A kit for quantitative detection of COVID-19 IFN-γ using peripheral blood and its application.

Abstract

The application discloses a kit for quantitatively detecting COVID-19 IFN-gamma by using peripheral blood and application thereof, and belongs to the technical field of biomedical testing. The kit provided by the application comprises blood stimulating reagents and an IFN-gamma detection kit, wherein the blood stimulating reagents comprise T cell epitope peptides, activators for enhancing the immune stimulation of T cells, and inhibitors for inhibiting the negative regulation of Treg cells. The kit can accurately detect whether a COVID-19 patient is effectively immunized after being cured and after being inoculated with a COVID-19 vaccine, has the advantages of good accuracy, high sensitivity, strong specificity, high stability, full-automatic detection and the like of the detection result, and has a good commercial application prospect in cell immune response detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing technology, and in particular to a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood and its application. Background Technology

[0002] COVID-19 is an enveloped beta-type single-stranded RNA coronavirus, whose main structures include: nucleocapsid protein (N), envelope protein (E), membrane protein (M), and spike protein (S). The S protein is a transmembrane protein and an important marker protein on the viral surface. Each S monomer consists of an N-terminal S1 subunit and a proximal membrane S2 subunit. The S1 subunit includes a receptor-binding domain (RBD), which binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. The RBD in the S protein can generate neutralizing antibodies, providing a key target for diagnosis and vaccines.

[0003] After spreading, the original COVID-19 strain mutated, forming COVID-19 variants such as Alpha (B1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B1.1.529). Some COVID-19 variants can evade the immune system and infect people who have been vaccinated against COVID-19, posing challenges to the prevention and diagnosis of COVID-19. Diagnosis is a prerequisite for effective treatment.

[0004] Currently, there are three main methods for detecting COVID-19 infection: PCR nucleic acid testing, rapid antigen testing, and antibody testing. ① Nucleic acid testing: The purpose is to detect viral genes (RNA). It typically involves collecting nasopharyngeal or nasal swabs, has high accuracy, and can detect low viral loads. The detection limit is 10^6 / mL. 2 -10 3 ① Genome copying: This method is time-consuming, relatively expensive, and requires specialized personnel and tools. ② Antigen detection: This method aims to detect viral proteins (such as nucleocapsid N or spike protein S). It typically involves collecting nasopharyngeal or nasal swabs and is rapid, simple, and inexpensive. However, its accuracy is lower than molecular detection, and it is difficult to detect low viral loads. The detection limit is 10^6 / mL. 5 -10 6③ Serological (antibody) testing: This aims to detect IgG and IgM antibodies. Blood samples are typically collected, and results are more reliable two weeks after symptom onset. It can be used for retrospective analysis, but false positives are possible. The accuracy of the above three methods is also affected by sampling quality and the proficiency of the sampling procedure. Furthermore, these three methods cannot accurately detect whether effective immunity has been acquired in the body after recovery from COVID-19 or after vaccination with a COVID-19 vaccine.

[0005] In view of the above problems, it is urgent to provide a testing method or product that can be used to assess vaccine efficacy and revaccination in COVID-19 vaccine recipients, as well as to assess the prognosis of COVID-19 patients. Summary of the Invention

[0006] To address the above technical problems, this invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood and its application. This kit has the advantages of high sensitivity, good specificity, and simple operation. It can be used to detect the content of IFN-γ in peripheral blood, and can then be used for vaccine efficacy evaluation and revaccination evaluation in COVID-19 vaccinated individuals, as well as for disease prognosis evaluation in COVID-19 patients.

[0007] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0008] A kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, the kit comprising a blood stimulation reagent and an IFN-γ detection kit, wherein the blood stimulation reagent comprises a negative control reagent, a positive control reagent, and a test culture reagent, each of the blood stimulation reagents comprising an activator that enhances the immune stimulation of T cells and an inhibitor that inhibits the negative regulatory effect of Treg cells (regulatory T cells), wherein the positive control reagent further comprises phytohemagglutinin (PHA), and the test culture reagent further comprises T cell epitope peptides.

