Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration

Patent metadata

Field	Value
Publication number	US-9709573-B2
Application number	US-201314384848-A
Country	US
Kind code	B2
Filing date	Mar 13, 2013
Priority date	Mar 13, 2012
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

The present invention relates to the field of biomarkers. More specifically, the present invention relates to biomarkers useful in diagnosing brain injury or neurodegeneration. In one embodiment, a method for diagnosing brain injury in a patient comprises the steps of (a) obtaining a sample from the patient; (b) determining the ratio of citrullinated to unmodified arginine residues at one or more arginine residues of one or more brain injury biomarker proteins; and (c) correlating the ratio to a patient having brain injury or to a patient not having brain injury, thereby providing the diagnosis.

First claim

We claim:

1. A method for detecting citrullinated brain injury biomarker proteins in a sample from a patient, the method comprising:
detecting in a patient sample citrullinated arginine residues in citrullinated brain injury biomarker protein neurogranin (NRGN) and in one or more citrullinated brain injury biomarker proteins selected from the group consisting of tubulin beta-4B chain, tubulin alpha-1B chain, CNPase, PPIA, Septin-7, Elongation factor1-alpha2, TPPP, TPPP3, Ermin Isoform 2, NDRG2Isoform 2, astrotactin 1 (ASTN1), brain angiogenesis inhibitor 3 (BAI3), carnosine dipeptidase 1 (CNDP1), ERMIN, glial fibrillary acidic protein (GFAP), glutamate receptor metabotropic 3 (GRM3), kelch-like protein 32 (KLH32), melanoma antigen family E,2 (MAGE2), myelin basic protein (MBP), neuregulin 3 (NRG3), oligodendrocyte myelin glycoprotein (OMG), solute carrier family 39 (zinc transporter), reticulon 1 (RTN1), MT3, and peptidylarginine deiminase (types 1-4 and 6) (PAD) by contacting the citrullinated arginine residues in the citrullinated brain injury biomarker proteins with an antibody that binds to said citrullinated arginine residues and detecting binding of the antibody to the residues or by detecting the citrullinated arginine residues using mass spectrometry, thereby detecting citrullinated brain injury biomarker proteins in the patient's sample.

2. The method of claim 1 , wherein the patient sample is selected from the group consisting of blood, peripheral blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, stool and synovial fluid.

3. The method of claim 2 , wherein the patient sample is blood, plasma serum, cerebrospinal fluid (CSF), or urine.

4. The method of claim 3 , wherein the sample is cerebrospinal fluid (CSF).

5. The method of claim 3 , wherein the sample is blood.

6. The method of claim 3 , wherein the sample is serum.

7. The method of claim 1 , wherein the detecting by mass spectrometry is accomplished using multiple reaction monitoring mass spectrometry (MRM-MS).

8. The method of claim 1 , wherein the detecting of antibody binding step is accomplished using an immunoassay.

9. The method of claim 1 , wherein the brain injury biomarker proteins are NRGN and MBP.

10. The method of claim 1 , wherein the brain injury biomarker proteins are NRGN and GFAP.

11. The method of claim 1 , wherein the subject has or is suspected of having a brain injury.

12. The method of claim 11 , wherein the brain injury is a non-traumatic brain injury selected from the group consisting of stroke, neurodegenerative disease and brain hemorrhage.

13. The method of claim 1 , wherein the brain injury biomarker proteins are citrullinated NRGN, citrullinated GFAP and citrullinated MBP.

14. A method for detecting autoantibodies against a citrullinated brain injury biomarker protein in a sample from a subject, the method comprising the steps of:
(a) contacting a biological sample obtained from the subject with citrullinated brain injury biomarker protein neurogranin (NRGN) and one or more citrullinated brain injury biomarker proteins selected from the group consisting of tubulin beta-4B chain, tubulin alpha-1B chain, CNPase, PPIA, Septin-7, Elongation factorl-alpha2, TPPP, TPPP3, Ermin Isoform 2, NDRG2 Isoform 2, astrotactin 1 (ASTN1), brain angiogenesis inhibitor 3 (BAI3); carnosine dipeptidase 1 (CNDP1); ERMIN; glial fibrillary acidic protein (GFAP); glutamate receptor metabotropic 3 (GRM3); kelch-like protein 32 (KLH32); melanoma antigen family E,2(MAGE2); myelin basic protein (MBP); neuregulin 3 (NRG3); oligodendrocyte myelin glycoprotein (OMG); solute carrier family 39 (zinc transporter); reticulon 1 (RTN1); MT3, and peptidylarginine deiminase (types 1-4 and 6) (PAD)); and
(b) detecting the binding of the citrullinated brain injury biomarker proteins to autoantibodies in the sample which are specific for the citrullinated brain injury biomarker proteins, wherein the detection of binding is indicative of the presence of citrullinated brain injury biomarker protein autoantibodies.

15. The method of claim 14 , wherein the binding is detected by immunosorbent assay, by immunoprecipitation, or by immunoblotting.

16. The method of claim 14 , wherein the subject has or is suspected of having a brain injury.

17. The method of claim 16 , wherein the brain injury is a non-traumatic brain injury selected from the group consisting of stroke, neurodegenerative disease and brain hemorrhage.

18. The method of claim 14 , wherein autoantibodies against citrullinated NRGN and citrullinated MBP are detected.

19. The method of claim 14 , wherein autoantibodies against citrullinated NRGN and citrullinated GFAP are detected.

20. The method of claim 14 , wherein autoantibodies against citrullinated NRGN, citrullinated MBP and citrullinated GFAP are detected.

Assignees

• Univ Johns Hopkins

Inventors

• Van Eyk Jennifer E
• Everett Allen Dale
• Jin Zhicheng

Classifications

• G01N33/6842 Primary

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• H01J49/0077

  specific reactions other than fragmentation · CPC title

• H01J49/0027

  Methods for using particle spectrometers · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N2800/28

  Neurological disorders · CPC title