Methods for detecting oncofetal fibronectin

• US 20060024723A1
• Filed: 07/29/2005
• Published: 02/02/2006
• Est. Priority Date: 07/30/2004
• Status: Active Grant

• Current Assignee: Cytyc Corporation (Hologic Incorporated)
• Original Assignee: Cytyc Corporation (Hologic Incorporated)
• Inventors: Hussa, Robert, Shorter, Simon, Fischer-Colbrie, Mark, Lapointe, Jerome P., Senyei, Andrew `
• Granted Patent: US 7,943,294 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 15/04 for inducing labour or abortion; Uterotonics
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: G01N 2333/471 Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2800/36 Gynecology or obstetrics
• CPC Class Codes: G01N 2800/368 Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/57476 involving oncofetal proteins
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: G01N 33/689 : related to pregnancy or the gonads

Abstract

Methods and products for the detection of oncofetal fibronectin indicating molecules in samples are provided. Methods for imaging of oncofetal fibronectin are provided. In some methods provided herein, the sample is treated with a reagent and/or contacted with a non-specific binder. Provided are methods for testing subjects to ascertain health and disease status and to assess the risk of developing a disease or condition. Methods for detecting the presence of oncofetal fibronectin indicating molecules by a variety of methods such as immunoassays and mass spectrometry also are provided. Methods and products for detection of oncofetal fibronection for selection of concepti are provided.

Claims

1. A method for detecting an oncofetal fibronectin indicating molecule or fragment thereof in a sample, comprising:

treating a sample under conditions that separate one or more first sample components from one or more second sample components, wherein an oncofetal fibronectin indicating molecule or fragment thereof, if present, is among the one or more first sample components; and

detecting the oncofetal fibronectin indicating molecule or fragment thereof by its molecular weight.

2. The method of claim 1, wherein:

treating further comprises contacting the sample with a fibronectin or oncofetal fibronectin binding partner immobilized on a solid support to form a complex thereof;

treating the solid support to release the oncofetal fibronectin indicating molecule or fragment thereof from the complex; and

detecting further comprises detecting the released oncofetal fibronectin indicating molecule or fragment thereof by its molecular weight.

3. The method of claim 1, further comprising:

calculating the molecular weight of the detected oncofetal fibronectin indicating molecule or fragment thereof.

4. The method of claim 1, wherein:

detecting further comprises comparing the detected first sample components to one or more references, wherein a reference that matches a detected first sample component corresponds to a fibronectin indicating molecule or fragment thereof.

5. The method of claim 2, further comprising:

prior to treating the solid support to release oncofetal fibronectin indicating molecule or fragment thereof from the binding partner, treating the solid support under conditions that separate the solid support from sample components not specifically bound to the binding partner.

6. The method of claim 2, wherein:

prior to detecting, and subsequent to, contacting the sample with a fibronectin or oncofetal fibronectin binding partner, the method further comprises contacting the sample with a fragmentation reagent.

7. The method of claim 6, wherein the fragmentation reagent is a protease or a nuclease.

8. The method of claim 7, wherein the fragmentation reagent is immobilized onto a second solid support;

and

contacting the sample with a fragmentation reagent further comprises contacting the sample with the second solid support.

9. The method of claim 8, wherein:

prior to detecting, and subsequent to, contacting the sample with a fragmentation reagent, the method further comprises treating the sample under conditions whereby the oncofetal fibronectin indicating molecule or fragment thereof is released from the second solid support.

10. The method of claim 9, wherein:

release of the oncofetal fibronectin indicating molecule or fragment thereof from the second solid support is accomplished by matrix-assisted laser desorption or electrospray desorption.

11. The method of claim 1, further comprising separating DNA from RNA in a sample.

12. The method of claim 11, further comprising:

contacting an RNA sample with a primer complementary to an oncofetal fibronectin-encoding nucleotide sequence; and

treating the sample with one or more nucleic acid synthesis steps.

13. The method of claim 12, wherein the primer is complementary to mRNA encoding oncofetal fibronectin.

14. The method of claim 13, wherein a first nucleic acid synthesis step includes nucleic acid synthesis by reverse transcriptase.

15. The method of claim 1, wherein the oncofetal fibronectin indicating molecule is selected from among an oncofetal fibronectin protein or fragment thereof, a nucleic acid molecule encoding oncofetal fibronectin or fragment thereof, a nucleic acid molecule complementary to a nucleic acid molecule encoding oncofetal fibronectin a fragment thereof, an autoantibody for oncofetal fibronectin protein or fragment thereof, and an autoantibody for a nucleic acid molecule encoding oncofetal fibronectin or fragment thereof.

16. The method of claim 2, wherein the oncofetal fibronectin indicating molecule is detected by measuring products of a nucleic acid synthesis reaction.

17. The method of claim 1, wherein the sample comprises a sample selected from among a tissue sample, a cell sample and a liquid sample.

18. The method of claim 17, wherein:

the tissue or cell sample is selected from among;

lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, pituitary, eye, brain, oral, skin, head and neck cancer, lymphoma, leukemia, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, sarcoma neuroblastoma, semen, stool and a fraction or component thereof.

