High-sensitivity and high-specificity serum SARS-CoV-2 IgM and IgG antibody detection method

Abstract

The invention discloses a method for detecting serum SARS-CoV-2IgM and IgG antibodies with high sensitivity and high specificity, and belongs to the technical field of antibody detection. The method comprises the following steps: marking an SARS-CoV-2 recombinant protein, then enabling the SARS-CoV-2 recombinant protein to react with a serum sample to be detected, detecting SARS-CoV-2IgM and IgG antibodies through combination of a plurality of epitopes in the recombinant protein, and judging whether the SARS-CoV-2IgM and IgG antibodies are contained or not according to a detection result, wherein the SARS-CoV-2 recombinant protein comprises at least one of S1 protein, S2 protein, RBD protein, N protein and E protein. According to the novel method based on GICA multi-epitope joint detection, interference of RF-IgM can be remarkably reduced, higher diagnostic specificity is shown, and the screening sensitivity of the novel method is remarkably higher than that of CLIA, single-epitope antibody detection and various commercial antibody detection methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection, and in particular relates to a method for detecting serum SARS-CoV-2 IgM and IgG antibodies with high sensitivity and specificity. Background Art

[0002] The novel coronavirus infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Typical early clinical symptoms include fever, cough, respiratory symptoms, and muscle pain. Severe cases can develop into acute respiratory distress syndrome, sepsis, septic shock, multi-organ and systemic failure, and even death in a short period of time. The recovery time of SARS-CoV-2 infected people may vary depending on the severity of the infection, which may cause persistent lung damage. The infection may also cause persistent or new symptoms in multiple organs and is associated with a significantly increased risk of developing various autoimmune diseases. The emergence of new genetic variants of SARS-CoV-2 increases its infectivity, immune evasion ability, and the potential to cause reinfection.

[0003] Currently, several technologies have been developed for the diagnosis of SARS-CoV-2 infection. Real-time polymerase chain reaction (RT-PCR) is the most widely used nucleic acid detection technology; however, in addition to its low sensitivity, it has several limitations. It requires well-trained personnel and strict requirements for laboratory settings and environment, which limits its application to specific laboratories. Collecting nasopharyngeal swab samples increases the risk of viral transmission and may be difficult for patients in special circumstances. In addition, the low viral load of patients in the early stage increases the possibility of false-negative test results. In order to improve the accuracy of the diagnosis of new coronavirus infection, SARS-CoV-2 antibody detection is usually combined with nucleic acid detection, and serum SARS-CoV-2 specific antibodies are considered to be one of the indicators for diagnosing new coronavirus infection.

[0004] Enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA) are two common methods for detecting SARS-CoV-2 antibodies in serum. These methods are more sensitive than lateral flow immunochromatography. However, ELISA and CLIA require professionals and equipment, have high testing costs, long report issuance time, and use plasma / serum for testing, while GICA testing, which belongs to the lateral flow immunochromatography family, can be tested with whole blood and does not require time-consuming sample preparation. In addition, CLIA test kits have a specified opening time, which may cause reagent waste when processing small samples. In contrast, GICA is simple, safe, and rapid to operate, and has the advantage of not requiring professionals or instruments; it is suitable for serum SARS-CoV-2 antibody testing in hospitals and communities at all levels. Antigen testing mainly uses throat swabs as samples, and the sampling quality has a significant impact on the test, the same as PCR. In contrast, antibody testing uses serum samples, which are less affected by sampling and viral load.

