PROTEIN MICROARRAY, DETECTION METHOD THEREOF AND EVALUATION METHOD THEREOF

Abstract

A protein microarray, a detection method thereof, and an evaluation method thereof are provided. The protein microarray includes a carrier including a protein array block on a surface thereof and at least one protein immobilized on the protein array block. The at least one protein includes a spike protein and a nucleocapsid protein, and can bind to a first antibody in a to-be-tested sample. A method for detecting virus infection or evaluating the ability of a bioagent to block virus infection includes the steps of respectively adding blood, serum, plasma or the bioagent and a second antibody to the protein microarray and detecting an optical signal. The protein microarray can be used to detect a coronavirus and an influenza virus, and achieve the effect of quickly, sensitively, and accurately confirming whether a subject is infected by the virus.

Claims

1. A protein microarray, comprising: a substrate including a plurality of protein array blocks on a surface of the substrate; and at least one protein immobilized on each of the plurality of protein array blocks, wherein the at least one protein is derived from a virus and comprises a spike protein and a nucleocapsid protein, the at least one protein comprises an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 4, and the at least one protein specifically binds to a first antibody in a to-be-tested sample or a bioagent.

2. The protein microarray according to claim 1, wherein the substrate is a glass slide or a nylon film substrate.

3. The protein microarray according to claim 1, wherein the surface of the substrate comprises an aldehyde modified layer or an amino modified layer.

4. The protein microarray according to claim 1, wherein the at least one protein further comprises a 51 domain of the spike protein, a hemagglutinin protein or a combination thereof.

5. The protein microarray according to claim 1, wherein the at least one protein further comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

6. The protein microarray according to claim 4, wherein the at least one protein further comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

7. The protein microarray according to claim 1, wherein the virus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARSC-CoV), human coronavirus HKU (HKU-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), influenza A virus subtype H1N1, influenza A virus subtype H3N2, and influenza B virus.

8. The protein microarray according to claim 1, wherein the to-be-tested sample is a blood sample, a serum sample or a plasma sample from a subject.

9. The protein microarray according to claim 1, wherein the first antibody is human immunoglobulin G, human immunoglobulin A or human immunoglobulin M.

10. The protein microarray according to claim 1, wherein the bioagent is a monoclonal antibody drug or a receptor blocker.

11. The protein microarray according to claim 10, wherein the monoclonal antibody drug is a murine-derived monoclonal antibody or a rabbit-derived monoclonal antibody.

12. The protein microarray according to claim 10, wherein the monoclonal antibody drug is a monoclonal antibody against the spike protein of the SARS-CoV-2, a monoclonal antibody against the S1 domain of the spike protein of the SARS-CoV-2 or a monoclonal antibody against the nucleocapsid protein of the SARS-CoV-2.

13. The protein microarray according to claim 10, wherein the receptor blocker is a human angiotensin-converting enzyme 2 on a cell surface.

14. A method for in vitro detection of virus infection in a to-be-tested sample, comprising steps of: providing a protein microarray according to claim 1; adding a non-protein blocking reagent to the plurality of protein array blocks of the protein microarray, and reacting for 5 to 10 minutes to obtain a first protein microarray; providing a to-be-tested sample from a subject, adding the to-be-tested sample to the first protein microarray, and reacting for 50 to 70 minutes followed by washing to obtain a second protein microarray; providing a second antibody, adding the second antibody to the second protein microarray, and reacting for 25 to 35 minutes followed by washing to obtain a third protein microarray, wherein the second antibody is fluorescently labeled or enzyme-labeled; and reading an optical signal generated from the third protein microarray by a signal reader.

15. The method according to any one of claim 14, after reading the optical signal generated from the third protein microarray by the signal reader, further comprising a step of determining whether the subject is infected by a virus, wherein the virus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARSC-CoV), human coronavirus HKU (HKU-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), influenza A virus subtype H1N1, influenza A virus subtype H3N2, and influenza B virus.