[0009] When a person is vaccinated against COVID-19 or COVID-19 invades the body, the immune system first initiates an innate immune response to fight the virus. After the innate immune response is initiated, a specific immune response, namely the T-cell immune response, is triggered. Some asymptomatic COVID-19 infected individuals, although lacking obvious antibody immune responses, possess memory T cells against COVID-19. Receptor molecules on the surface of T cells recognize COVID-19 antigens that have been degraded into short peptides, thereby inducing an immune response. CD8+ T cells, in particular, exhibit long-lasting antigen-specific immune activity and play a crucial role in the immune defense against COVID-19. Furthermore, most T cells also possess good recognition capabilities for mutated viral antigens. Therefore, the T-cell immune response plays a key role in assessing vaccine efficacy and evaluating the prognosis of COVID-19 patients, and the strength of the T-cell immune response directly reflects the sensitivity of diagnostic reagents in detecting lower levels of immune activity.

[0010] When the body's T-cell immune response is activated, T cells secrete interferon-gamma (IFN-γ), which plays an antiviral role. Memory T cells, upon encountering COVID-19 again, also secrete IFN-γ, again exerting an antiviral effect. The amount of antigen-specific IFN-γ produced is closely related to whether the body has been infected with COVID-19 and whether it has been vaccinated.

[0011] The blood stimulation reagent in this invention induces an immune response through T-cell epitope peptides and modulates T-cell function through a combination of activators that enhance T-cell immune stimulation and inhibitors that suppress the negative regulatory effect of Treg cells. This allows the reagent to induce high levels of IFN-γ in the peripheral blood of vaccinated individuals or recovered patients during quantitative detection of COVID-19, while unvaccinated individuals or those not infected with COVID-19 cannot produce IFN-γ upon antigen stimulation. By combining this blood stimulation reagent with an IFN-γ detection kit and employing a double-antibody sandwich chemiluminescence method to detect the peripheral blood sample, accurate detection of whether effective immunity has been acquired after recovery from COVID-19 or after COVID-19 vaccination can be achieved. This method offers advantages such as high accuracy, high specificity, and high sensitivity, and can be used for vaccine efficacy assessment and re-vaccination evaluation in COVID-19 vaccinated individuals, as well as for disease prognosis assessment in COVID-19 patients. It can also be used to detect individuals with low cellular immune activity.

[0012] In conjunction with the first aspect, the T-cell epitope peptide comprises a CO VID-19 nucleocapsid protein (2019-nCoV NP Protein, hereinafter referred to as NP) T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO. 1) and a CO VID-19 S protein receptor-binding domain antigen (2019-nCoV Spike RBD Protein, hereinafter referred to as S-RBD) T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO. 2).

[0013] The RBD fragment of the S protein contains multiple B-cell and T-cell epitopes and can induce strong protective antiviral immunity; the N protein also has high immunogenicity and is one of the potential vaccine targets. Therefore, this invention selects a composition of the COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence described above and the COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment, so that T cells in the blood of individuals vaccinated against COVID-19 or infected with COVID-19 produce high levels of IFN-γ, while T cells in unvaccinated individuals or non-COVID-19 infected individuals cannot be stimulated to produce IFN-γ, thereby achieving the purpose of accurately detecting whether effective immunity has been acquired after the recovery of COVID-19 patients and after vaccination with COVID-19 vaccine.

[0014] Preferably, the concentration of the COVID-19 nucleocapsid protein T-cell epitope peptide fragment is 0.6–0.8 μg / mL, more preferably 0.7 μg / mL; and the concentration of the COVID-19S protein receptor-binding domain antigen T-cell epitope peptide fragment is 0.9–1.2 μg / mL, more preferably 1.0 μg / mL.

[0015] In conjunction with the first aspect, the activator that enhances the immune stimulation of T cells is a Toll-like receptor agonist (TLR).

[0016] Preferably, the Toll-like receptor agonist comprises at least one of the imidazoquinone compound R848 and an oligonucleotide fragment of unmethylated CpG (CpG ODN), wherein the sequence of the unmethylated CpG oligonucleotide fragment is 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO. 3). R848 is an imidazoquinone compound with potent antiviral activity that can activate immune cells through the TLR7 / TLR8 / MyD88-dependent signaling pathway. The aforementioned unmethylated CpG oligonucleotide fragment exists only in microbial DNA and not in mammalian DNA, and can be specifically recognized by TLR9 to stimulate the production of IFN-γ by plasma dendritic cells (pDCs).