[0005] Although the detection of SARS-CoV-2 using GICA is not affected by hemolysis, lipolysis, jaundice, or respiratory pathogens other than novel coronavirus infection. However, there are still a variety of factors that cause the accuracy of SARS-CoV-2 detection kits in clinical practice to be much lower than the accuracy marked on the kit. The presence of heterophilic antibodies and medium to high concentrations of rheumatoid factor (RF) can affect the GICA results of SARS-CoV-2 detection and increase the false positive detection rate. The time of sample collection, the type of anticoagulant in the sample, previous infection, and vaccination can also affect the accuracy of the test. Specifically:

[0006] 1. It takes time for the patient's serum antibodies to develop; however, they can only be detected within a specific time window. 2. Patients may lack IgG antibodies and cannot avoid false negative diagnoses of previous infections. 3. Because recent vaccinations can induce antibody production, patients must exclude recent vaccine history before using serum anti-tests to diagnose SARS-CoV-2 infection. 4. Some studies have shown that there may be cross-reactions between other viruses and SARS-CoV-2, especially with regard to the "N" antigen. Due to difficulties in sample collection, no other new coronavirus infections have been tested and evaluated. 5. The new method uses the naked eye to observe the results; weak positive results may be affected by subjective factors. 6. Testing multiple reagent strips will increase the cost and complexity of the testing process.

[0007] In addition, the accuracy of SARS-CoV-2 antibody detection is also affected by the target design of the test kit. The sensitivity of detecting SARS-CoV-2 IgM antibodies is lower than that of IgG antibodies. Kits targeting S-IgM are more specific than N-IgM, while the sensitivity of detecting SARS-CoV-2 IgM antibodies is lower than that of IgG antibodies. Commercially available S protein kits have higher sensitivity than N protein kits. When the kit targets multi-epitope antigens, its sensitivity is higher than that of single-epitope kits. When testing SARS-CoV-2 serum antibodies in patients infected with the new variant virus, the sensitivity of kits targeting S, S1, or RBD was significantly reduced. In contrast, kits targeting the N protein showed no significant difference in detecting new variants and wild-type strains. One researcher has demonstrated that several commercial immunoassays lack sensitivity for detecting antibodies caused by Omicron infection. Summary of the invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention provides a highly sensitive and specific method for detecting serum SARS-CoV-2 IgM and IgG antibodies, which can effectively solve the problem that the prior art has insufficient sensitivity for detecting serum SARS-CoV-2 IgM and IgG antibodies and is prone to false positives.

[0009] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0010] A highly sensitive and specific method for detecting serum SARS-CoV-2 IgM and IgG antibodies, wherein a SARS-CoV-2 recombinant protein is labeled and then reacted with a serum sample to be tested, SARS-CoV-2 IgM and IgG antibodies are detected by combining multiple antigenic epitopes in the SARS-CoV-2 recombinant protein, and whether SARS-CoV-2 IgM and IgG antibodies are contained therein is determined based on the test results;

[0011] The SARS-CoV-2 recombinant protein includes at least one of S1, S2, RBD, N and E proteins.

[0012] The serum SARS-CoV-2 IgM and IgG antibody detection method provided by the present invention is a detection method for non-diagnostic or therapeutic purposes, for example, it can be used for laboratory research, evaluation of the protective effect of vaccines against novel coronavirus infection, epidemiological surveys of novel coronavirus infection, etc.

[0013] The S1, S2, RBD, N and E proteins used in the present invention are all commercially available products purchased from Tianjin Medisai Biotechnology Co., Ltd.

[0014] Furthermore, the SARS-CoV-2 recombinant protein includes N, and S2 and / or RBD proteins.

[0015] Furthermore, the SARS-CoV-2 recombinant protein includes N and S2 proteins, and the antigenic epitopes of the N and S2 proteins are used to jointly detect SARS-CoV-2 IgM antibodies in serum.

[0016] Furthermore, the SARS-CoV-2 recombinant protein includes N and RBD proteins, and the antigenic epitopes of N and RBD proteins are used to jointly detect SARS-CoV-2 IgG antibodies in serum.

[0017] Furthermore, colloidal gold was used to label the SARS-CoV-2 recombinant protein.

[0018] Furthermore, when both the test line and the quality control line show red strips, it means that the test result is positive, indicating that the serum contains SARS-CoV-2 IgM and / or IgG antibodies.

[0019] The use of the above-mentioned highly sensitive and highly specific serum SARS-CoV-2 IgM and IgG antibody detection method in constructing the GICA method for detecting serum SARS-CoV-2 IgM and IgG antibodies.