16. A method for evaluating the ability of a bioagent to block virus infection, comprising steps of: providing a protein microarray according to claim 1, adding a non-protein blocking reagent to the plurality of protein array blocks of the protein microarray, and reacting for 5 to 10 minutes to obtain a fourth protein microarray; providing a bioagent, adding the bioagent to the fourth protein microarray, and reacting for 50 to 70 minutes followed by washing to obtain a fifth protein microarray; providing a second antibody, adding the second antibody to the fifth protein microarray, and reacting for 25 to 35 minutes followed by washing to obtain a sixth protein microarray, wherein the second antibody is fluorescently labeled or enzyme-labeled; and reading an optical signal generated from the sixth protein microarray by a signal reader.

17. The method according to claim 16, after reading the optical signal generated from the sixth protein microarray by the signal reader, further comprising a step of evaluating the ability of the bioagent to block virus infection, wherein the virus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARSC-CoV), human coronavirus HKU (HKU-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), human coronavirus OC43 (OC43-CoV), influenza A virus subtype H1N1, influenza A virus subtype H3N2, and influenza B virus.

18. The method according to claim 16, wherein the bioagent is a monoclonal antibody drug or a receptor blocker.

19. The method according to claim 18, wherein the monoclonal antibody drug is a monoclonal antibody against the spike protein of the SARS-CoV-2, a monoclonal antibody against the S1 domain of the spike protein of the SARS-CoV-2 or a monoclonal antibody against the nucleocapsid protein of the SARS-CoV-2.

20. The method according to claim 18, wherein the receptor blocker is a human angiotensin-converting enzyme 2 on a cell surface.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1A is a resultant graph of the binding specificity between a protein microarray of the present disclosure and a human angiotensin-converting enzyme 2 (ACE2) on a cell surface. Data are analyzed by t-test, * p<0.05 and *** p<0.001, compared with MERS-CoV.

[0046] FIG. 1B is a resultant graph of the binding specificity between a protein microarray of the present disclosure and a monoclonal antibody against a 51 domain of a spike protein of SARS-CoV-2 (αS protein mAb). Data are analyzed by t-test, * p<0.05 and *** p<0.001, compared with MERS-CoV.

[0047] FIG. 1C is a resultant graph of the binding specificity between a protein microarray of the present disclosure and a monoclonal antibody against a nucleocapsid protein of SARS-CoV-2 (αN protein mAb). Data are analyzed by t-test, * p<0.05 and *** p<0.001, compared with MERS-CoV.

[0048] FIG. 2A is a resultant graph of the Cy3 background intensity of the protein microarray of the present disclosure after blocking with 5% bovine serum albumin (BSA) for 1 hour or a non-protein reagent (HyBlock) for 10 minutes, respectively. Data are analyzed by t-test, ****p<0.0001, compared with BSA-1 hour.

[0049] FIG. 2B is a resultant graph of the Cy5 background intensity of the protein microarray of the present disclosure after blocking with 5% bovine serum albumin (BSA) for 1 hour or a non-protein reagent (HyBlock, Hycell International Co. Ltd., Taiwan) for 10 minutes, respectively. Data are analyzed by t-test, ****p<0.0001, compared with BSA-1 hour.

[0050] FIG. 3A is a resultant graph of the serum IgG reactivity to the spike protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from a healthy subject or a patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0051] FIG. 3B is a resultant graph of the serum IgG reactivity to the nucleocapsid protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0052] FIG. 3C is a resultant graph of the serum IgG reactivity to the spike protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0053] FIG. 3D is a resultant graph of the serum IgG reactivity to the nucleocapsid protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0054] FIG. 3E is a resultant graph of a serum IgA reactivity to the spike protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0055] FIG. 3F is a resultant graph of the serum IgA reactivity to the nucleocapsid protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0056] FIG. 3G is a resultant graph of the serum IgA reactivity to the spike protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0057] FIG. 3H is a resultant graph of the serum IgA reactivity to the nucleocapsid protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the healthy subject or the patient suffering from COVID-19. Data are analyzed by t-test, ****p<0.0001, compared with the healthy subject.

[0058] FIG. 4A is a resultant graph of the serum IgG cross-reactivity to the spike protein of SARS-CoV-2 and the S1 domain of the spike protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0059] FIG. 4B is a resultant graph of the relationship of the serum IgG cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.005.