[0017] Preferably, the concentration of the imidazoquinoline compound R848 is 0.05–0.2 μg / mL, and more preferably, the concentration is 0.1 μg / mL.

[0018] Preferably, the concentration of the oligonucleotide fragment of the unmethylated CpG is 1–10 μmol / mL, and more preferably 10 μmol / mL.

[0019] Preferably, the concentration of the phytohemagglutinin is 10 μg / mL.

[0020] More preferably, the Toll-like receptor agonist is the imidazoquinone compound R848. The imidazoquinone compound R848 does not increase the background N concentration and can stimulate T cells to secrete IFN-γ, exhibiting higher specificity.

[0021] In conjunction with the first aspect, the inhibitors suppressing the negative regulatory effects of Treg cells include at least one of CD25 antibodies and CD127 antibodies. Treg cells exert inhibitory effects on various immune cells. CD25, also known as the interleukin-2 receptor-α subunit (IL-2Ra), is highly expressed in Treg cells and, as one of the specific markers on the surface of Treg cells, is a potential target for Treg cells. Utilizing CD25 antibodies to specifically kill Treg cells can relieve immunosuppression of T cells. CD127, part of the IL-7 receptor, is a Treg cell marker molecule that can exert a highly inhibitory effect on Treg cell function.

[0022] Preferably, the inhibitor that suppresses the negative regulatory effect of Treg cells is a combination of CD25 antibody and CD127 antibody, and the combination of the two can more significantly suppress the negative regulatory effect of Treg cells.

[0023] In conjunction with the first aspect, each of the blood stimulating reagents also includes PBS buffer and trehalose.

[0024] Preferably, the concentration of the PBS buffer is 0.01M and the pH is 7.4; the concentration of the trehalose is 2mg / mL.

[0025] In conjunction with the first aspect, the reagents in the IFN-γ detection kit include magnetic bead coating working solution, enzyme-labeled antibody working solution, luminescent colorimetric solution, and washing solution.

[0026] For example, the magnetic bead coating working solution consists of IFN-γ monoclonal antibody coated with carboxyl magnetic beads and 0.01M Tris buffer at pH 7.4, with the concentration of IFN-γ monoclonal antibody coated with carboxyl magnetic beads in the magnetic bead coating working solution being 0.25 mg / mL; the enzyme-labeled antibody working solution consists of IFN-γ monoclonal antibody labeled with alkaline phosphatase and 0.01M Tris buffer at pH 7.4, with the concentration of IFN-γ monoclonal antibody labeled with alkaline phosphatase in the enzyme-labeled antibody working solution being 0.5 μg / mL; the luminescent developing solution consists of AMPPD and Diox etane Buffer, with the AMPPD concentration being 0.1 mg / mL; and the washing solution is 0.01M PBS T buffer at pH 7.4.

[0027] Preferably, the IFN-γ detection kit further includes calibrators. Exemplarily, the calibrators are recombinant IFN-γ antigen proteins diluted in PB buffer containing 0.1% ProClin 300 and 5% BSA at concentrations of 50 ± 10 pg / mL and 500 ± 100 pg / mL.

[0028] Furthermore, the present invention also provides the application of the above-mentioned kit for quantitative detection of COVID-19 IFN-γ in peripheral blood for non-diagnostic purposes: after the peripheral blood sample to be tested is co-cultured with each of the blood stimulating reagents, centrifuged, the supernatant is taken, and the supernatant is quantitatively detected by the IFN-γ detection kit.

[0029] Quantitative detection of COVID-19 IFN-γ in peripheral blood for non-diagnostic purposes can assess vaccine efficacy and provide data support for vaccine development, efficacy evaluation, and revaccination evaluation.

