Detection of intraamniotic and/or infection
The present invention concerns the identification of biomarkers and groups or combinations of biomarkers that can be used for non-invasive diagnosis of intra-amniotic infection, and diagnostic assays using such biomarkers.
1. A method of diagnosing intra-amniotic infection in a pregnant female mammalian subject comprising:
(a) obtaining a test a sample of cervical-vaginal fluid from the subject, wherein the pregnant female mammalian subject has intact membranes, is in pre-term labor, and is from about 22 weeks to about 36 weeks gestation;
(b) detecting the levels of interleukin-6 (IL-6) and alpha-fetoprotein (AFP) in the test cervical-vaginal fluid sample by contacting the sample with detecting agents specific for IL-6 and AFP and detecting binding between the detecting agents and the IL-6 and AFP proteins;
(c) diagnosing said subject with intra-amniotic infection when each of said levels of each of said proteins in said sample is determined to show a statistically significant difference relative to the corresponding levels of each of said proteins in said normal cervical-vaginal fluid; and
(d) performing amniocentesis on the diagnosed subject or treating said subject that is not diagnosed in step (c) for preterm labor.
2. The method of claim 1 wherein the subject is a human patient.
3. The method of claim 1 wherein said levels of said IL-6 and AFP are determined by methods comprising the use of an immunoassay, a protein array, an immunochromatographic test, mass spectrometry, or combinations thereof.
4. The method of claim 3 wherein said levels are determined using an immunochromtaographic test employing a lateral flow device.
5. The method of claim 4 , wherein the lateral flow device is an immunochromatographic strip (ICS) test.
6. The method of claim 3 wherein said immunoassay employs antibodies and reagents for the detection of IL-6 and AFP.
7. The method of claim 6 , wherein the antibodies and reagents comprise a capture antibody and a detector antibody.
8. The method of claim 7 , wherein the capture antibody and detector antibody are monoclonal antibodies.
9. The method of claim 7 , wherein the capture antibody and detector antibody are polyclonal antibodies.
10. The method of claim 7 , wherein the capture antibody and detector antibody are either monoclonal or polyclonal antibodies.
11. The method of claim 8 , wherein the antibodies comprise monoclonal antibodies having binding specificity for IL-6 and AFP, and wherein the method further comprises use of an anti-antibody immunoglobulin.
12. The method of claim 11 , wherein the monoclonal antibodies and the anti-antibody immunoglobulin are provided in an amount of about 0.001 mg to about 100 grams.
13. The method of claim 12 , wherein the monoclonal antibodies and the anti-antibody immunoglobulin are provided in an amount of about 0.01 mg to about 1 gram.
14. The method of claim 11 , wherein the anti-antibody immunoglobulin is selected from the group consisting of a polyclonal immunoglobulin, protein A and protein G, or functional fragments thereof.
15. The method of claim 14 , wherein the anti-antibody immunoglobulin is labeled.
16. The method of claim 6 , further comprising use of agents for reducing background interference in a test or agents for increasing signal.
17. The method of claim 6 , further comprising use of software and algorithms for combining and interpolating marker values to produce a prediction of clinical outcome of interest.
18. The method of claim 6 , further comprising use of an apparatus for conducting a test.
19. A method for determining signs and symptoms indicating intra-amniotic infection comprising
(a) providing a test a sample of cervical-vaginal fluid from the subject, wherein the subject:
i) is a pregnant female mammalian subject;
ii) has intact membranes;
iii) is in pre-term labor; and
iv) is from about 22 weeks to about 36 weeks gestation;
(b) detecting the levels of interleukin-6 (IL-6) and alpha-fetoprotein (AFP) in the test cervical-vaginal fluid sample by contacting the sample with detecting agents specific for IL-6 and AFP and detecting binding between the detecting agents and the IL-6 and AFP proteins;
(c) diagnosing said subject with intra-amniotic infection when each of said levels of IL-6 and AFP in said sample are determined to show a statistically significant difference relative to the corresponding levels of IL-6 and AFP in said normal cervical-vaginal fluid; and
(d) performing amniocentesis on the diagnosed subject or treating said subject that is not diagnosed in step (c) for preterm labor.
20. The method of claim 19 , wherein the signs and symptoms comprise maternal fever, maternal leukocytosis, maternal and/or fetal tachycardia, and/or uterine tenderness.
21. The method of claim 19 , wherein said protein levels are determined by methods comprising the use of an immunoassay, a protein array, an immunochromatographic test, mass spectrometry, or combinations thereof.
22. The method of claim 20 , wherein the maternal fever is >37.8° C.
23. The method of claim 20 , wherein the maternal leukocytosis is >15,000/mm 3 .
24. The method of claim 19 , wherein said protein levels are determined by methods comprising the use of an immunoassay, a protein array, an immunochromatographic test, mass spectrometry, or combinations thereof.