[0020] Beneficial effects of the present invention:

[0021] The new method based on GICA multi-epitope combined detection developed by the present invention can significantly reduce the interference of RF IgM and show higher diagnostic specificity. Its screening sensitivity is also significantly higher than CLIA, single epitope antibody detection and various commercial antibody detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of the GICA IgM combined test developed for the present invention;

[0023] Figure 2 The results of the GICA IgG combined test developed by the present invention. DETAILED DESCRIPTION

[0024] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0025] The invention was approved by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (2022ER539-1). A total of 282 serum samples of patients newly infected with SARS-CoV-2 were collected from December 2022 to March 2023, and these samples were confirmed by PCR to form the experimental group.

[0026] Among them, 84 sera were collected from patients 5-7 days after symptom onset and were divided into the IgM experimental group; 198 samples were collected from patients 14-21 days after symptom onset to form the IgG experimental group. To ensure accurate results, sera positive for influenza A / B IgM, RF IgM, or antinuclear antibodies (ANA) were excluded from the experimental group because they may interfere with the detection of SARS-CoV-2 serum antibodies. 16 cases with positive influenza A / B IgM antibodies before April 2019, 32 cases with positive rheumatoid (RF) IgM antibodies, 12 cases with positive ANA, and 88 cases of physical examination people who were not vaccinated and not infected before June 2020, a total of 148 cases were used as the control group for serum SARS-CoV-2 IgM and IgG detection.

[0027] All blood samples were centrifuged at 4000 rpm for 5 min. Serum was then transferred to polyethylene tubes and stored at -80°C until assayed, thawed again at 37°C, mixed, and centrifuged again at 4000 rpm for 5 min, and the supernatant was collected for analysis.

[0028] Example 1 Construction of GICA detection method based on combined epitopes

[0029] 1. Preparation of GICA reagent

[0030] (1) Preparation of colloidal gold markers

[0031] The pH of colloidal gold was adjusted to 9.0 using a 0.1 mol / L K2CO3 solution. Recombinant proteins (two combinations of S1, S2, RBD, N, or E) were bound to 15 μg / mL colloidal gold nanoparticles, and chicken IgY was bound to 5 μg / mL colloidal gold nanoparticles. The solution was mixed quickly and incubated for 30 minutes. Next, 10% bovine serum albumin was added to the mixture to obtain a final concentration of 1%, and the mixture was incubated for 30 minutes. The reaction was terminated and the mixture was centrifuged at 8500 rpm for 25 minutes. The supernatant was then discarded, and the remaining recombinant SARS-CoV-2 protein mixture solution was colloidal gold labeled with a T line. The chicken IgY solution consisted of colloidal gold labeled with a C line.

[0032] (2) Preparation of colloidal gold binding pad

[0033] The mixture was diluted with T-line and C-line colloidal gold in a ratio of 9:1 to produce a mixture, which was mixed with a colloidal gold complex solution (0.05 mol / L Tris-HCl buffer (pH=9.0), 0.5% casein sodium salt, and 0.02% TW-20) in a volume ratio of 9:1. The mixture was diluted to the required volume and then applied to the glass fiber membrane at a concentration of 40 μL per square centimeter. Drying in an oven at 37°C gave a colloidal gold conjugate pad.

[0034] (3) NC membrane preparation

[0035] Anti-human IgM / IgG and goat anti-chicken IgY were diluted to a concentration of 1.5 mg / mL using 0.01 mol / L Tris buffer (pH = 9.0). The T and C lines of the nitrocellulose membrane were coated with the diluted solution using a volume of 0.75 μl / cm2 of the encapsulation solution. The membrane was dried in an oven at 55°C and treated as a conjugate pad.

[0036] (4) Sample pad and buffer preparation

[0037] The sample pad treatment solution (0.05 mol / L Tris buffer (pH=7.2), 0.05% casein sodium salt, 0.1% bovine serum albumin and 0.1 Traton X-100) was uniformly coated on the glass fiber membrane and then fixed on a polyvinyl chloride adhesive plate. Dry at 37°C to obtain a sample pad. 0.1% Tween-20 (0.05 mol / L, pH 7.5) was added to the buffered saline to prepare a sample diluent.