[0030] When using this kit for testing, the test results should be interpreted according to the following criteria:

[0031]

[0032] The N value represents the IFN-γ level of the peripheral blood sample to be tested after co-culturing with the negative control reagent, that is, the IFN-γ level exhibited by the peripheral blood sample to be tested without any antigen stimulation; the P value represents the IFN-γ level of the peripheral blood sample to be tested after co-culturing with the positive control reagent, that is, the level of non-specific IFN-γ released by lymphocytes in the peripheral blood sample to be tested after stimulation by non-specific antigens (phytohemagglutinins), reflecting the basic cellular immune status of the donor of the peripheral blood sample to be tested and the viability of lymphocytes in the peripheral blood sample to be tested; the T value represents the IFN-γ level of the peripheral blood sample to be tested after co-culturing with the test culture reagent, that is, the level of specific IFN-γ released by lymphocytes in the peripheral blood sample to be tested after stimulation by COVID-19 antigen T cell epitope peptide fragments.

[0033] The beneficial effects of this invention are as follows:

[0034] The kit provided by this invention contains a test culture reagent in the blood stimulation reagent that can stimulate T cells in the peripheral blood of COVID-19 infected individuals or those vaccinated against COVID-19 to produce high levels of IFN-γ. However, unvaccinated individuals or those not infected with COVID-19 cannot produce IFN-γ through antigen stimulation. By combining the IFN-γ detection kit with the detection of IFN-γ, it is possible to accurately detect whether COVID-19 patients have acquired effective immunity after recovery or after vaccination against COVID-19. The kit has the advantages of high accuracy, high sensitivity, high specificity, high stability, and fully automated detection capabilities, and has good commercial application prospects in the detection of cellular immune responses. Attached Figure Description

[0035] Figure 1 This is the linear fitting curve in Example 5 of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] Unless otherwise specified, the reagents used in the following examples are all commercially available or obtained using methods known in the art.

[0038] Example 1

[0039] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0040] 1. Blood irritant:

[0041] This includes negative control reagents, positive control reagents, and test culture reagents.

[0042] 1.1 Negative control reagent

[0043] A 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, and 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition;

[0044] 1.2 Positive control reagent

[0045] The following ingredients were prepared: 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition, and 10 μg / mL phytohemagglutinin.

[0046] 1.3 The test culture reagent is:

[0047] A mixture of a 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, and 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition, and a mixture of a COVID-19 nucleocapsid protein (NP) T cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen (S-RBD) T cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0048] 2. IFN-γ detection kit

[0049] The apparatus includes magnetic bead coating working solution, enzyme-labeled antibody working solution, luminescent reagent, washing solution, and calibrators. Specifically: the magnetic bead coating working solution consists of IFN-γ monoclonal antibody coated with carboxyl magnetic beads and 0.01M Tris buffer at pH 7.4, with a concentration of 0.25 mg / mL for the carboxyl magnetic bead-coated IFN-γ monoclonal antibody; the enzyme-labeled antibody working solution consists of IFN-γ monoclonal antibody labeled with alkaline phosphatase and 0.01M Tris buffer at pH 7.4, with a concentration of 0.5 μg / mL for the alkaline phosphatase-labeled IFN-γ monoclonal antibody; the luminescent reagent consists of AMPPD and Dioxetane Buffer, with an AMPPD concentration of 0.1 mg / mL; the washing solution is 0.01M PBST buffer at pH 7.4; and the calibrators are prepared using a solution containing 0.1% ProClin... Recombinant IFN-γ antigen protein diluted in PB buffer of 300 and 5% BSA at concentrations of 50 ± 10 pg / mL and 500 ± 100 pg / mL.

[0050] The preparation method of this IFN-γ detection kit includes the following steps:

[0051] 2.1 Preparation of Mother Solution and Working Solution for Magnetic Bead Coating

[0052] 2.1.1 Preparation of Mother Liquor for Magnetic Bead Coating

[0053] 1) Washing: Take 900 μL of 0.05M MES buffer (pH 6.0) and 100 μL of carboxylated magnetic beads with a solid content of 1% (final solid content is 0.1%) into a centrifuge tube, shake to mix, so that the carboxylated magnetic beads are evenly dispersed in the buffer, and then place on a magnetic rack and let stand for 2 minutes to separate the magnetic beads.

[0054] 2) Activation: Remove the supernatant, add 1 mL of 0.05 M MES buffer (pH 6.0) to reconstitute, and vortex to mix. Add 10 μL of NHS solution (20 mg / mL) and 5 μL of EDC solution (20 mg / mL) respectively, vortex to mix, and place in a rotary reactor. React at 25 °C and 40 rpm for 20 min.