[0060] FIG. 4C is a resultant graph of the relationship of the serum IgG cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of HKU-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0061] FIG. 4D is a resultant graph of the relationship of the serum IgG cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of OC43-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.005.

[0062] FIG. 4E is a resultant graph of the relationship of the serum IgG cross-reactivity to the spike protein of SARS-CoV-2 and a hemagglutinin protein of influenza A virus subtype H3N2 by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.001.

[0063] FIG. 4F is a resultant graph of the relationship of the serum IgG cross-reactivity to the nucleocapsid protein of SARS-CoV-2 and the nucleocapsid protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgG is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0064] FIG. 5A is a resultant graph of the serum IgA cross-reactivity to the spike protein of SARS-CoV-2 and the S1 domain of the spike protein of SARS-CoV-2 by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0065] FIG. 5B is a resultant graph of the relationship of the serum IgA cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0066] FIG. 5C is a resultant graph of the relationship of the serum IgA cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of HKU-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0067] FIG. 5D is a resultant graph of the relationship of the serum IgA cross-reactivity to the spike protein of SARS-CoV-2 and the spike protein of OC43-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0068] FIG. 5E is a resultant graph of the relationship of the serum IgA cross-reactivity to the nucleocapsid protein of SARS-CoV-2 and the nucleocapsid protein of SARS-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.0001.

[0069] FIG. 5F is a resultant graph of the relationship of the serum IgA cross-reactivity to the nucleocapsid protein of SARS-CoV-2 and the spike protein of 229E-CoV by use of the protein microarray of the present disclosure for detection. The serum IgA is obtained from the patient suffering from COVID-19. Data are analyzed by t-test, p<0.001.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0070] The following provides specific embodiments to illustrate the implementation of the present disclosure. A person having ordinary skill in the art can understand other advantages and effects of the present disclosure from the contents disclosed in the present specification. However, the exemplary embodiments disclosed in the present disclosure are only for illustrative purposes and should not be regarded as limiting the scope of the present disclosure. In other words, the present disclosure can also be implemented or applied by other different specific embodiments, and various details in the present specification can also be modified and changes based on different viewpoints and applications without departing from the concept of the present disclosure.

[0071] Unless otherwise indicated herein, the singular forms “one” and “the” used in the specification and the appended claims of the present disclosure include the plural. Unless otherwise indicated herein, the term “or” used in the specification and the appended claims of the present disclosure includes the meaning of “and/or”.

Example 1: Preparation of Protein Microarray

[0072] The proteins from different viruses with histidine tag (His-tag) listed in Table 1 are purchased from Sino Biological Inc. (mainland China), and each of the proteins is used as a biomarker for detecting virus infection. Spike proteins (hereinafter referred to as “S protein”) shown in SEQ ID NO: 1 ′ SEQ ID NO: 2, and SEQ ID NO: 3 are respectively from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARSC-CoV); nucleocapsid proteins (hereinafter referred to as “N protein”) shown in SEQ ID NO: 4 ′ SEQ ID NO: 5, and SEQ ID NO: 6 are respectively from SARS-CoV-2, MERS-CoV, and SARSC-CoV; and 51 domains of the spike proteins (hereinafter referred to as “51 domain of S protein”) shown in SEQ ID NO: 7 ′ SEQ ID NO: 8, and SEQ ID NO: 9 are respectively from SARS-CoV-2, MERS-CoV, and SARSC-CoV; the S proteins shown in SEQ ID NO: 10 ′ SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 are respectively from human coronavirus HKU (HKU-CoV), human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), and human coronavirus OC43 (OC43-CoV); the N proteins shown in SEQ ID NO: 14 ′ SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 are respectively from HKU-CoV, 229E-CoV, NL63-CoV, and OC43-CoV; and hemagglutinin proteins (hereinafter referred to as “HA protein”) shown in SEQ ID NO: 18 ′ SEQ ID NO:19, and SEQ ID NO: 20 are respectively from influenza A virus subtype H1N1, influenza A virus subtype H3N2, and influenza B virus.