[0038] 2. GICA detection method

[0039] 7μL serum and 70μL sample diluent were added to the sample well of the test card in sequence. The colloidal gold-labeled SARS-CoV-2 recombinant protein was added to the test card binding pad. If there are SARS-CoV-2 specific IgM / IgG antibodies in the serum sample, they will specifically bind to the recombinant protein in the binding pad, chromatograph to the T line of the nitrocellulose membrane under capillary action, and be captured by the anti-human IgM secondary antibody / anti-human IgG secondary antibody to form a red "colloidal gold-recombinant protein-specific antibody-secondary antibody" complex; if there is no SARS-CoV-2 antibody in the specimen, only the colloidal gold-labeled chicken IgY will move and bind to the sheep anti-chicken IgY at the C line to form a red immune complex. The results were observed 15 minutes after the sample addition was completed.

[0040] When both the C line and the T line show red strips, the result is positive, indicating that SARS-CoV-2 IgM and / or IgG antibodies are present; when only the C line shows red strips, the antibody test is negative, indicating that SARS-CoV-2 IgM and / or IgG antibodies are not present.

[0041] Example 2 SARS-CoV-2 combined epitope detection and single epitope detection

[0042] 1. Perform single epitope detection on serum SARS-CoV-2 as follows:

[0043] (1) Serum SARS-CoV-2 single epitope IgM antibody detection

[0044] Five S1-IgM positive cases were detected in 148 serum specimens of the control group. Their distribution was as follows: 1 in 16 influenza A / B IgM positive cases, 1 in 32 RF-IgM positive patients, and 3 in 12 ANA-IgG positive cases. In addition, 7 S2-IgM positive cases were detected, and their distribution was as follows: 1 in 16 influenza A / B IgM positive cases, 4 in 32 RF-IgM positive patients, 1 in 12 ANA-IgG positive cases, and 1 in 88 healthy individuals. Six RBD-IgM positive cases were detected, including 1 in 16 influenza A / B IgM positive cases, 1 in 32 RF-IgM positive patients, 2 in 12 ANA-IgG positive cases, and 2 in 88 healthy individuals. In addition, 3 N-IgM-positive cases were detected, including 1 in 16 influenza A / B IgM-positive cases, 1 in 12 ANA-IgG-positive cases, and 1 in 88 healthy individuals. One E-IgM-positive case was detected among 88 healthy individuals (Table 1).

[0045] Among the 84 serum samples in the IgM experimental group, the numbers of S1-IgM, S2-IgM, RBD-IgM, N-IgM, and E-IgM positive sera detected by the GICA method were 18, 49, 39, 75, and 0, respectively ( Table 1 ).

[0046] Table 1. Sensitivity and specificity of GICA in detecting SARS-CoV-2 single epitope IgM in serum of different populations

[0047]

[0048]

[0049] Note: Influenza A and B IgM; RF-IgM: Rheumatoid factor IgM; ANA-IgG: Antinuclear antibody IgG; SARS-CoV-2: Severe acute respiratory syndrome coronavirus type 2; GICA: Colloidal gold immunochromatography.

[0050] (2) Serum SARS-CoV-2 single epitope IgG antibody detection

[0051] In the 148 serum samples of the control group, no S1-IgG cases were detected. Three S2-IgG positive cases were detected: 1 of 32 RF-IgM positive cases, 1 of 12 ANA-IgG negative cases, and 1 of 88 healthy individuals. Eleven RBD-IgG positive cases were detected: 1 of 16 influenza A / B IgM positive cases, 3 of 32 RF-IgM positive cases, 5 of 12 ANA-IgG positive cases, and 2 of 88 healthy individuals. One N-IgG and E-IgG positive case was detected among 88 healthy individuals (Table 2).

[0052] Among the 174 serum samples in the IgG experimental group, the numbers of S1-IgG, S2-IgG, RBD-IgG, N-IgG, and E-IgG positive sera detected by GICA were 121, 163, 165, 160, and 0, respectively ( Table 2 ).