[0055] 3) Washing: After activation, place the magnetic beads on a magnetic rack and let stand for 2 minutes to separate them. Discard the supernatant and reconstitute with 1 mL of 0.05 MME S buffer (pH = 6.0).

[0056] 4) Washing: Repeat step 3) once and shake to mix.

[0057] 5) Conjugation: Add IFN-γ mouse monoclonal antibody to achieve a final antibody concentration of 50 μg / mL. Place in a rotary reactor and react at 25°C and 40 rpm for 3 h.

[0058] 6) Sealing: Let stand on a magnetic rack for 2 minutes, separate the magnetic beads, discard the supernatant, and add 50 μL of 10% BSA for sealing. Place on a rotary reactor at 25°C and 40 rpm for 1 hour. After sealing, separate the magnetic beads on a magnetic rack.

[0059] 7) Washing: Discard the supernatant, redissolve in 0.05M Tris-HCl (containing 0.2% BSA), shake to mix, and wash twice with magnetic separation.

[0060] 8) Storage: Remove the supernatant from step 7), add 100 μL of 0.05 M Tris-HCl (containing 0.2% BSA) solution, shake to mix, and store at 2℃~8℃ for later use.

[0061] 2.1.2 Preparation of Magnetic Bead Coating Working Solution

[0062] Preparation of coating antibody dilution buffer: 0.01M Tris buffer (pH 7.4), 1% trehalose, 0.5% BSA, 0.01% mouse IgG antibody, 0.1% Tween 20, 0.1% Triton X-100, 0.1% ProClin 300.

[0063] For the preparation of the magnetic bead coating working solution: Dilute the magnetic bead coating stock solution with the coating antibody diluent at a certain ratio so that the final concentration of IFN-γ monoclonal antibody coated with carboxyl magnetic beads in the magnetic bead coating working solution is 0.25 mg / mL.

[0064] 2.2 Preparation of enzyme-labeled antibody stock solution and working solution

[0065] 2.2.1 Preparation of enzyme-labeled antibody stock solution

[0066] 1) Ultrafiltration: Add 300 μL of 0.1 M borate buffer (pH = 9.0) and 200 μL of IFN-γ monoclonal antibody to the inner tube of the ultrafiltration tube, and cap it. Centrifuge at 5000 rpm for 10 minutes at 4°C. Add another 300 μL of 0.1 M borate buffer (pH = 9.0) and centrifuge again. After centrifugation, remove the inner tube, invert it into a clean microcentrifuge tube, and centrifuge at 5000 rpm for 2 minutes at 4°C to transfer the IFN-γ monoclonal antibody (after buffer replacement) from the ultrafiltration inner tube to the collection tube for later use.

[0067] 2) Activation: Take 250 μg of ALP, add 222.5 μL of 0.05 M MES buffer (pH = 5.6) to dissolve it, then add 2.5 μL of NHS solution (20 mg / mL) and 25 μL of EDC solution (20 mg / mL), shake to mix well, and place on a rotary reactor to react at 25°C and 40 rpm for 1 h.

[0068] 3) Washing: Add 300 μL of 0.1 M borate buffer (pH = 9.0) to an ultrafiltration tube (30 K ultrafiltration tube) containing activated ALP solution and perform ultrafiltration. Centrifuge at 25 °C and 5000 rpm for 10 minutes. Repeat the washing twice and collect the ultrafiltration ALP solution.

[0069] 4) Conjugation: Add IFN-γ monoclonal antibody to achieve a final antibody concentration of 20 μg / mL. Place in a rotary reactor and react at 25°C and 40 rpm for 3 hours.

[0070] 5) Blocking: Add 50 μL of blocking solution (10% BSA, 0.01 M ethanolamine, pH 8.0), place in a rotary reactor, and block at 25°C and 40 rpm for 1 h.

[0071] 6) Washing: After blocking, add 300 μL of 0.01M PBS buffer (pH=7.4, containing 0.2% BSA) to an ultrafiltration tube (30K ultrafiltration tube) containing enzyme-labeled solution for ultrafiltration. Centrifuge at 25°C, 5000 rpm for 10 minutes. Repeat washing twice.