[0073] The sequence number shown in SEQ ID NO: 1 is renumbered from 1 to 1198 according to Val 16 to Pro1213 of Uniprot ID P0DTC2 sequence. The sequence number shown in SEQ ID NO: 7 is renumbered from 1 to 670 according to Val 16 to Arg685 of Uniprot ID P0DTC2 sequence. The sequence number shown in SEQ ID NO: 11 is renumbered from 1 to 1100 according to Cys16-Trp1115 of Uniprot ID P15423 sequence.

TABLE-US-00001 TABLE 1 List of the proteins from different viruses with His-tag protein virus amino acid Uniprot ID S SARS-CoV-2 SEQ ID NO: 1 P0DTC2 (Val 16-Pro1213) protein MERS-CoV SEQ ID NO: 2 K9N5Q8 (Met1-Trp1297) SARS-CoV SEQ ID NO: 3 P59594 (Met1-Pro1195) N SARS-CoV-2 SEQ ID NO: 4 P0DTC9 (Met1-A1a419) protein MERS-CoV SEQ ID NO: 5 R9UM87 (Met1-Asp413) SARS-CoV SEQ ID NO: 6 P59595 (Met1-A1a422) S1 SARS-CoV-2 SEQ ID NO: 7 P0DTC2 (Val 16-Arg685) domain MERS-CoV SEQ ID NO: 8 K9N5Q8 (Met1-G1u725) of S SARS-CoV SEQ ID NO: 9 P59594 (Met1-Arg667) protein S HKU-CoV SEQ ID NO: 10 Q0ZME7 (Met1-Pro1295) protein 229E-CoV SEQ ID NO: 11 P15423 (Cys16-Trp1115) NL63-CoV SEQ ID NO: 12 Q6Q152 (Met1-Pro1296) OC43-CoV SEQ ID NO: 13 P36334 (Met1-Pro1304) N HKU-CoV SEQ ID NO: 14 Q5MQC6 (Met1-Ala441) protein 229E-CoV SEQ ID NO: 15 P15130 (Met1-Asn389) NL63-CoV SEQ ID NO: 16 Q6Q1R8 (Met1-His377) OC43-CoV SEQ ID NO: 17 P33469 (Met1-I1e448) HA influenza SEQ ID NO: 18 Q9WFX3 (Met 1-Gln 529) protein A virus subtype H1N1 influenza SEQ ID NO: 19 P03437 (Met 1-Trp 530) A virus subtype H3N2 influenza SEQ ID NO: 20 P03460 (Met 1-Ala 556) B virus

[0074] After rinsing a glass slide with water, ethanol, acetone and methanol in sequence, wash the glass slide with 20% KOH solution at 65° C. for 2 hours, followed by wishing with H.sub.2SO.sub.4/H.sub.2O.sub.2 with a volume ratio of 3:1 for 12 minutes to obtain a cleaned glass slide. The cleaned glass slide is then coated by the steps of incubating the cleaned glass slide with 2.5% 3-aminopropyl triethoxysilane (dissolved in alcohol) for 5 minutes, then washing and being dried; incubating with 0.5% glutaraldehyde (dissolved in 0.05 M sodium borate pH 8.5) for 16 hours, then washing and being dried to obtain a surface-treated glass slide. The surface-treated glass slide is stored in a sealed vacuum bag at 4° C.

[0075] Each surface-treated glass slide has 14 blocks. Triplicates of each of the proteins shown in Table 1 and each of the 11 control group samples shown in Table 2 are printed in each block (9×10 format) on the surface-treated glass slide by a microarray spotter (CapitalBio SmartArrayer™ 136, mainland China) to obtain a protein microarray. The protein microarray is immobilized overnight at room temperature, then vacuum sealed, and stored at 4° C. for short-term (less than 6 months) or at −80° C. for long-term storage (6 months to a few years).

Table 2 List of the control group samples

Example 2 Analysis of Receptor Specificity and Antibody Specificity of the Protein Microarray

[0076] The receptor specificity of the protein microarray is analyzed by a human angiotensin-converting enzyme 2 (ACE2) on a cell surface purchased from Sino Biological Inc. (mainland China). The antibody specificity of the protein microarray is analyzed by antibody drugs, such as a monoclonal antibody against the S1 domain of S protein of SARS-CoV-2 (hereinafter referred to as αS protein mAb) purchased from Sino Biological Inc. (mainland China) and a monoclonal antibody against the N protein of SARS-CoV-2 (hereinafter referred to as αN protein mAb) purchased from Sino Biological Inc. (mainland China).

