Samples for detection of oncofetal fibronectin and uses thereof
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.
1 . A method for detecting the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:
contacting a sample with a substance that reduces non-specific binding of background material to a fibronectin or oncofetal fibronectin binding partner;
contacting the sample with a fibronectin or oncofetal fibronectin binding partner; and
detecting any complex formed between an oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner, whereby detection of complex is indicative of the presence of oncofetal fibronectin indicating molecule in the sample.
2 . The method of claim 1 , wherein:
the fibronectin or oncofetal fibronectin binding partner is a fibronectin or oncofetal fibronectin binding protein; and
the oncofetal fibronectin indicating molecule is an oncofetal fibronectin protein or fragment thereof.
3 . The method of claim 1 , wherein the substance is in a liquid mixture immobilized on a solid support.
4 . The method of claim 1 , further comprising contacting the sample with a second fibronectin or oncofetal fibronectin binding partner, wherein:
the second fibronectin or oncofetal fibronectin binding partner is conjugated to a detectable or bindable moiety or is immobilized on a solid support; and
detecting includes detecting complexes of the first binding partner, the second binding partner and the oncofetal fibronectin indicating molecule.
5 . The method of claim 4 , wherein:
the sample is selected from among urine, lymph, blood, plasma, serum, saliva, seminal fluid, ravages, cervical fluid, labial fluid, lower vaginal fluid, cervicovaginal fluid, vaginal fluid, breast fluid, breast milk, synovial fluid, semen, seminal fluid, stool, sputum, cerebral spinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum and a fraction or component of any of the preceding samples.
6 . The method of claim 4 , wherein:
the sample is selected from among urine, lavage, breast milk, a labial swab, a cervicovaginal swab, saliva, serum, plasma, blood and interstitial fluid.
7 . The method of claim 6 , wherein:
the sample is urine; and
the urine is neat or frozen.
8 . The method of claim 1 , wherein:
the substance that reduces non-specific binding of background material to a fibronectin or oncofetal fibronectin binding partner is a non-specific binding compound.
9 . The method of claim 8 , wherein:
the non-specific binding compound is selected from among albumin, casein, fetal calf serum, gelatin, and an antibody that does not specifically bind an oncofetal fibronectin indicating molecule.
10 . The method of claim 4 , wherein the second binding partner is immobilized to the solid support of a test strip.
11 . The method of claim 10 , wherein:
the second binding partner is immobilized to a first region of the test strip;
the non-specific binding compound is immobilized to a second region of the test strip; and
the first region is downstream of the sample fluid flow pathway relative to the second region.
12 . The method of claim 1 , further comprising:
normalizing the amount of oncofetal fibronectin indicating molecule in the sample according to the concentration of one or more normalization analytes in the sample.
13 . The method of claim 12 , wherein the normalization analyte is creatinine.
14 . The method of claim 8 , further comprising:
separating the sample from the non-specific binding compound;
contacting the separated sample with a solid support whereby one or more components of the sample are immobilized on the solid support; and
contacting the solid support with a fibronectin or oncofetal fibronectin binding partner.
15 . The method of claim 14 , wherein the one or more components are selected from among a protein component, a nucleic acid component and a combination thereof.
16 . The method of claim 1 , wherein the non-specific binder is immobilized on a solid support.
17 . The method of claim 1 , wherein a sample positive for oncofetal fibronectin identifies the subject from whom the sample was collected as having cancerous cells.
18 . The method of claim 17 , wherein the cancerous cells originate from an organ selected from among breast, bladder, kidney, prostate, cervix and ovary.
19 . The method of claim 1 , wherein:
a sample positive for oncofetal fibronectin identifies the subject from whom the sample was collected as having an increased risk of imminent or preterm delivery.
20 . The method of claim 1 , wherein:
a sample positive for oncofetal fibronectin identifies the subject from whom the sample was collected as one for whom induction of labor is likely to be successful.
21 . The method of claim 1 , wherein fibronectin or oncofetal fibronectin binding partner is detectably labeled.
22 . The method of claim 21 , wherein:
the detectable label is selected from among colloidal metal, photodetectable latex bead, chromophore, fluorescent moiety, quantum dot and detectable enzyme.