[0072] 7) Storage: Centrifuge the inverted ultrafiltration tube, collect about 150 μL of enzyme-labeled solution, and store at 2℃~8℃ for later use.

[0073] 2.2.2 Preparation of enzyme-labeled antibody working solution

[0074] Preparation of enzyme-labeled antibody dilution buffer: 0.01M Tris buffer (pH 7.4), 1% trehalose, 0.5% BSA, 0.01% mouse IgG antibody, 0.1% Tween 20, 0.1% Triton X-100, 0.1% ProClin 300.

[0075] For the preparation of enzyme-labeled antibody working solution: Dilute the enzyme-labeled antibody stock solution with enzyme-labeled antibody diluent at a certain ratio so that the final concentration of IFN-γ monoclonal antibody labeled with alkaline phosphatase in the enzyme-labeled antibody working solution is 0.5 μg / mL.

[0076] 2.3 Preparation of luminescent substrates

[0077] Dissolve 2.5 mg of AMPPD (3-(2-spirodaradinane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane disodium salt solution) in 25 mL of Dioxetane Buffer to prepare a 0.1 mg / mL AMPPD substrate solution, which is AMPPDDioxetane Buffer (luminescent colorimetric solution) for color development.

[0078] 2.4 Preparation of cleaning solution

[0079] Preparation of 0.01M PBST washing solution: Take a 1L clean beaker, add 800mL purified water, then add 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 in sequence, and stir thoroughly until all solids are dissolved; adjust the pH to 7.4 with 6M / L HCl, add 1mL ProClin 300 and 500μL Tween-20, and add water to make up to 1L. Filter through a 0.22μm membrane to obtain 0.01MPBST, and store at room temperature for later use.

[0080] 2.5 Calibrator Preparation

[0081] Calibrator antigen: Recombinant IFN-γ antigen protein with a purity ≥95% is used; or the IFN-γ biological reference standard (1 IU / mL ≈ 50 pg / mL) of the National Institute for Biological Standards and Control (NIBSC) No. 87 / 586 is used as a control.

[0082] Antigen dilution solution: PB buffer containing 5% BSA, containing 0.1% ProClin 300;

[0083] The IFN-γ antigen was diluted with antigen diluent to prepare low-value calibrator 1 and high-value calibrator 2. After being dispensed and lyophilized, the concentrations of the calibrators were assigned as follows: Calibrator 1 (Cal 1): 50±10 pg / mL; Calibrator 2 (Cal 2): ​​500±100 pg / mL.

[0084] 2.6 Packaging Combination

[0085] The above-mentioned magnetic bead coating working solution, enzyme-labeled antibody working solution, luminescent reagent, washing solution, and calibrators are aliquoted, sealed, and assembled into qualified single-use reagent kits. Detection is performed using a double-antibody sandwich chemiluminescence assay.

[0086] Example 2

[0087] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0088] 1. Blood irritant

[0089] This includes negative control reagents, positive control reagents, and test culture reagents.

[0090] 1.1 Negative control reagent

[0091] A 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.2 μg / mL imidazoquinone R848, and 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition;

[0092] 1.2 Positive control reagent

[0093] The following ingredients were prepared: 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.2 μg / mL imidazoquinone R848, 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition, and 10 μg / mL phytohemagglutinin.

[0094] 1.3 The test culture reagent is:

[0095] A mixture consisting of a 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.2 μg / mL imidazoquinone R848, and a 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a mixture of a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0096] 2. IFN-γ detection kit: Same as in Example 1.

[0097] Example 3

[0098] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0099] 1. Blood irritant

[0100] This includes negative control reagents, positive control reagents, and test culture reagents.

[0101] 1.1 Negative control reagent

[0102] A 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.05 μg / mL imidazoquinone R848, and 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition;

[0103] 1.2 Positive control reagent

[0104] 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.05 μg / mL imidazoquinone R848, 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition, 10 μg / mL phytohemagglutinin;

[0105] 1.3 The test culture reagent is:

[0106] A mixture consisting of a 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.05 μg / mL imidazoquinone R848, and a 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0107] 2. IFN-γ detection kit: Same as in Example 1.