19. The method of claim 17, wherein:

the liquid sample comprises a body fluid selected from among;

urine, blood, plasma, serum, saliva, lavage, cervical fluid, cervicovaginal fluid, vaginal fluid, breast fluid, breast milk, synovial fluid, seminal fluid, sputum, cerebral spinal fluid, tears, interstitial fluid, follicular fluid, anmiotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, and a fraction or component thereof.

20. The method of claim 19, wherein the sputum is lung sputum.

21. The method of claim 2, further comprising:

removing sample components not bound to the binding partner prior to the step of removing the oncofetal fibronectin indicating molecule or fragment thereof from the complex.

22. The method of claim 21, wherein the binding partner contains an energy absorbing moiety.

23. The method of claim 22, wherein:

removing is accomplished by a method selected from among ultraviolet Matrix-Assisted Laser Desorption/Ionization, infrared Matrix-Assisted Laser Desorption/Ionization, and electrospray ionization.

24. The method of claim 23, wherein:

the oncofetal fibronectin indicating molecule or fragment thereof is detected by a mass spectrometry method selected from among time-of-flight, Fourier transform, and magnetic sector/magnetic deflection.

25. The method of claim 24, further comprising enhancing a signal from the oncofetal fibronectin indicating molecule bound to the binding partner.

26. The method of claim 25, further comprising quantitating the amount of oncofetal fibronectin indicating molecule in the sample.

27. The method of claim 1, wherein:

presence of the oncofetal fibronectin indicating molecule in a sample determines a risk or identifies a health problem associated with oncofetal fibronectin.

28. The method of claim 1, wherein:

presence of the oncofetal fibronectin indicating molecule in a sample indicates an increased risk of imminent or preterm delivery.

29. The method of claim 1, wherein:

the oncofetal fibronectin indicating molecule is autoantibodies specific for oncofetal fibronectin; and

the presence of anti-oncofetal fibronectin autoantibodies in the sample indicates the presence of an oncofetal fibronectin-associated health problem in the subject.

30. The method of claim 1, wherein:

the oncofetal fibronectin indicating molecule is autoantibodies specific for oncofetal fibronectin; and

the presence of anti-oncofetal fibronectin autoantibodies in the sample indicates the presence of an oncofetal fibronectin in the sample.

31. The method of claim 30, wherein:

detecting further comprises contacting the sample with an anti-oncofetal fibronectin autoantibody binding partner; and

detecting complexes formed between the binding partner and an anti-oncofetal fibronectin autoantibody.

32. The method of claim 2, further comprising:

contacting the sample with a binding partner that specifically binds the first fibronectin or oncofetal fibronectin binding partner.

33. The method of claim 32, wherein:

the binding partner that specifically binds the first fibronectin or oncofetal fibronectin binding partner is selected from among an antibody and a nucleic acid molecule.

34. The method of claim 33, wherein:

the fibronectin or oncofetal fibronectin binding partner is an anti-fibronectin antibody, or antigen-binding fragment thereof.

35. The method of claim 1, wherein presence of an oncofetal fibronectin indicating molecule equal to or above a threshold level classifies the sample as oncofetal fibronectin positive.

36. The method of claim 35, wherein:

the amount of oncofetal fibronectin indicating molecule detected is compared to one or more thresholds, wherein the sample is classified according to the highest threshold that is less than or equal to the detected amount of oncofetal fibronectin indicating molecule.

37. The method of claim 36, wherein:

the subject from whom the sample is collected is pregnant and the one or more threshold levels correspond to increasing likelihood of imminent pregnancy termination.

38. The method of claim 2, wherein:

the complex between the oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner is measured by detecting the oncofetal fibronectin indicating molecule or a fragment thereof.

39. The method of claim 38, wherein:

the oncofetal fibronectin indicating molecule is detected by mass spectrometry or gel electrophoresis.

40. The method of claim 2, wherein:

the complex between the oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner is measured by detecting the fibronectin or oncofetal fibronectin binding partner.

41. A method for indicating an oncofetal fibronectin indicating molecule in a subject comprising:

detecting the presence of autoantibodies specific for oncofetal fibronectin in a sample from a subject, wherein;

the presence of anti-oncofetal fibronectin autoantibodies in the sample indicates the presence oncofetal fibronectin in the subject.

42. The method of claim 41, wherein:

detecting further comprises contacting the sample with an anti-oncofetal fibronectin autoantibody binding partner; and

detecting complexes formed between the binding partner and an anti-oncofetal fibronectin autoantibody.

43. The method of claim 41, wherein,:

the presence of autoantibodies specific for oncofetal fibronectin in a sample from a subject indicates the presence of an oncofetal fibronectin-associated health problem in the subject.

44. The method of claim 41, further comprising:

contacting a sample with a solution that reduces non-specific binding of background material to a fibronectin or oncofetal fibronectin binding partner.

