DETECTION ASSAY FOR ANTI-SARS-COV-2 ANTIBODIES
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.
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, and
c) 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; and
iii) 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, and
the second fusion protein comprises a second SARS-CoV-2 N protein domain andthe second peptide fragment of the split reporter protein; and
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.
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, and
the second fusion protein comprises a second SARS-CoV-2 SpikeRBD domain, and the second peptide fragment of the split reporter protein; and
wherein 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, and
wherein 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, and
wherein 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, and
ii) 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, and
ii) 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, and
ii) 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, 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.
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, and
iii) 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; and
iii) 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.