[0108] Comparative Example 1

[0109] This comparative example provides a kit for the quantitative detection of COVID-19 IFN-γ using peripheral blood, consisting of a blood stimulating reagent and an IFN-γ detection kit.

[0110] 1. Blood irritant

[0111] include:

[0112] Negative control reagent, positive control reagent, and test culture reagent.

[0113] 1.1 Negative control reagent

[0114] A 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.01 μg / mL imidazoquinone R848, and 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition.

[0115] 1.2 Positive control reagent

[0116] The following ingredients were prepared: 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.01 μg / mL imidazoquinone R848, 0.5 μg / mL (CD25 and CD127 mixed in a 1:1 mass ratio) antibody composition, and 10 μg / mL phytohemagglutinin.

[0117] 1.3 The test culture reagent is:

[0118] A mixture consisting of a 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.01 μg / mL imidazoquinone R848, and a 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0119] 2. IFN-γ detection kit: Same as in Example 1.

[0120] Example 4

[0121] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0122] 1. Blood irritant

[0123] This includes negative control reagents, positive control reagents, and test culture reagents.

[0124] 1.1 Negative control reagent

[0125] The composition consisted of 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 10 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), and 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio).

[0126] 1.2 Positive control reagent

[0127] The following are the contents of the following: 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 10 μmol / mL oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and 10 μg / mL phytohemagglutinin;

[0128] 1.3 The test culture reagent is:

[0129] The mixture consists of 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 10 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a mixture of a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0130] 2. IFN-γ detection kit: Same as in Example 1.

[0131] Example 5

[0132] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0133] 1. Blood irritant

[0134] This includes negative control reagents, positive control reagents, and test culture reagents.

[0135] 1.1 Negative control reagent

[0136] The composition consisted of 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 1 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), and 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio).

[0137] 1.2 Positive control reagent

[0138] The following are the contents of the following: 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 1 μmol / mL oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and 10 μg / mL phytohemagglutinin;

[0139] 1.3 The test culture reagent is:

[0140] The mixture consists of 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 1 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a mixture of a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0141] 2. IFN-γ detection kit: Same as in Example 1.

[0142] Comparative Example 2

[0143] This comparative example provides a kit for the quantitative detection of COVID-19 IFN-γ using peripheral blood, consisting of a blood stimulating reagent and an IFN-γ detection kit.

[0144] 1. Blood irritant

[0145] This includes negative control reagents, positive control reagents, and test culture reagents.

[0146] 1.1 Negative control reagent

[0147] The composition consisted of 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), and 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio).

[0148] 1.2 Positive control reagent

[0149] The following are the ingredients: 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.1 μmol / mL oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and 10 μg / mL phytohemagglutinin;

[0150] 1.3 The test culture reagent is:

[0151] The mixture consists of 0.01M PBS buffer at pH 7.4, 2 mg / mL trehalose, 0.1 μmol / mL of an oligonucleotide fragment of unmethylated CpG with the sequence 5'-ggGGGACGATCGTCggggg-3' (as shown in SEQ ID NO.3), 0.5 μg / mL of an antibody composition (CD25 and CD127 mixed in a 1:1 mass ratio), and a mixture of a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0152] 2. IFN-γ detection kit: Same as in Example 1.

[0153] Example 6

[0154] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0155] 1. Blood irritant

[0156] This includes negative control reagents, positive control reagents, and test culture reagents.

[0157] 1.1 Negative control reagent

[0158] 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, and 0.5 μg / mL LCD25 antibody;

[0159] 1.2 Positive control reagent

[0160] 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, 0.5 μg / mL LCD25 antibody, and 10 μg / mL phytohemagglutinin;

[0161] 1.3 The test culture reagent is:

[0162] The mixture consisted of 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, 0.5 μg / mL LCD25 antibody, and a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0163] 2. IFN-γ detection kit: Same as in Example 1.

[0164] Example 7

[0165] This invention provides a kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, which consists of a blood stimulating reagent and an IFN-γ detection kit.

[0166] 1. Blood irritant

[0167] This includes negative control reagents, positive control reagents, and test culture reagents.