23 . The method of claim 4 , wherein:
complexes are detected by determining if any first binding partner is in spatial proximity to the second binding partner, whereby detection of any first and second binding partners in spatial proximity indicates presence of an oncofetal fibronectin indicating molecule in a sample.
24 . The method of claim 1 , wherein presence of an oncofetal fibronectin indicating molecule equal to or above a threshold amount classifies the sample as oncofetal fibronectin positive.
25 . The method of claim 1 , wherein the amount of oncofetal fibronectin detected is compared to two 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 two or more threshold amounts are a function of pregnancy gestational duration.
26 . The method of claim 1 , wherein the fibronectin or oncofetal fibronectin binding partner is selected from among an anti-oncofetal fibronectin antibody or antigen-binding fragment thereof and an anti-fibronectin antibody or antigen-binding fragment thereof.
27 . The method of claim 4 , wherein the second binding partner is an anti-fibronectin antibody or fragment thereof.
28 . The method of claim 1 , wherein:
the oncofetal fibronectin indicating molecule is selected from among an oncofetal fibronectin protein or a fragment indicative of an oncofetal fibronectin protein, a nucleic acid molecule encoding oncofetal fibronectin or a fragment indicative of an oncofetal fibronectin protein, a nucleic acid molecule complementary to a nucleic acid molecule encoding oncofetal fibronectin or a fragment indicative of an oncofetal fibronectin protein, autoantibody for oncofetal fibronectin protein or a fragment indicative of an oncofetal fibronectin protein, and an autoantibody for a nucleic acid molecule encoding oncofetal fibronectin or a fragment indicative of an oncofetal fibronectin protein.
29 . The method of claim 1 , wherein:
presence of an oncofetal fibronectin indicating molecule below a threshold amount classifies the sample as oncofetal fibronectin negative.
30 . The method of claim 1 , wherein:
the complex is detected by measuring the oncofetal fibronectin indicating molecule that bound to the fibronectin or oncofetal fibronectin binding partner, or a fragment of the oncofetal fibronectin indicating molecule that bound to the fibronectin or oncofetal fibronectin binding partner.
31 . The method of claim 30 , wherein the oncofetal fibronectin indicating molecule is detected by a molecular weight of the molecule or a fragment thereof.
32 . The method of claim 31 , wherein the indicating molecule is detected by mass spectrometry or gel electrophoresis.
33 . The method of claim 1 , wherein at least one fibronectin or oncofetal fibronectin binding partner is immobilized to a test strip.
34 . A method for detecting the presence of an oncofetal fibronectin indicating molecule in a urine sample, comprising:
determining the amount of oncofetal fibronectin indicating molecule present in a buffer-treated urine sample, wherein:
600 ng/ml or more, or about 60 ng/ml or more of oncofetal fibronectin indicating molecule present in the sample identifies the sample as positive for oncofetal fibronectin.
35 . The method of claim 34 , further comprising:
contacting the sample with a first fibronectin or oncofetal fibronectin binding partner;
contacting the sample with a second fibronectin or oncofetal fibronectin binding partner, wherein: the second fibronectin or oncofetal fibronectin binding partner is conjugated to a detectable or bindable moiety, or is immobilized on a solid support; and
detecting complexes of the first binding partner, oncofetal fibronectin indicating molecule and the second binding partner.
36 . The method of claim 34 , further comprising contacting the sample with a non-specific binding compound.
37 . The method of claim 34 , wherein a sample positive for oncofetal fibronectin identifies the subject from whom the sample was collected as having cancerous cells.
38 . The method of claim 37 , wherein the cancerous cells originate from an organ selected from among endometrium, uterus, breast, bladder, kidney, prostate, cervix and ovary.
39 . A method for determining the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:
collecting the sample,
treating the sample under conditions for fragmentation of an oncofetal fibronectin indicating molecule; and
detecting one or more fragments of the oncofetal fibronectin indicating molecule in the sample; whereby detection of oncofetal fibronectin indicating molecule fragments is indicative of the presence of oncofetal fibronectin indicating molecule in the sample.
40 . The method of claim 39 , wherein an amount of oncofetal fibronectin indicating molecule in the sample is determined.