45. The method of claim 41, wherein:

the sample is selected from among urine, lavage, breast milk, cervicovaginal swab, saliva, serum, plasma, blood and interstitial fluid.

46. The method of claim 45, wherein the sample is urine and one or more filtering and/or non-specific binding steps is performed on the urine sample prior to the detecting step.

47. The method of claim 42, further comprising:

contacting the sample with a binding partner selected from among an antibody and a nucleic acid molecule that specifically binds the anti-oncofetal fibronectin autoantibody.

48. The method of claim 41, wherein the oncofetal fibronectin indicating molecule is selected from among an oncofetal fibronectin protein or fragment thereof, a nucleic acid molecule encoding oncofetal fibronectin or fragment thereof, a nucleic acid molecule complementary to a nucleic acid molecule encoding oncofetal fibronectin a fragment thereof, an autoantibody for oncofetal fibronectin protein or fragment thereof, and an autoantibody for a nucleic acid molecule encoding oncofetal fibronectin or fragment thereof.

49. The method of claim 41, wherein presence of an oncofetal fibronectin indicating molecule equal to or above a threshold level classifies the sample as oncofetal fibronectin positive.

50. The method of claim 49, wherein:

the amount of oncofetal fibronectin indicating molecule detected is compared to one or more thresholds; and

the sample is classified according to the highest threshold that is less than or equal to the detected amount of oncofetal fibronectin indicating molecule, whereby the subject from whom the sample is collected is pregnant and the one or more threshold levels correspond to increasing likelihood of imminent pregnancy termination.

51. The method of claim 41, wherein:

the complex between the oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner is measured by detecting the oncofetal fibronectin indicating molecule or a fragment thereof.

52. The method of claim 51, wherein:

the oncofetal fibronectin indicating molecule is detected by mass spectrometry or gel electrophoresis.

53. The method of claim 42, wherein:

the complex between the oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner is measured by detecting the fibronectin or oncofetal fibronectin binding partner.

54. A method for classifying the level of oncofetal fibronectin in a sample, comprising:

measuring the amount of an oncofetal fibronectin indicating molecule in a sample;

comparing the amount of oncofetal fibronectin indicating molecule in a sample to two or more threshold levels; and

classifying the amount of oncofetal fibronectin indicating molecule in a sample according to the highest threshold level that is less than or equal to amount of oncofetal fibronectin indicating molecule in the sample, whereby classification of oncofetal fibronectin indicating molecule in the sample according to the highest threshold identifies an indication selected from among a higher risk of preterm, impending delivery, imminent delivery, increased ability to predict delivery date, decreased likelihood of maintaining pregnancy, increased benefit from using methods of preventing preterm delivery, increased likelihood of success in inducing delivery, increased likelihood of delivery within a predetermined time, presence of cancerous cells, an increased risk of developing cancer or recurrence of cancer, a faster progression of cancer, a more aggressive cancer and a decreased efficacy of cancer therapy.

55. A method for classifying a sample as oncofetal fibronectin positive or negative, comprising:

measuring the amount of an oncofetal fibronectin indicating molecule in a sample;

comparing the measured sample oncofetal fibronectin indicating molecule amount to a threshold level of 150 ng/ml;

classifying the sample as oncofetal fibronectin positive if the amount of oncofetal fibronectin indicating molecule is equal to or greater than the threshold level; and

classifying the sample as oncofetal fibronectin negative if the amount of oncofetal fibronectin indicating molecule is equal to or less than the threshold level.

56. A method for detecting the presence of an oncofetal fibronectin indicating molecule or analog thereof in a sample, comprising:

contacting a sample with a first fibronectin or oncofetal fibronectin binding partner and a second fibronectin or oncofetal fibronectin binding partner; and

measuring formation of a complex of oncofetal fibronectin, the first binding partner and the second binding partner by detecting fluorescence from non-radioactive energy transfer selected from among fluorescence energy transfer (FET), fluorescence resonance energy transfer (FRET), or homogeneous time-resolved fluorescence (HTRF).

57. The method of claim 56, wherein a binding partner is conjugated to a fluorescent dye or a quantum dot.

58. The method of claim 56, wherein:

the oncofetal fibronectin indicating molecule or analog thereof is conjugated to a fluorescent dye or a quantum dot; and

signal dissipation or change indicates complex formation of a sample oncofetal fibronectin indicating molecule and the binding partner.

59. A method for detecting the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:

contacting a sample with a fibronectin or oncofetal fibronectin binding partner;

measuring formation of a complex of an oncofetal fibronectin indicating molecule and the binding partner by detecting fluorescence polarization indicative of complex formation.

60. The method of claim 59, wherein:

the fluorescence polarization measurement indicates the mass of the complex; and

the binding partner is conjugated to a fluorescent dye or a quantum dot.

61. A method for detecting an oncofetal fibronectin indicating molecule in a sample, comprising:

detecting the molecular weight of an oncofetal fibronectin indicating molecule or fragment thereof by mass spectrometry to thereby detect the presence of an oncofetal fibronectin indicating molecule.