Detection of Isotype Profiles as Signatures for Disease
The invention provides a non-invasive technique for the detection and quantification of immunoglobulin isotypes, in a biological sample containing a plurality of distinct cell populations. Methods are conducted using sequencing technology to detect and enumerate immunoglobulin isotype profiles within a heterogeneous biological sample.
1 . A method of determining immunoglobulin isotype in a whole blood sample comprising:
a. isolating a plurality of nucleic acids from a biological sample comprising a plurality of cell types obtained from a subject,
b. detecting sequences specific for the constant regions of immunoglobulin in the plurality of nucleic acids; thereby determining the immunoglobulin isotype.
2 . The method of claim 1 , wherein the nucleic acid is RNA.
3 . The method of claim 2 , further comprising obtaining cDNA is from the RNA prior to step (b).
4 . The method of claim 1 , wherein the whole blood sample size is 100 ul or less.
5 . A method for determining an immunoglobulin isotype profile indicative of a biological condition in a subject, the method comprising the steps of:
a. isolating a plurality of nucleic acids from a biological sample comprising a plurality of cell types obtained from a subject,
b. detecting sequences specific for one or more regions of immunoglobulin in the plurality of nucleic acids; and
c. comparing the levels of different sequences to generate a profile of immunoglobulin isotypes.
6 . The method of claim 1 , wherein the biological sample is selected from the group consisting of blood, a blood fraction, saliva, sputum, urine, semen, transvaginal fluid, cerebrospinal fluid, stool, a cell or a tissue biopsy.
7 . The method of claim 6 , wherein the biological sample is blood or a fraction thereof.
8 . The method of claim 7 , wherein the blood is peripheral whole blood.
9 . The method of claim 8 , wherein whole blood sample size is 100 ul or less.
10 . The method of claim 6 , wherein the blood fraction comprises peripheral blood mononuclear cells.
11 . The method of claim 5 , wherein the nucleic acid is DNA.
12 . The method of claim 11 , wherein the DNA is cDNA.
13 . The method of claim 5 , wherein the nucleic acid is RNA.
14 . The method of claim 13 , further comprising obtaining cDNA is from the RNA prior to step (b)
15 . The method of claim 5 , wherein the detection step is performed using hybrid capture.
16 . The method of claim 5 , wherein the detection step is performed using sequencing technology.
17 . The method of claim 16 , wherein the sequencing technology is sequencing-by-synthesis technology.
18 . The method of claim 17 , wherein the sequencing-by-synthesis technology is single molecule sequencing.
19 . The method of claim 16 , wherein the sequencing-by-synthesis technology is massively parallel sequencing.
20 . The method of claim 5 , wherein the one or more immunoglobulin regions comprise the immunoglobulin VDJ region.
21 . The method of claim 5 , wherein the one or more immunoglobulin regions comprise the Ig constant region.
22 . The method of claim 5 , wherein the immunoglobulin isotype profile is indicative of a normal, healthy state.
23 . The method of claim 5 , wherein the immunoglobulin isotype profile is indicative of a diseased state.
24 . The method of claim 23 , wherein the diseased state is selected from the group consisting of an autoimmune disease, cancer, and infectious disease.
25 . The method of claim 24 , wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), and ankylosing spondylitis.
26 . The method of claim 25 , wherein the autoimmune disease is systemic lupus erythematosus (SLE).
27 . The method of claim 5 , wherein the immunoglobulin isotype profile is indicative of transplant rejection or immune aging.