[0077] The ACE2 receptors of 25 ng, 50 ng, 75 ng, 100 ng, and 125 ng, which have been serially diluted, are reacted with the protein microarray for 1 hour, and then the protein microarray is reacted with a Cy3 labeled anti-human IgG antibody for 30 minutes. Referring to FIG. 1A, the result shows that nanogram-level of ACE2 may significantly bind to the S protein of SARS-CoV-2 and the S protein of SARS-CoV on the protein microarray but may not bind to the S protein of MERS-CoV.

[0078] The αS protein mAb of 100 pg, 200 pg, 300 pg, and 400 pg, which have been serially diluted, are reacted with the protein microarray for 1 hour, and then the protein microarray is reacted with a Cy5 labeled anti-rabbit IgG antibody for 30 minutes. Referring to FIG. 1B, the result shows that picogram-level of αS protein mAb may significantly bind to the S protein of SARS-CoV-2 and the S protein of SARS-CoV on the protein microarray, but may not bind to the S protein of MERS-CoV.

[0079] The αN protein mAb of 10 pg, 20 pg, 30 pg, 40 pg, and 50 pg which have been serially diluted, are reacted with the protein microarray for 1 hour, and then the protein microarray is reacted with a Cy5 labeled anti-rabbit IgG antibody for 30 minutes. Referring to FIG. 1C, the result shows that picogram-level of αN protein mAb may significantly bind to the N protein of SARS-CoV-2 and the N protein of SARS-CoV on the protein microarray, but may not bind to the N protein of MERS-CoV.

[0080] Based on the above, the results show that the protein microarray may effectively and specifically bind to the ACE2 receptor, the αS protein mAb and the αN protein mAb. Moreover, the minimum detection limits of the αS protein mAb and the αN protein mAb are 200 pg and 10 pg, respectively. Therefore, it is demonstrated that the protein microarray may be used to evaluate the abilities of a ACE2 receptor blocker and a monoclonal antibody drug against a virus to block virus infection.

Example 3: Analysis of the Reactivity of the Protein Microarray with an Immunoglobulin G (IgG) and an Immunoglobulin a (IgA)

[0081] In order to confirm the influence of a blocking buffer on the accuracy of the reading of the protein microarray, each of the protein microarrays is respectively blocked with 5% BSA blocking reagent for 1 hour and HyBlock reagent (purchased from Hycell International Co. Ltd., Taiwan) which is a non-protein blocking reagent for 10 minutes, then each of the protein microarrays is reacted with 0.1 μl serum from COVID-19 patients for 1 hour, followed by washing with Tris-buffered saline with 0.1% Tween® 20 (TBST) buffer, and then each of the protein microarrays is reacted with Cy3 labeled anti-human IgG antibody or Cy5 labeled anti-human IgA antibody for 30 minutes. Finally, each of the protein microarrays is washed with TBST buffer, and then the background fluorescence of each of the protein microarrays is detected by a fluorescence detection system (“Caduceus” SpinScan Microarray Scanner HC-BS01, Caduceus Biotechnology Inc., Taiwan). Referring to FIG. 2A and FIG. 2B, the results show that blocking the non-specific antigen on the protein microarray with the HyBlock reagent shows a cleaner background with superior blocking time, thereby shortening the analysis time compared to 5% BSA blocking reagent.

[0082] To profile the reactivities of serum IgG and serum IgA from a subject to each protein on the protein microarray, each of the protein microarrays is blocked with HyBlock reagent for 10 minutes, and then reacted with 0.1 μl of serum from 32 patients suffering from COVID-19 and from 32 healthy subjects for 1 hour, followed by washing with TBST buffer. Finally, each of the protein microarrays is reacted with a Cy3 labeled anti-human IgG antibody or a Cy5 labeled anti-human IgA antibody for 30 minutes, followed by washing with TBST buffer. Cy3 fluorescence or Cy5 fluorescence of each of the protein microarrays is detected by the fluorescence detection system microarray (“Caduceus” SpinScan Microarray Scanner HC-BS01, Caduceus Biotechnology Inc., Taiwan).