41 . A method for determining the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:
collecting the sample,
treating the sample under conditions for fragmentation of an oncofetal fibronectin indicating molecule; and
detecting one or more fragments of the oncofetal fibronectin indicating molecule in the sample; whereby detection of oncofetal fibronectin indicating molecule fragments is indicative of the presence of oncofetal fibronectin indicating molecule in the sample, wherein the sample is selected from among urine, lymph, blood, plasma, serum, saliva, seminal fluid, lavages, cervical fluid, labial fluid, lower vaginal fluid, cervicovaginal fluid, vaginal fluid, breast fluid, breast milk, a labial swab, a cervicovaginal swab, saliva, serum, plasma, blood, interstitial fluid, synovial fluid, semen, seminal fluid, stool, sputum, cerebral spinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum and a fraction or component of any of the preceding samples.
42 . A test strip for detecting the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:
a non-specific binding region; and
an analyte binding region containing a first fibronectin or oncofetal fibronectin binding partner immobilized thereon;
wherein the analyte binding region is downstream of the sample fluid flow pathway relative to the non-specific binding region.
43 . The test strip of claim 42 , further comprising:
a conjugate pad, which serves as a sample application component;
an absorbent pad, which serves to draw liquid continuously through the device, wherein the materials of the membrane system form a single fluid flow pathway; and
a porous or bibulous member in fluid communication with the absorbent pad and conjugate pad, which porous or bibulous member accommodates a liquid sample and contains the analyte binding region.
44 . The test strip of claim 43 , further comprising:
a mobilization region containing a second fibronectin or oncofetal fibronectin binding partner, wherein:
the second fibronectin or oncofetal fibronectin binding partner is mobilized upon contact with the sample; and
the mobilization region is upstream of the analyte binding region.
45 . The test strip of claim 44 , further comprising:
a control region containing a biomolecule that specifically binds the second fibronectin or oncofetal fibronectin binding partner, wherein:
the control region is downstream of the analyte binding region.
46 . The test strip of claim 42 , wherein the first fibronectin or oncofetal fibronectin binding partner binds oncofetal fibronectin in preference to fibronectin.
47 . The test strip of claim 42 , wherein:
the non-specific binding region contains a non-specific binding protein immobilized thereon; and
the non-specific binding protein is selected from among BSA, methylated BSA, W632 and mouse IgG.
48 . A combination, comprising: a fibronectin or oncofetal fibronectin binding partner and a non-specific binder.
49 . The combination of claim 48 , wherein:
the non-specific binder is a non-specific binding compound or a non-specific binding surface of a solid support.
50 . The combination of claim 49 , wherein the solid support contains a non-specific binding surface.
51 . The combination of claim 50 , wherein the solid support is a test strip with the non-specific binding compound immobilized thereto.
52 . The combination of claim 48 , further comprising a sample collection device.
53 . A kit comprising the combination of claim 48 , and optionally instructions for use.
54 . The kit of claim 53 , wherein the combination further contains a sample collection device.
55 . The kit of claim 54 , wherein:
the sample collection device is selected from among a urine collection device, a dipstick, a swab and a passive cervicovaginal fluid collection device; and
and, optionally, one or more instructions for collecting and/or measuring the oncofetal fibronectin indicating molecule, and reagents therefor.
56 . The kit of claim 53 , further comprising a system for classifying the subject with respect to multiple thresholds.
57 . The kit of claim 53 , wherein:
the combination further comprises a test strip with a non-specific binding compound immobilized thereto; and
the test strip reader is configured to indicate a positive result when the amount of oncofetal fibronectin indicating molecule in the sample is above a threshold level.
58 . A combination, comprising a fibronectin or oncofetal fibronectin binding partner and a solid support containing a non-specific binding surface.
59 . The combination of claim 58 , wherein:
the solid support is a test strip with a non-specific binding compound immobilized thereto.
60 . The combination of claim 59 , further comprising:
a test strip reader configured to indicate a positive result when the amount of oncofetal fibronectin indicating molecule in the sample is above a threshold level.
61 . The combination of claim 58 , further comprising a sample collection device.
62 . A kit comprising the combination of claim 58 , and optionally instructions for use.
63 . The kit of claim 62 , wherein the combination further contains a sample collection device.
64 . The kit of claim 63 , wherein:
the sample collection device is selected from among a urine collection device, a dipstick, a swab and a passive cervicovaginal fluid collection device; and
optionally one or more of instructions for collecting and/or measuring the oncofetal fibronectin indicating molecule, and reagents therefor.
