Compositions for detecting human interferon-alpha subtypes and methods of use

Abstract

The invention provides highly sensitive, specific and efficient quantitative real-time PCR compositions, methods and assay kits to detect at least one IFN subtype and/or IFN subtype allotypic variants. Primer/probe sets complementary to the coding sequence of an IFN subtype of interest avoid spurious detection of degraded mRNA and enhances the correlation between the IFN subtype that is measured by the assays of the invention and the protein that is actually expressed. The invention also provides methods for designing primers and methods of using the compositions and assay kits. The compositions, kits, and methods of the invention may be used, for example, to monitor vaccine efficacy, autoimmune disease, chronic infections, or tumor therapy.

1 - 49 . (canceled) 
     
     
         50 . A method of detecting an inflammatory condition in an individual in need thereof, comprising:
 a) contacting a biological sample from the individual with a composition comprising a first oligonucleotide pair and a second oligonucleotide pair, wherein each oligonucleotide pair is capable of specifically binding to a single subtype of IFN transcript thereby simultaneously obtaining IFN transcript accumulation levels;   b) determining a pattern of IFN transcript levels in the sample;   c) comparing the pattern of IFN transcript levels of the individual to a pattern of IFN transcript levels of a known inflammatory condition; and   d) categorizing the individual as having the condition if the pattern of IFN transcript levels of the individual substantially match the pattern of IFN transcript levels of the known inflammatory condition; or   e) categorizing the individual as not having the condition if the pattern of IFN transcript levels of the individual does not substantially match the pattern of IFN transcript levels of the known inflammatory condition.   
     
     
         51 . The method of  claim 50 , wherein the first oligonucleotide pair is selected from the list of oligonucleotide pairs consisting of: SEQ ID NO: 30 and SEQ ID NO: 45; SEQ ID NO: 31 and SEQ ID NO: 46; SEQ ID NO: 32 and SEQ ID NO: 47; SEQ ID NO: 33 and SEQ ID NO: 48; SEQ ID NO: 34 and SEQ ID NO: 49; SEQ ID NO: 35 and SEQ ID NO: 50; SEQ ID NO: 36 and SEQ ID NO: 51; SEQ ID NO: 37 and SEQ ID NO: 52; SEQ ID NO: 38 and SEQ ID NO: 53; SEQ ID NO: 39 and SEQ ID NO: 54; SEQ ID NO: 40 and SEQ ID NO: 55; SEQ ID NO: 41 and SEQ ID NO: 56; SEQ ID NO: 42 and SEQ ID NO: 57; and SEQ ID NO: 43 and SEQ ID NO: 58. 
     
     
         52 . The method of  claim 50 , wherein the set of IFN subtype transcripts comprises IFN-alpha1 transcripts. 
     
     
         53 . The method of  claim 50 , wherein the IFN subtype transcript comprises a set of at least four different IFN subtype transcripts. 
     
     
         54 . The method of  claim 53 , wherein the set comprises at least six different IFN subtype transcripts. 
     
     
         55 . The method of  claim 53 , wherein the set comprises at least eight different IFN subtype transcripts. 
     
     
         56 . The method of  claim 53 , wherein the set comprises at least eleven different IFN subtype transcripts. 
     
     
         57 . The method of  claim 50 , wherein the composition further comprises a probe selected from the list of probes consisting of SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; and SEQ ID NO: 72. 
     
     
         58 . The method of  claim 50 , wherein the sample is selected from the list consisting of a tissue sample, a cell sample, a bodily fluid, urine, blood, serum, plasma, a leukocyte, a monocyte, a peripheral blood leukocyte (PBL), lymph, saliva, cerebrospinal fluid (CSF), synovial fluid, bronchoalveolar lavage (BAL), pericardial fluid, spinal fluid, pleural fluid, pleural effusion, mucus, breast milk, amniotic fluid, vaginal fluid, semen, prostatic fluid, ascites, ascitic fluid, peritoneal fluid, aqueous humor, vitreous humor, tears, rheum, perspiration, cystic fluid, gastric acid and a tumor tissue. 
     
     
         59 . The method of  claim 50 , wherein the inflammatory condition is selected from the group consisting of a cancer, a viral infection, and an autoimmune disorder. 
     
     
         60 . The method of  claim 59 , wherein the cancer, viral infection, or autoimmune disorder is at least one of lupus, rheumatoid arthritis, multiple sclerosis, or Sjogren's syndrome. 
     
     
         61 . The method of  claim 50 , wherein the condition is lupus or a lupus-mediated condition. 
     