[0083] Referring to FIG. 3A and FIG. 3B, the results respectively show that the protein microarray may significantly distinguish the reactivity of the S protein of SARS-CoV-2 to the serum IgG from COVID-19 patients from that of the healthy subjects, and the reactivity of the N protein of SARS-CoV-2 to the serum IgG from COVID-19 patients from that of the healthy subjects. Referring to FIG. 3C and FIG. 3D, the results respectively show that the protein microarray may significantly distinguish the reactivity of the S protein of SARS-CoV to the serum IgG from COVID-19 patients from that of the healthy subjects, and the reactivity of the N protein of SARS-CoV to the serum IgG from COVID-19 patients from that of the healthy subjects.

[0084] Referring to FIG. 3E and FIG. 3F, the results respectively show that the protein microarray may significantly distinguish the reactivity of the S protein of SARS-CoV-2 to the serum IgA from COVID-19 patients from that of the healthy subjects, and the reactivity of the N protein of SARS-CoV-2 to the serum IgA from COVID-19 patients from that of the healthy subjects. Referring to FIG. 3G and FIG. 3H, the results respectively show that the protein microarray may significantly distinguish the reactivity of the S protein of SARS-CoV to the serum IgA from COVID-19 patients from that of the healthy subjects, and the reactivity of the N protein of SARS-CoV to the serum IgA from COVID-19 patients from that of the healthy subjects.

[0085] Based on the above, it is demonstrated that the protein microarray may significantly distinguish the reactivity of each protein such as the S protein or N protein from SARS-CoV-2 and SARS-CoV to the serum IgG and serum IgA from COVID-19 patients from that of the healthy subjects.

Example 4: Evaluation of the Specificity and Sensitivity of Protein Microarrays with a Single Biomarker or a Combination of Two Biomarkers for Detecting the Serum IgG or the Serum IgA from the Patients Suffering from COVID-19

[0086] The protein microarrays with a single biomarker or a combination of two biomarkers are blocked with HyBlock regent for 10 minutes, and each of the protein microarrays is reacted with 0.1 μl serum of 36 patients suffering from a COVID-19 for 1 hour, followed by washing with TBST buffer, and hybridized with Cy3 labeled anti-human IgG antibody or Cy5 labeled anti-human IgA antibody for 30 minutes. Finally, each of the protein microarrays is reacted with a Cy3 labeled anti-human IgG antibody or a Cy5 labeled anti-human IgA antibody for 30 minutes, followed by washing with TBST buffer. Cy3 fluorescence or Cy5 fluorescence of each of the protein microarrays is detected by the fluorescence detection system microarray (“Caduceus” SpinScan Microarray Scanner HC-BS01, Caduceus Biotechnology Inc., Taiwan).

[0087] Referring to Table 4, the results show that the sensitivity and specificity of the protein microarray with a single biomarker of the S protein from SARS-CoV-2 for the detection of serum IgG in patients suffering from COVID-19 are 90.6% and 97.2%, respectively. Moreover, the sensitivity and specificity of the protein microarray with a single biomarker of the S protein from SARS-CoV-2 for detection of serum IgA in patients suffering from COVID-19 are 84.4% and 100%, respectively. Furthermore, the sensitivity and specificity of the protein microarray with a combination of two biomarkers, the S protein of SARS-CoV-2 and the N protein of SARS-CoV-2 for the detection of serum IgG in COVID-19 patients are as high as 97%.