65 . The kit of claim 62 , further comprising a system for classifying the subject with respect to multiple thresholds.
66 . The kit of claim 65 , wherein:
the combination further comprises a test strip with a non-specific binding compound immobilized thereto; and
the test strip reader is configured to indicate a positive result when the amount of oncofetal fibronectin indicating molecule in the sample is above one or more thresholds.
67 . A combination, comprising a sample collection device and a fibronectin or oncofetal fibronectin binding partner.
68 . The combination of claim 67 , wherein:
the sample collection device is selected from among a urine collection device, a dipstick, a swab and a passive cervicovaginal fluid collection device.
69 . The combination of claim 68 , wherein the swab is long enough to insert into the vagina, but not long enough to contact the cervix.
70 . The combination of claim 67 , wherein the fibronectin or oncofetal fibronectin binding partner is immobilized onto the sample collection device.
71 . The combination of claim 67 , further comprising a second fibronectin or oncofetal fibronectin binding partner.
72 . The combination of claim 71 , wherein the second fibronectin or oncofetal fibronectin binding partner is detectably labeled.
73 . The combination of claim 67 , which is configured to indicate a positive result when the amount of oncofetal fibronectin indicating molecule in the sample is above a threshold level.
74 . A kit comprising the combination of claim 67 , and optionally instructions for use.
75 . The kit of claim 74 , wherein:
the sample collection device is selected from among a urine collection device, a dipstick, a swab and a passive cervicovaginal fluid collection device; and
and optionally one or more of instructions for collecting and/or measuring the oncofetal fibronectin indicating molecule, and reagents therefor.
76 . The kit of claim 74 , further comprising a system for classifying the subject with respect to multiple thresholds.
77 . The kit of claim 76 , wherein:
the combination further comprises a test strip with a non-specific binding compound immobilized thereto; and
the test strip reader is configured to indicate a positive result when the amount of oncofetal fibronectin indicating molecule in the sample is above one or more thresholds.
78 . A probe for detecting an oncofetal fibronectin indicating molecule, comprising
a mass spectrometry substrate; and
a fibronectin or oncofetal fibronectin binding partner immobilized on the mass spectrometry substrate.
79 . The probe of claim 78 , wherein:
the substrate contains a substance selected from among glass, metal, ceramic, Teflon coated magnetic material, organic polymer, biopolymer and inorganic polymer.
80 . The probe of claim 79 , wherein the molecular weight of the oncofetal fibronectin indicating molecule is detected by mass spectrometry.
81 . A conjugate, comprising a fibronectin or oncofetal fibronectin binding partner linked directly or via a linker to a therapeutic agent selected from among a cytokine, a growth factor, a photosensitizing agent, a toxin, an anticancer antibiotic, a chemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, a signaling modulator and a bioluminescent compound or to a detectable moiety selected from among a fluorescent moiety, a radionuclide, a magnetically detectable isotope or compound, a sonographic imaging agent, a chromophore, a latex microsphere, or a quantum dot.
82 . The conjugate of claim 81 , wherein the therapeutic agent is an angiogenesis inhibitor.
83 . The conjugate of claim 81 , wherein the therapeutic agent is a signaling modulator.
84 . The conjugate of claim 83 , wherein the signaling modulator is selected from among an inhibitor of macrophage inhibitory factor, a toll-like receptor agonist and a stat 3 inhibitor.
85 . The conjugate of claim 81 , wherein binding partner is an antibody selected from among FDC-6, BC-1, ME4C and L19.
86 . A method for detecting the presence of an oncofetal fibronectin indicating molecule in a sample, comprising:
contacting the sample with a fibronectin or oncofetal fibronectin binding partner; and
detecting any complex formed between an oncofetal fibronectin indicating molecule and the fibronectin or oncofetal fibronectin binding partner, whereby detection of complex is indicative of the presence of oncofetal fibronectin indicating molecule in the sample, wherein the sample is selected from among lymph, blood, plasma, serum, saliva, seminal fluid, lavages, cervical fluid, labial fluid, lower vaginal fluid, vaginal fluid, breast fluid, breast milk, synovial fluid, semen, seminal fluid, stool, sputum, cerebral spinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum and a fraction or component of any of the preceding samples.
87 . The method of claim 86 , wherein the presence of an oncofetal fibronectin indicating molecule or it presence above a threshold level is indicative of cancer.