Method for the detection of an albumin isoform

Patent metadata

Field	Value
Publication number	US-10126309-B2
Application number	US-201514799778-A
Country	US
Kind code	B2
Filing date	Jul 15, 2015
Priority date	Jun 6, 2011
Publication date	Nov 13, 2018
Grant date	Nov 13, 2018

Abstract

The present invention provides methods for detecting, analyzing, and identifying biomolecules used to identifying patient with dengue-like symptom who are at risk of DHF. The inventive method comprises detecting in a sample from a subject dengue infected patient one or more biomarkers selected from the group consisting of IL-10, fibrinogen, C4A, immunoglobulin, tropomyosin, and three isoforms of albumin, and which are used in a predictive MARS model to detect patients with risk of developing DHF.

First claim

What is claimed is:

1. A sample fractionation method for albumin biomarker identification comprising:
(a) fractionating a sample derived from blood by size exclusion chromatography, wherein the sample has protein/peptide complexes dissociated by denaturation and the sample is spiked with a labeled protein of about 200 amino acid residues for differentiating protein and peptide pools;
(b) collecting a (i) protein pool comprising the fractions preceding the end of the labeled protein peak, and (ii) a peptide pool comprising the fractions after the end of the labeled protein peak and before the free dye peak;
(c) incubating the collected protein pool to allow for renaturation of the collected proteins in the protein pool;
(d) depleting the protein pool by exposing the protein pool to an antibody depletion column and collecting column flow through to produce a depleted sample;
(e) labeling the depleted sample with an uncharged thiol reactive label forming a saturated fluorescence labeled sample;
(f) conducting two dimensional (2D) gel electrophoresis on the saturated fluorescence labeled sample, producing a 2D gel;
(g) imaging the 2D gel and identifying a protein(s) of interest, wherein the protein of interest is an albumin isoform; and
(h) conducting mass spectrometry analysis of the protein(s) of interest.

2. The method of claim 1 , wherein the size exclusion chromatography uses a size exclusion column having a separation range between molecular weights of 3,000 to 70,000 daltons.

3. The method of claim 1 , wherein the spiked labeled protein is a labeled thaumatin.

4. The method of claim 1 , wherein the antibody depletion column is an IgY antibody depletion column.

5. The method of claim 1 , wherein the thiol reactive label is bodipy FL-maleimide.

Assignees

• Univ Texas
• The Board Of Regents Of The Univ Of Texas Systems

Inventors

• Wiktorowicz John E

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• G01N2333/185

  Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue · CPC title

• G01N2333/4716

  Complement proteins, e.g. anaphylatoxin, C3a, C5a · CPC title

• G01N33/6869 Primary

  Interleukin · CPC title

• G01N33/564

  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 title