TABLE-US-00002 TABLE 4 Results of the specificity and sensitivity of protein microarrays with a single biomarker or a combination of two biomarkers for detecting the serum IgG or the serum IgA from the patients suffering from COVID-19 Biomarker and serum sensitivity specificity S protein of SARS-CoV-2, IgG 90.6 97.2 S protein of SARS-CoV-2, IgA 84.4 100 N protein of SARS-CoV-2, IgG 65.6 100 N protein of SARS-CoV, IgG 65.6 100 S protein of SARS-CoV, IgG 65.6 97.2 S protein of SARS-CoV-2, IgG + N protein of 96.9 97.2 SARS-CoV-2, IgG S protein of SARS-CoV-2, IgG + S protein of 93.8 97.2 SARS-CoV-2, IgA S protein of SARS-CoV-2, IgG + N protein of 93.8 97.2 SARS-CoV, IgG S protein of SARS-CoV-2, IgG + S protein of 90.6 97.2 SARS-CoV, IgG

Example 5 Analysis of the Cross-Reactivity Between the S Protein and the N Protein of SARS-CoV-2 on the Protein Microarray and Different Types of Viruses

[0088] The protein microarrays are used to detect the reactivity of the IgG in the serum from the patients suffering from COVID-19 with the S protein of SARS-CoV-2, the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, the S protein of OC43-CoV, the HA protein of influenza A H3N2 subtype, the N protein of SARS-CoV-2, and the N protein of SARS-CoV, and further analyze the cross-reactivity of the S protein of SARS-CoV-2 with the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, the S protein of OC43-CoV, and the HA protein of influenza A H3N2 subtype, as well as the cross-reactivity between the N protein of SARS-CoV-2 and the N protein of SARS-CoV.

[0089] Referring to FIG. 4A to FIG. 4E, the results show, in the serum from the patients suffering from COVID-19, a positive cross-reactive correlation of the IgG against the S protein of SARS-CoV-2 with the IgGs against the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, the S protein of OC43-CoV, and the HA protein of influenza A H3N2 subtype. Moreover, as shown in FIG. 4F, the result shows, in the serum from the patients suffering from COVID-19, a positive cross-reactive correlation of the IgG against the N protein of SARS-CoV-2 with the IgG against the N protein of SARS-CoV.

[0090] The protein microarrays are used to detect the reactivity of the IgA in the serum from the patients suffering from COVID-19 with the S protein of SARS-CoV-2, the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, the S protein of OC43-CoV, the N protein of SARS-CoV-2, the N protein of SARS-CoV, and the S protein of OC43-CoV, and further analyze the cross-reactivity of the S protein of SARS-CoV-2 with the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, and the S protein of OC43-CoV, as well as the cross-reactivity between the N protein of SARS-CoV-2 and the N protein of SARS-CoV, and the S protein of 229E-CoV.

[0091] Referring to FIG. 5A to FIG. 5D, the results show, in the serum from the patients suffering from COVID-19, a positive cross-reactive correlation of the IgA against the S protein of SARS-CoV-2 with the IgGs against the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, and the S protein of OC43-CoV. Moreover, as shown in FIG. 5E and FIG. 5F, the results show, in the serum from the patients suffering from COVID-19, a positive cross-reactive correlation of the IgA against the N protein of SARS-CoV-2 with the IgGs against the N protein of SARS-CoV and the S protein of 229E-CoV.

[0092] Based on the above, the results of the detection of using the IgG or IgA in the serum from the patients suffering from COVID-19 show a positive cross-reactivity correlation of the S protein of SARS-CoV-2 with the S1 domain of S protein of SARS-CoV-2, the S protein of SARS-CoV, the S protein of HKU-CoV, the S protein of OC43-CoV, and the HA protein of influenza A H3N2 subtype. Moreover, a positive cross-reactivity correlation of the N protein of SARS-CoV-2 with the N protein of SARS-CoV and the S protein of 229E-CoV. It is demonstrated that the protein microarray with the S protein of SARS-CoV-2 and the N protein of SARS-CoV may be used to detect different types of coronaviruses and influenza viruses.

[0093] The technical solutions of the present disclosure will be described clearly and completely in combined with the drawings of the present disclosure. Obviously, the described embodiments are only one part of the embodiments of the present disclosure, but not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by a person skilled in the art without making creative efforts fall within the claim scope of the present disclosure.

[0094] Although the present disclosure has been disclosed in preferred embodiments, it is not intended to limit the present disclosure. A person having ordinary skill in the art can make various changes and modifications without departing from the concept and scope of the present disclosure. Therefore, the claimed scope of the present disclosure shall be based on the scope defined by the attached claims of the patent disclosure.