perlecan, neurocan, brevican, fibromodulin, serglycin, syndecan, betaglycan, vimentin, or C-reactive protein naturally present in a biofluid sample obtained from a patient. An above normal elevation of the measured protein fragments in the patient is associated with the presence or extent of fibrosis.

Claims (230)

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

obtaining a patient biofluid sample,

conducting an immunoassay to measure fragments of collagen type III having an N- or C-terminal neo epitope formed by cleavage of intact collagen type III by MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, CathepsinK, CathepsinS, ADAMTS1, ADAMTS4, ADAMTS5, or ADAMTS8, said fragments being naturally present in said 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 fragments of collagen type III having said N- or C-terminal neo epitope 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 intact collagen type III, and

measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, wherein said immunological binding partner specifically binds to an N-terminal neo epitope amino acid sequence formed by cleavage of collagen type III by MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, CathepsinK, CathepsinS, ADAMTS1, ADAMTS4, ADAMTS5, or ADAMTS8, said N-terminal amino acid sequence selected from the group consisting of:

Collagen type III

GIPGAP . . . SEQ ID NO: 372

IAGITG . . . SEQ ID NO: 375

KGDAGQ . . . SEQ ID NO: 381

GKSGDR . . . SEQ ID NO: 383

DGTSGH . . . SEQ ID NO: 135

GPPGVA . . . SEQ ID NO: 158

GARGLA . . . SEQ ID NO: 111

KGESGK . . . SEQ ID NO: 374

QPGVMG . . . SEQ ID NO: 144

GPPGPT . . . SEQ ID NO: 391

GLPGPP . . . SEQ ID NO: 394

GINGSP . . . SEQ ID NO: 397

LMGARG . . . SEQ ID NO: 400

DKGEPG . . . SEQ ID NO: 403

PGMKGH . . . SEQ ID NO: 406

FPGMKG . . . SEQ ID NO: 409

GQPGDK . . . SEQ ID NO: 412

GSDGQP . . . SEQ ID NO: 405

GFPGAP . . . SEQ ID NO: 420

PGPQGH . . . SEQ ID NO: 426

PGPPGI . . . SEQ ID NO: 432

GPPGSN . . . SEQ ID NO: 423

PQGLQG . . . SEQ ID NO: 440

GAPGFR . . . SEQ ID NO: 435

GAPGPQ . . . SEQ ID NO: 445

GPTGPI . . . SEQ ID NO: 448

KGSPGA . . . SEQ ID NO: 444

GSRGAP . . . SEQ ID NO: 462

NTGAPG . . . SEQ ID NO: 437

HAGAQG . . . SEQ ID NO: 434

PGPQGP . . . SEQ ID NO: 453

AGQPGE . . . SEQ ID NO: 454

VKGERG . . . SEQ ID NO: 159

GPPGAP . . . SEQ ID NO: 461

GSPGAQ . . . SEQ ID NO: 464

PGAPGL . . . SEQ ID NO: 467

ESCPTG . . . SEQ ID NO: 433

GPAGIP . . . SEQ ID NO: 441

GDPGPP . . . SEQ ID NO: 373

IKGHRG . . . SEQ ID NO: 376

ITGARG . . . SEQ ID NO: 379

LQGLPG . . . SEQ ID NO: 384

IGSPGP . . . SEQ ID NO:

