A PROTEOLIPOSOME-BASED ZNT8 SELF-ANTIGEN FOR TYPE 1 DIABETES DIAGNOSIS

• US 20200132698A1
• Filed: 07/05/2018
• Published: 04/30/2020
• Est. Priority Date: 07/12/2017
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University, Stanford University
• Inventors: Fu, Dax, Merriman, Chengfeng, Dai, Hongjie, Wan, Hao
• Granted Patent: US 11,892,457 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 33/564 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 Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Methods of detecting ZNT8 antibodies in serum are described. The methods include proteoliposomes comprising a transmembrane domain (TMD) and a cytosolic domain (CTD) of ZnT8 proteins exposed on the exterior of the proteoliposome; serum comprising antibodies targeting the ZnT8 proteins; and labelled captured autoantibodies that bind to ZnT8 antibodies.

Claims

1. A method of detecting ZNT8 antibodies in serum comprising:

providing a collection of proteoliposomes having an interior and an exterior wherein some proteoliposomes have protein sequences of a transmembrane domain (TMD) of a ZnT8 on its exterior and other proteoliposomes comprise protein sequences of a cytosolic domain (CTD) of a ZnT8 protein exposed on its exterior;

adding serum comprising antibodies targeting the protein sequences of TMD and/or CTD forming proteoliposomes bound to the antibodies;

adding a labelled captured autoantibody that binds to the antibodies bound to the proteoliposomes; and

measuring the amount of antibodies targeting the protein sequences of TMD and/or CTD bound to the proteoliposomes.

2. The method of claim 1 wherein the protein sequences of a TMD and a CTD are part of a full length ZnT8 protein.

3. The method of claim 2 wherein the ZnT8 protein is a human protein.

4. The method of claim 2 wherein the full length ZnT8 protein is a 325R polymorphic variant of human ZnT8.

5. The method of claim 1 wherein the collection of proteoliposomes comprises inside out proteoliposomes and right side out proteoliposomes.

6. The method of claim 5 wherein inside out proteoliposomes comprise CTD protein sequences on its exterior.

7. The method of claim 6 wherein the CTD protein sequences are part of a full length ZnT8 protein.

8. The method of claim 5 wherein the right side out proteoliposomes comprise TMD protein sequences on its exterior.

9. The method of claim 8 wherein the TMD protein sequences are part of a full length ZnT8 protein.

10. The method of claim 1 wherein the collection of proteoliposomes is on a chip.

11. The method of claim 10 wherein the chip is a pGOLD chip.

12. The method of claim 11 comprising one or more secondary proteins bound to the chip.

13. The method of claim 12 wherein the one or more secondary proteins is selected from the group consisting of IA2, GADA, and a combination thereof.

14. The method of claim 1 wherein the serum is human.

15. The method of claim 14 wherein the human serum is diluted with fetal bovine serum (FBS).

16. The method of claim 15 wherein the human serum is diluted in the range of 10 to 30 fold with the fetal bovine serum (FBS).

17. The method of claim 1 wherein the labelled captured autoantibody comprises a fluorescent label.

18. The method of claim 17 wherein the measuring comprises determining the amount of the antibodies bound to the ZnT8 proteins by analyzing the fluorescence intensity of the fluorescent label.

19. The method of claim 11 wherein the measuring the antibodies bound to the ZnT8 proteins comprises an infrared scanner.

20. The method of claim 1 wherein the proteoliposomes comprises lipids selected from the group comprising 1,2-dioleoyl-sn-glycero-3-phophocholine;

1,2-dioleoyl-sn-glycero-3-phospho-(1′

-rac-glycerol);

1,2-dioleoyl-sn-glycero-3-phophoethaolamine; and

a combination thereof.

21. The method of claim 1 wherein the detection has a detection performance of greater than 70% sensitivity and greater than 90% specificity.

22. The method of claim 10 wherein the collection of proteoliposomes is printed on a chip.

23. A method of detecting ZNT8 antibodies in serum comprising:

forming a mixture by combining proteoliposomes having an interior and an exterior comprising protein sequences of a transmembrane domain (TMD) and a cytosolic domain (CTD) of a ZnT8 proteins exposed on the exterior of the proteoliposomes;

serum comprising antibodies targeting the protein sequences;

a labelled captured autoantibody that binds to the antibodies targeting the protein sequences; and

measuring the amount of antibodies bound to the protein sequences.