     
         62 . A method of determining whether an individual in need thereof has a disease, the method comprising:
 a) contacting a biological sample from the individual with a composition comprising a first oligonucleotide pair and a second oligonucleotide pair, wherein each oligonucleotide pair is capable of specifically binding to a single subtype of IFN transcript thereby simultaneously obtaining IFN transcript accumulation levels;   b) determining a pattern of IFN transcript levels in the sample;   c) comparing the pattern of IFN transcript levels of the individual to a pattern of IFN transcript levels of a known immune response, wherein the known immune response is associated with the disease; and   d) categorizing the individual as having the disease if the pattern of IFN transcript levels of the individual substantially match the pattern of IFN transcript levels of the known immune response; or   e) categorizing the individual as not having the disease if the pattern of IFN transcript levels of the individual does not substantially match the pattern of IFN transcript levels of the known immune response.   
     
     
         63 . The method of  claim 62 , wherein the first oligonucleotide pair is selected from the list of oligonucleotide pairs consisting of: SEQ ID NO: 30 and SEQ ID NO: 45; SEQ ID NO: 31 and SEQ ID NO: 46; SEQ ID NO: 32 and SEQ ID NO: 47; SEQ ID NO: 33 and SEQ ID NO: 48; SEQ ID NO: 34 and SEQ ID NO: 49; SEQ ID NO: 35 and SEQ ID NO: 50; SEQ ID NO: 36 and SEQ ID NO: 51; SEQ ID NO: 37 and SEQ ID NO: 52; SEQ ID NO: 38 and SEQ ID NO: 53; SEQ ID NO: 39 and SEQ ID NO: 54; SEQ ID NO: 40 and SEQ ID NO: 55; SEQ ID NO: 41 and SEQ ID NO: 56; SEQ ID NO: 42 and SEQ ID NO: 57; and SEQ ID NO: 43 and SEQ ID NO: 58. 
     
     
         64 . The method of  claim 62 , wherein the IFN subtype transcript comprises an IFN-alpha1 transcript. 
     
     
         65 . The method of  claim 62 , wherein the IFN subtype transcript comprises a set of at least four different IFN subtype transcripts. 
     
     
         66 . The method of  claim 65 , wherein the set comprises at least six different IFN subtype transcripts. 
     
     
         67 . The method of  claim 62 , wherein the composition further comprises a probe selected from the list of probes consisting of SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; and SEQ ID NO: 72. 
     
     
         68 . The method of  claim 62 , wherein the sample is selected from the list consisting of a tissue sample, a cell sample, a bodily fluid, urine, blood, serum, plasma, a leukocyte, a monocyte, a peripheral blood leukocyte (PBL), lymph, saliva, cerebrospinal fluid (CSF), synovial fluid, bronchoalveolar lavage (BAL), pericardial fluid, spinal fluid, pleural fluid, pleural effusion, mucus, breast milk, amniotic fluid, vaginal fluid, semen, prostatic fluid, ascites, ascitic fluid, peritoneal fluid, aqueous humor, vitreous humor, tears, rheum, perspiration, cystic fluid, gastric acid and a tumor tissue. 
     
     
         69 . The method of  claim 62 , wherein the disease is lupus or a lupus mediated condition. 
     
     
         70 . A kit for diagnosing an individual with a disease, wherein the kit comprises a composition comprising a first oligonucleotide pair and a second oligonucleotide pair, wherein each oligonucleotide pair is capable of specifically binding to a single subtype of IFN transcript and instructions for use in the method of  claim 62 . 
     
     
         71 . The kit of  claim 70 , wherein the first oligonucleotide pair is selected from the list of oligonucleotide pairs consisting of: SEQ ID NO: 30 and SEQ ID NO: 45; SEQ ID NO: 31 and SEQ ID NO: 46; SEQ ID NO: 32 and SEQ ID NO: 47; SEQ ID NO: 33 and SEQ ID NO: 48; SEQ ID NO: 34 and SEQ ID NO: 49; SEQ ID NO: 35 and SEQ ID NO: 50; SEQ ID NO: 36 and SEQ ID NO: 51; SEQ ID NO: 37 and SEQ ID NO: 52; SEQ ID NO: 38 and SEQ ID NO: 53; SEQ ID NO: 39 and SEQ ID NO: 54; SEQ ID NO: 40 and SEQ ID NO: 55; SEQ ID NO: 41 and SEQ ID NO: 56; SEQ ID NO: 42 and SEQ ID NO: 57; and SEQ ID NO: 43 and SEQ ID NO: 58.