DETECTION ASSAY FOR ANTI-SARS-COV-2 ANTIBODIES

Abstract

Protein biosensors and methods of using these sensors to detect anti-SARS-CoV-2 patient antibodies (Abs) in a solution-based, rapid, and quantitative COVID-19 serological assay are provided. In certain aspects, the sensors each comprise a first fusion protein that comprises a first SARS-CoV-2 viral protein and a first peptide fragment of a split reporter protein, and a second fusion protein that comprises a second fusion protein that comprises a second SARS-CoV-2 viral protein domain and a second peptide fragment of the split reporter protein. Only if the test sample comprise SARS-CoV- antibodies, the first and second peptide fragments associate to produce an enzymatically active reporter protein.

Description

Claims

1. A method for detecting antibodies against a SARS-CoV-2 viral protein in a biological sample comprising i) combining a) the biological sample;b) a first fusion protein that comprises a first SARS-CoV-2 viral protein domain and a first peptide fragment of a split reporter protein, andc) a second fusion protein that comprises a second SARS-CoV-2 viral protein domain and a second peptide fragment of the split reporter protein to produce a mixture;ii) maintaining the mixture under conditions in which, only if the biological sample comprises individual antibodies, at least one of which binds the first and the second SARS-CoV-2 viral protein domain simultaneously, the first peptide fragment and the second peptide fragment associate to produce an enzymatically active reporter protein; andiii) detecting the association of the first peptide fragment and the second peptide fragment if the biological sample comprises antibodies against the SARS-CoV-2 viral protein.

2. The method of claim 1 wherein the first and second SARS-CoV-2 viral protein domains are the same.

3. The method of claim 1, wherein the SARS-CoV-2 viral protein is the a SARS-CoV-2 N protein, wherein the first fusion protein comprises a first SARS-CoV-2 N protein domain andthe first peptide fragment of a split reporter protein, andthe second fusion protein comprises a second SARS-CoV-2 N protein domain andthe second peptide fragment of the split reporter protein; andwherein each of the first SARS-CoV-2 N protein domain and the second SARS-CoV-2 N protein domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

4. The method of claim 3 wherein the first and second SARS-Cov-2 N protein domains are the same.

5. The method of claim 1, wherein the SARS-CoV-2 viral protein is a Spike protein, wherein the first fusion protein comprises a first SARS-CoV-2 Spike RBD (SpikeRBD) domain and the first peptide fragment of a split reporter protein, andthe second fusion protein comprises a second SARS-CoV-2 SpikeRBD domain, and the second peptide fragment of the split reporter protein; andwherein each of the first SARS-CoV-2 SpikeRBD domain and the second SARS-CoV-2 SpikeRBD domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

6. The method of claim 5 wherein the first and second SARS-Cov-2 SpikeRBD domains are the same.

7. The method of claim 1, wherein the antibodies detected are neutralizing antibodies.

8. The method of claim 1, wherein the split reporter protein is a split-luciferase.

9. The method of claim 1, wherein the first peptide fragment of the split reporter protein comprises a sequence of SEQ ID NO: 4 (LgBiT) and second peptide fragment of the split reporter protein comprises a sequence of SEQ ID NO: 3 (SmBiT).

10. The method of claim 3, wherein the first peptide fragment of the split reporter protein is fused to the C-terminus of the SARS-CoV-2 N protein domain, and wherein the second peptide fragment of the split reporter protein is fused to the C-terminus of the second SARS-CoV-2 N protein domain.

11. The method of claim 1, wherein the first peptide fragment of the split reporter protein is fused to the first SARS-CoV-2 viral protein domain via a first flexible linker and/or the second peptide fragment of the split reporter protein is fused to the second SARS-CoV-2 viral protein domain via a second flexible linker.

12. The method of claim 11, wherein each of the first and second flexible linkers has a length in the range of one to 50 amino acids.

13. The method of claim 5, wherein the first peptide fragment of the split reporter protein is fused to the first SARS-CoV-2 SpikeRBD domain via a first flexible linker and/or the second peptide fragment of the split reporter protein is fused to the second SARS-CoV-2 SpikeRBD domain via a second flexible linker, andwherein the first flexible linker has a length of 15 amino acids and the second flexible linker has a length of 25 amino acids.

14. The method of claim 3, wherein the first peptide fragment of the split reporter protein is fused to the first SARS-CoV-2 N protein domain via a first flexible linker and/or the second peptide fragment of the split reporter protein is fused to the second SARS-CoV-2 N protein domain via a second flexible linker, andwherein the first flexible linker and the second flexible linker has each have a length of 10 amino acids.

15. The method of claim 1, wherein the first fusion protein is present in the mixture at a concentration in the range from 0.3 nM to 10 nM, and/or the second fusion protein is present in the mixture at a concentration in the range from 0.3 nM to 10 nM.

