Proteoliposome-based ZnT8 self-antigen for type 1 diabetes diagnosis
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.
1. A method of detecting ZnT8 antibodies in serum comprising:
(a) providing a collection of proteoliposomes having an interior and an exterior wherein some proteoliposomes have protein sequences of a transmembrane domain (TMD) of a zinc transporter 8 (ZnT8) on its exterior and other proteoliposomes comprise protein sequences of a cytosolic domain (CTD) of a ZnT8 protein exposed on its exterior,
wherein the protein sequences of a TMD and a CTD are part of a full length ZnT8 protein,
wherein the full length ZnT8 protein is a 325R polymorphic variant of human ZnT8,
wherein the collection of proteoliposomes is prepared by dehydrating soluble ZnT8-containing proteoliposomes followed by rehydration of the dehydrated ZnT8-containing proteoliposomes, and
wherein the collection of proteoliposomes is bound to a plate or chip;
(b) adding serum comprising antibodies targeting the protein sequences of TMD and/or CTD forming proteoliposomes bound to the antibodies;
(c) adding a labelled secondary antibody that binds to the antibodies bound to the proteoliposomes; and
(d) 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 collection of proteoliposomes comprises inside out proteoliposomes and right side out proteoliposomes.
3. The method of claim 2 wherein inside out proteoliposomes comprise CTD protein sequences on its exterior.
4. The method of claim 2 wherein the right side out proteoliposomes comprise TMD protein sequences on its exterior.
5. The method of claim 1 , wherein the collection of proteoliposomes is printed on the chip.
6. The method of claim 5 , wherein the chip is a plasmonic gold (pGOLD) chip.
7. The method of claim 6 comprising one or more secondary proteins bound to the chip.
8. The method of claim 7 wherein the one or more secondary proteins is selected from the group consisting of islet antigen 2 (IA2), glutamic acid decarboxylase (GAD), and a combination thereof.
9. The method of claim 1 wherein the serum is human.
10. The method of claim 9 wherein the human serum is diluted with fetal bovine serum (FBS).
11. The method of claim 10 wherein the human serum is diluted in the range of 10 to 30 fold with the fetal bovine serum (FBS).
12. The method of claim 1 wherein the labelled secondary antibody comprises a fluorescent label.
13. The method of claim 12 , wherein the measuring comprises determining the amount of the antibodies bound to the ZnT8 protein by analyzing the fluorescence intensity of the fluorescent label.
14. The method of claim 11 , wherein the measuring the antibodies bound to the ZnT8 protein comprises an infrared scanner.
15. The method of claim 1 wherein the collection of proteoliposomes comprises lipids selected from the group consisting of 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.
16. The method of claim 1 wherein the detection has a detection performance of greater than 70% sensitivity and greater than 90% specificity.