[0053] Table 2. Sensitivity and specificity of GICA in detecting SARS-CoV-2 single epitope IgG in serum of different populations

[0054]

[0055] Flu A / B IgM: influenza A and B IgM; RF-IgM: rheumatoid factor IgM; ANA-IgG: antinuclear antibody IgG; SARS-CoV-2: severe acute respiratory syndrome coronavirus type 2; GICA: colloidal gold immunochromatography.

[0056] 2. The GICA detection method constructed in Example 1 was used to detect serum SARS-CoV-2 combined epitope antibodies, as follows:

[0057] (1) GICA detection of serum SARS-CoV-2 combined epitope IgM antibodies

[0058] Among the 84 serum samples in the IgM experimental group, 42 were positive for the combined detection of S2-IgM and N-IgM, and 41 were positive for the single epitope detection. Among the 148 sera in the control group, only one serum from an ANA-IgG-positive case was positive for the combined detection of S2-IgM and N-IgM.

[0059] Among all the IgM combined detection items, the combined detection of S2-IgM and N-IgM had the highest screening and diagnostic sensitivity; the screening sensitivity was higher than that of the most sensitive N-IgM in the GICA single epitope IgM antibody detection (98.81% vs. 89.29%; P = 0.022). The diagnostic specificity of the S2-IgM and N-IgM combined detection system was 99.32%, which was similar to the highest specificity of E-IgM based on the GICA single epitope IgM antibody detection. It was slightly higher than the second highest specificity N-IgM (97.97%; P = 0.615), and slightly lower than the combined detection of S1-IgM and N-IgM (100.00%; P>0.05) (Table 3).

[0060] Taking into account the diagnostic specificity and screening sensitivity, the combined detection of S2-IgM and N-IgM should be the optimal detection combination for SARS-CoV-2 IgM ( Figure 1 , among which A, S2-IgM and N-IgM were all negative; B and C, only one of S2-IgM and N-IgM tests was positive; D, S2-IgM and N-IgM were both positive).

[0061] Table 3. Performance evaluation of GICA combined epitope detection for SARS-CoV-2 IgM

[0062]

[0063] (2) GICA detection of serum SARS-CoV-2 combined epitope IgG antibodies

[0064] Of the 174 serum samples in the IgG experimental group, 151 were positive for the combined detection of RBD-IgG and N-IgG, and 23 were positive for individual epitopes. Of the 148 serum samples in the control group, none were positive for the combined detection of RBD-IgG and N-IgG.

[0065] The combined detection of RBD-IgG and N-IgG had the highest screening sensitivity and diagnostic specificity among all combined IgG detection procedures (both 100.00%); the screening sensitivity was higher than that of RBD-IgG, which had the highest sensitivity of GICA single epitope IgG antibody detection (94.83%; P = 0.004). The diagnostic sensitivity of the combined detection system of RBD-IgG and N-IgG was slightly lower than that of the combined detection system of S2-IgG and RBD-IgG (86.78% vs. 91.95%; P>0.05). The diagnostic specificity of the combined detection system of RBD-IgG and N-IgG was the same as that of S1-IgG, which had the highest specificity of single epitope antibody detection (100.00% vs. 100.00%; P>0.05) (Table 4).

[0066] Taking into account the diagnostic specificity and screening sensitivity, the combination of RBD-IgG and N-IgG is the best combination for detecting SARS-CoV-2 IgG ( Figure 2 , among which, A, RBD-IgG and N-IgG were both negative; B and C, only one of the RBD-IgG and N-IgG tests was positive; D, RBD-IgG and N-IgG were both positive).

[0067] Table 4. Performance evaluation of GICA combined detection of SARS-CoV-2 IgG

[0068]

[0069] Comparative Example CLIA Detection of Serum SARS-CoV-2 Antibodies

[0070] Among the 148 sera in the control group, 24 SARS-CoV-2 IgM-positive cases were detected by CLIA, including 3 of 16 influenza A / B IgM-positive cases, 6 of 32 RF IgM-positive patients, 15 of 88 normal cases, and no IgG-positive cases were detected. Among the 84 sera in the IgM experimental group, 69 were IgM-positive. Among the 198 serum samples in the IgG experimental group, 192 were IgG-positive.