16. The method of claim 15, wherein the first fusion protein and the second fusion protein are present in the mixture at about equal molar concentrations.

17. A kit for detecting antibodies against a SARS-CoV-2 viral protein in a biological sample, wherein the kit comprises: i) a first fusion protein that comprises a first SARS-CoV-2 viral protein domain and a first peptide fragment of a split reporter protein, andii) a second fusion protein that comprises a second SARS-CoV-2 viral protein domain and a second peptide fragment of the split reporter protein,wherein the first SARS-CoV-2 viral protein domain shares at least 90% sequence identity with the second viral protein domain.

18. The kit of claim 17, wherein i) the first fusion protein comprises a first SARS-CoV-2 N protein domain and the first peptide fragment of a split reporter protein, andii) the second fusion protein comprises a second SARS-CoV-2 N protein domain and the second peptide fragment of the split reporter protein,wherein each of the first SARS-CoV-2 N protein domain and the second SARS-CoV-2 N protein domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.

19. The kit of claim 17, wherein i) the first fusion protein comprises a first SARS-CoV-2 SpikeRBD domain and the first peptide fragment of a split reporter protein, andii) the second fusion protein comprises a second SARS-CoV-2 SpikeRBD domain and the second peptide fragment of the split reporter protein,wherein each of the first and the second SARS-CoV-2 SpikeRBD domains comprise a sequence that is at least 90% identical to SEQ ID NO: 1.

20. The kit of claim 18, wherein the first peptide fragment of the split reporter protein is fused to the C- terminus of the first SARS-CoV-2 N protein domain, andwherein the second peptide fragment of the split reporter protein is fused to the C-terminus of the second SARS-CoV-2 N protein domain.

21. The kit of claim 17, wherein the split reporter protein is a split-luciferase.

22. The kit of claim 17, wherein the first fusion protein and the second fusion protein are lyophilized.

23. The kit of claim 21, wherein the kit further comprises a substrate for the split-luciferase.

24. The kit of claim 17, wherein the first peptide fragment of the split reporter protein comprises a sequence of SEQ ID NO: 4 and the second peptide fragment comprise a sequence of SEQ ID NO: 3.

25. The kit of claim 17, wherein the kit further comprises a negative control sample, optionally wherein the negative control sample comprises PBST and 4-10% PBS.

26. The kit of claim 19, wherein the kit further comprises a positive control sample, wherein the positive control sample comprises an antibody known to specifically bind to the SARS-CoV-2 SpikeRBD domain or to an ACE-Fc protein.

27. The kit of claim 18, wherein the kit further comprises a positive control sample, wherein the positive control sample comprises an antibody that is known to specifically bind to the SARS-CoV-2 N protein domain.

28. A reaction mixture comprising i) a test sample,ii) a first fusion protein that comprises a first SARS-CoV-2 viral protein domain of a SARS-CoV-2 viral protein and a first peptide fragment of a split reporter protein, andiii) a second fusion protein that comprises a second SARS-CoV-2 viral protein domain of the SARS-CoV-2 viral protein and a second peptide fragment of the split reporter protein,wherein, the first peptide fragment and the second peptide fragment of the split reporter protein associate to produce a detectable reporter protein if the test sample comprises antibodies that specifically bind the first and second SARS-CoV-2 viral protein domains.

29. The reaction mixture of claim 28, wherein the SARS-CoV-2 viral protein is the Spike (S) protein, and wherein each of the first SARS-CoV-2 viral protein domain and the second SARS-CoV-2 viral protein domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

30. The reaction mixture of claim 28, wherein the SARS-CoV-2 viral protein is the N protein, and wherein each of the first SARS-CoV-2 viral protein and the second SARS-CoV-2 viral protein comprise a sequence that is at least 90% identical to SEQ ID NO: 5.

31. A method of determining if an antibody in a test sample is competitive with a reference antibody against a SARS-CoV-2 viral protein domain, the method comprising: i) contacting in a first reaction mixture according to claim 28 a first aliquot of the test sample and a viral protein sensor, wherein the viral protein sensor comprises the first and second fusion proteins, and detecting a first signal produced from association of the first peptide fragment and the second peptide fragment in the viral protein sensor,ii) contacting in a second reaction mixture according to claim 28 a second aliquot of the test sample and an epitope-masked viral protein sensor, wherein the epitope-masked viral protein sensor comprises the first and second fusion proteins that are bound to a reference antibody that specifically binds to the SARS-CoV-2 viral protein domain at a known epitope, and detecting a second signal produced from association of the first peptide fragment and the second peptide fragment in the epitope-masked sensor; andiii) determining that the test sample comprises an antibody competitive with the reference antibody if the first signal is substantially higher than the second signal.

32. The kit of claim 17, wherein the first peptide fragment and the second peptide fragment associate to produce a detectable reporter protein only if the biological sample comprises an antibody that specifically binds to both the first and second SARS-CoV-2 viral protein domains simultaneously.