AGPPGM . . . SEQ ID NO: 145

GAPGEK . . . SEQ ID NO: 141

GLSGER . . . SEQ ID NO: 387

IPGAPG . . . SEQ ID NO: 117

INGSPG . . . SEQ ID NO: 392

KNGETG . . . SEQ ID NO: 395

PGENGK . . . SEQ ID NO: 398

GKDGES . . . SEQ ID NO: 418

GHAGAQ . . . SEQ ID NO: 404

FPGARG . . . SEQ ID NO: 407

PGDKGE . . . SEQ ID NO: 410

GPPGEN . . . SEQ ID NO: 413

PGVPGA . . . SEQ ID NO: 416

GPPGPP . . . SEQ ID NO: 100

GAAGEP . . . SEQ ID NO: 421

PGFPGM . . . SEQ ID NO: 427

GITGAR . . . SEQ ID NO: 430

RPGLPG . . . SEQ ID NO: 436

GPPGVA . . . SEQ ID NO: 158

PGFRGP . . . SEQ ID NO: 443

GFPGNP . . . SEQ ID NO: 446

GDAGQP . . . SEQ ID NO: 449

GSPGER . . . SEQ ID NO: 439

TGARGL . . . SEQ ID NO: 378

VGGLAG . . . SEQ ID NO: 155

PGAPGG . . . SEQ ID NO: 455

AGQQGA . . . SEQ ID NO: 457

GLAGPP . . . SEQ ID NO: 388

GGAGEP . . . SEQ ID NO: 463

SPGAQG . . . SEQ ID NO: 465

IKGPAG . . . SEQ ID NO: 169

GIPGQP . . . SEQ ID NO: 468

DAGAPG . . . SEQ ID NO: 469

LAGPPG . . . SEQ ID NO: 89

RGLAGP . . . SEQ ID NO: 377

VKGESG . . . SEQ ID NO: 380

LRGGAG . . . SEQ ID NO: 382

AIGSPG . . . SEQ ID NO: 143

LSGERG . . . SEQ ID NO: 176

PQGPPG . . . SEQ ID NO: 389

YQGPPG . . . SEQ ID NO: 401

FRGPAG . . . SEQ ID NO: 137

GPPGEP . . . SEQ ID NO: 393

LPGIAG . . . SEQ ID NO: 396

QQGAIG . . . SEQ ID NO: 390

GERGAP . . . SEQ ID NO: 402

GFPGAR . . . SEQ ID NO: 408

GDKGET . . . SEQ ID NO: 411

AAGFPG . . . SEQ ID NO: 417

GARGND . . . SEQ ID NO: 419

GGAGPP . . . SEQ ID NO: 425

GARGPP . . . SEQ ID NO: 422

GSPGGP . . . SEQ ID NO: 428

GIAGIT . . . SEQ ID NO: 431

GAPGPM . . . SEQ ID NO: 438

SGDRGE . . . SEQ ID NO: 429

GPVGPS . . . SEQ ID NO: 447

GPPGIN . . . SEQ ID NO: 470

NGEKGE . . . SEQ ID NO: 450

AIGPSG . . . SEQ ID NO: 368

ERGLPG . . . SEQ ID NO: 385

VAGPPG . . . SEQ ID NO: 452

GIPGFP . . . SEQ ID NO: 414

PGPPGP . . . SEQ ID NO: 458

GRNGEK . . . SEQ ID NO: 460

SPGGKG . . . SEQ ID NO: 459

PGVSGP . . . SEQ ID NO: 466

PGAPGQ . . . SEQ ID NO: 456

SRGAPG . . . SEQ ID NO: 451

GPKGDA . . . SEQ ID NO: 424

or wherein said immunological binding partner specifically binds to a C-terminal neo epitope amino acid sequence formed by cleavage of collagen type III by MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, CathepsinK, CathepsinS, ADAMTS1, ADAMTS4, ADAMTS5, or ADAMTS8, wherein said C-terminal amino acid sequence is selected from the group consisting of:

Collagen type III

. . . GPPGPA SEQ ID NO: 94

. . . GMPGPR SEQ ID NO: 473

. . . ERGAAG SEQ ID NO: 476

. . . ERGPPG SEQ ID NO: 147

. . . GLPGLA SEQ ID NO: 486

. . . GLAGTA SEQ ID NO: 488

. . . LAGPPG SEQ ID NO: 89

. . . IPGFPG SEQ ID NO: 492

. . . FPGPKG SEQ ID NO: 491

. . . GPPGIC SEQ ID NO: 2187

. . . PGPQGL SEQ ID NO: 497

. . . SPGPKG SEQ ID NO: 499

. . . LPGPPG SEQ ID NO: 72

. . . GHRGFD SEQ ID NO: 503

. . . GLPGIA SEQ ID NO: 507

. . . GLPGPP SEQ ID NO: 394

. . . TGARGL SEQ ID NO: 378

. . . PQGLPG SEQ ID NO: 508

. . . GTPGLQ SEQ ID NO: 521

. . . GMKGHR SEQ ID NO: 531

. . . EMGPAG SEQ ID NO: 534

. . . GVKGER SEQ ID NO: 538

. . . GPPGPR SEQ ID NO: 544

. . . AGPRGA SEQ ID NO: 547

. . . GRNGDP SEQ ID NO: 171

. . . AGIPGF SEQ ID NO: 496

. . . PPGPQG SEQ ID NO: 103

. . . IPGAPG SEQ ID NO: 117

. . . TSGHPG SEQ ID NO: 518

. . . PSGPPG SEQ ID NO: 483

. . . PPGPAG SEQ ID NO: 52

. . . FPGMKG SEQ ID NO: 409

. . . EKGPAG SEQ ID NO: 515

. . . MPGPRG SEQ ID NO: 523

. . . GIPGAP SEQ ID NO: 372

. . . NGDPGI SEQ ID NO: 471

. . . SPGPAG SEQ ID NO: 474

. . . PGPLGI SEQ ID NO: 477

. . . PGPPGT SEQ ID NO: 479

. . . APGLRG SEQ ID NO: 481

. . . GSPGPA SEQ ID NO: 484

. . . PGLMGA SEQ ID NO: 489

. . . GPPGPQ SEQ ID NO: 490

. . . GPAGIP SEQ ID NO: 441

. . . PPGPPG SEQ ID NO: 119

. . . GAPGLM SEQ ID NO: 498

. . . LPGAAG SEQ ID NO: 2188

. . . GPPGIN SEQ ID NO: 470

. . . PGLPGI SEQ ID NO: 504

. . . PGPKGD SEQ ID NO: 506

. . . GANGLP SEQ ID NO: 510

. . . GPPGIK SEQ ID NO: 512

. . . GAPGLR SEQ ID NO: 509

. . . GEVGPA SEQ ID NO: 514

. . . GKPGAN SEQ ID NO: 537

. . . PGAAGF SEQ ID NO: 539

. . . GDAGAP SEQ ID NO: 542

. . . GPAGPR SEQ ID NO: 545

. . . GGKGER SEQ ID NO: 548

. . . GPAGAN SEQ ID NO: 550

. . . VKGESG SEQ ID NO: 380

. . . TGPRGP SEQ ID NO: 177

. . . EPGPRG SEQ ID NO: 516

. . . GAPGPA SEQ ID NO: 519

. . . GTSGHP SEQ ID NO: 522

. . . GAPGLK SEQ ID NO: 525

. . . GEPGPR SEQ ID NO: 500

. . . PGPKGN SEQ ID NO: 527

. . . PPGAPG SEQ ID NO: 517

. . . TPGLQG SEQ ID 520

. . . SPGPQG SEQ ID NO: 472

. . . PGPQGV SEQ ID NO: 475

. . . AAGTPG SEQ ID NO: 478

. . . GNRGER SEQ ID NO: 480

. . . HPGSPG SEQ ID NO: 482

. . . GPAGPP SEQ ID NO: 485

. . . QGPPGP SEQ ID NO: 487

. . . GFPGMK SEQ ID NO: 493

. . . FPGAPG SEQ ID NO: 494

. . . FPGARG SEQ ID NO: 407

. . . GAIGPS SEQ ID NO: 495

. . . APGPLG SEQ ID NO: 2189

. . . IPGQPG SEQ ID NO: 501

. . . GAAGIK SEQ ID NO: 505

. . . GPPGVA SEQ ID NO: 158

. . . GPPGPS SEQ ID NO: 511

. . . TAGFPG SEQ ID NO: 513

. . . GPQGVK SEQ ID NO: 524

. . . QPGPPG SEQ ID NO: 536

. . . PGANGL SEQ ID NO: 529

. . . GPAGER SEQ ID NO: 543

. . . RGFDGR SEQ ID NO: 546

. . . APGLMG SEQ ID NO: 549

. . . PQGVKG SEQ ID NO: 541

. . . TGERGA SEQ ID NO: 540

. . . GSPGYQ SEQ ID NO: 526

. . . GAAGAR SEQ ID NO: 528

. . . TGAPGS SEQ ID NO: 502

. . . GTGGPP SEQ ID NO: 530

. . . GITGAR SEQ ID NO: 430

. . . GIAGPR SEQ ID NO: 535

. . . GLSGER SEQ ID NO: 387

. . . EGGPPG SEQ ID NO: 532

. . . GFPGAR SEQ ID NO: 408.

2. A method as claimed in claim 1 , wherein said immunological binding partner specifically binds fragments of collagen type III comprising an N-terminal sequence KNGETG (SEQ ID NO: 395).

3. A method as claimed in claim 1 , wherein said immunological binding partner specifically binds to N-terminal sequence KNGETGPQ . . . (SEQ ID NO: 2251).

4. A method as claimed in claim 1 , wherein said immunological binding partner specifically binds to N-terminal sequence GAPGFR . . . (SEQ ID NO: 435).

5. A method as claimed in claim 1 , wherein said immunological binding partner is a polyclonal antibody or a monoclonal antibody.

Priority Claims (3)
GB 0721713.6 · Nov 5, 2007 · national
GB 0722748.1 · Nov 20, 2007 · national
GB 0802814.4 · Feb 15, 2008 · national
Continuity (5)
Division 12749652 · Mar 30, 2010
Continuation In Part PCTEP2008064946 · Nov 4, 2008
Provisional Application 61289081 · Dec 22, 2009
Provisional Application 61211467 · Mar 30, 2009
Related Publication 20160091502A1 · Mar 31, 2016