[0071] According to the above test results, the sensitivity and specificity of serum SARS-CoV-2 IgM detected by CLIA were 82.10% and 83.78%, respectively, which were significantly lower than the screening sensitivity and diagnostic specificity of S2 IgM and N-IgM based on GICA combined epitope IgM antibody detection developed by the present invention (98.98% and 99.32%; both P<0.001). For SARS-CoV-2 IgG, the sensitivity of the CLIA method was lower than that of RBD IgG and N-IgG based on GICA combined epitope IgG antibody detection (96.50% vs. 100.00%, P=0.016), and the specificity of serum SARS-CoV-2 IgG detected by CLIA was the same as that of GICA (100.00% vs. 100.00%), P>0.05.

[0072] Since the detection of serum SARS-CoV-2 antibodies is easily affected by medium and high concentrations of RF and heterophilic antibodies, this application includes RF IgM-positive and ANA IgG-positive sera in the control group. At the same time, in order to observe the impact of other influenza viruses on the detection of serum antibodies using the new method, A / B influenza IgM-positive patients were included in the control group.

[0073] According to the test results of this application, the presence of RF IgM can affect the accuracy of IgM and IgG test results, especially when testing serum S2-IgM and RBD-IgG. In contrast, in the RF IgM positive control group, no cases showed double positivity in the combined test of S2-IgM and N-IgM; and the new GICA-based method developed by the present invention can significantly reduce the interference of RFIgM and show higher diagnostic specificity, and its screening sensitivity is also significantly higher than CLIA, single epitope antibody detection and various commercial antibody detection methods.

[0074] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A highly sensitive and specific method for detecting serum SARS-CoV-2 IgM and IgG antibodies, It is characterized in that Labeling the SARS-CoV-2 recombinant protein and then reacting it with the serum sample to be tested, detecting SARS-CoV-2 IgM and IgG antibodies through multiple antigenic epitopes in the SARS-CoV-2 recombinant protein, and determining whether SARS-CoV-2 IgM and IgG antibodies are contained therein based on the test results; The SARS-CoV-2 recombinant protein includes at least one of S1, S2, RBD, N and E proteins.

2. The highly sensitive and specific serum SARS-CoV-2 IgM and IgG antibody detection method according to claim 1, It is characterized in that The SARS-CoV-2 recombinant protein includes N, and S2 and / or RBD proteins.

3. The highly sensitive and specific serum SARS-CoV-2 IgM and IgG antibody detection method according to claim 2, It is characterized in that The SARS-CoV-2 recombinant protein includes N and S2 proteins, and the antigenic epitopes of the N and S2 proteins are used to jointly detect SARS-CoV-2 IgM antibodies in serum.

4. The highly sensitive and specific serum SARS-CoV-2 IgM and IgG antibody detection method according to claim 2, It is characterized in that The SARS-CoV-2 recombinant protein includes N and RBD proteins, and the antigenic epitopes of the N and RBD proteins are used to jointly detect SARS-CoV-2 IgG antibodies in serum.

5. The highly sensitive and specific serum SARS-CoV-2 IgM and IgG antibody detection method according to claim 1, It is characterized in that SARS-CoV-2 recombinant protein was labeled with colloidal gold.

6. The highly sensitive and specific serum SARS-CoV-2 IgM and IgG antibody detection method according to claims 1 to 5, It is characterized in that When both the test line and the quality control line show red strips, the test result is positive, indicating that the serum contains SARS-CoV-2 IgM and / or IgG antibodies.

7. Use of the highly sensitive and highly specific serum SARS-CoV-2 IgM and IgG antibody detection method according to any one of claims 1 to 6 in constructing a GICA method for detecting serum SARS-CoV-2 IgM and IgG antibodies.

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