[0168] 1.1 Negative control reagent

[0169] 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, and 0.5 μg / mL LCD127 antibody;

[0170] 1.2 Positive control reagent

[0171] 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, 0.5 μg / mL LCD127 antibody, and 10 μg / mL phytohemagglutinin;

[0172] 1.3 The test culture reagent is:

[0173] The mixture consisted of 0.01M PBS buffer (pH 7.4), 2 mg / mL trehalose, 0.1 μg / mL imidazoquinone R848, 0.5 μg / mL LCD127 antibody, and a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY (amino acid sequence as shown in SEQ ID NO.1) and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY (amino acid sequence as shown in SEQ ID NO.2), with the former at a concentration of 0.7 μg / mL and the latter at a concentration of 1.0 μg / mL.

[0174] 2. IFN-γ detection kit: Same as in Example 1.

[0175] Test Example 1

[0176] This test example provides the detection results of the kits used in Examples 1-3 and Comparative Example 1.

[0177] The negative control reagent, positive control reagent, and test culture reagent from Examples 1-3 and Comparative Example 1 were each placed into 4 mL culture tubes, designated as negative control tubes (N), positive control tubes (P), and test culture tubes (T), respectively, with 50 μL dispensed into each tube. Different colored screw caps were used for each culture tube to distinguish the different components. Specifically, the screw cap for the negative control tube (N) was white; the screw cap for the positive control tube (P) was red; and the screw cap for the test culture tube (T) was blue.

[0178] Take 15 mL each of negative samples (blood from subjects who have not been vaccinated against COVID-19 and have not been infected with COVID-19) and positive samples (blood from vaccinated individuals or patients who have recovered from COVID-19). Dispense 1 mL of each sample into N, P, and T tubes. Place the blood culture tubes vertically in a 37°C incubator and incubate for 22 hours. Remove the culture tubes, centrifuge at 5000 rpm for 10 minutes at 25°C, and collect 50 μL of the supernatant into an IFN-γ detection kit (same as in Example 1). Perform the detection on a fully automated chemiluminescence immunoassay analyzer. The results are shown in Table 1. Wherein: N value represents the IFN-γ level of the test sample (fresh blood) without any antigen stimulation; P value represents the level of non-specific IFN-γ release from lymphocytes in the test sample after stimulation by non-specific antigens (phytohemagglutinins), reflecting the subject's basic cellular immune status and the activity of lymphocytes in the sample; T value represents the level of specific IFN-γ release from lymphocytes in the test sample after stimulation by COVID-19 antigen T cell epitope peptide fragments. The control group did not include R848, and the remaining reagents were the same as those in the corresponding N-tube, P-tube, and T-tube.

[0179] Table 1. IFN-γ levels (μg / mL) at different concentrations of R848.

[0180]

[0181]

[0182] Table 1 shows that TN and (TN) / (PN) can be detected in Examples 1-3 compared to the control group. When the concentration of the imidazoquinoline compound R848 is 0.1 μg / mL, the TN and (TN) / (PN) values ​​of the positive samples are higher, which can be used to make a more accurate judgment on the detection results.

[0183] Test Example 2

[0184] This test example provides the detection results of the kits used in Examples 4, 5 and Comparative Example 2.

[0185] The negative control reagent, positive control reagent, and test culture reagent from Examples 4 and 5 and Comparative Example 2 were respectively placed into 4 mL culture tubes, serving as negative control tubes (N), positive control tubes (P), and test culture tubes (T), with 50 μL dispensed into each tube. Different colored inner screw caps were used for each culture tube to distinguish the different components. Specifically, the inner screw cap of the negative control tube (N) was white; the inner screw cap of the positive control tube (P) was red; and the inner screw cap of the test culture tube (T) was blue.

[0186] Take 12 mL each of negative samples (blood from subjects who have not been vaccinated against COVID-19 and are not infected with COVID-19) and positive samples (blood from vaccinated subjects), and aliquot 1 mL into N, P, and T tubes respectively. Place the blood culture tubes vertically in an incubator at 37°C and incubate for 22 hours. Remove the culture tubes, centrifuge at 25°C and 5000 rpm for 10 minutes, and take 50 μL of the supernatant into an IFN-γ detection kit (same as in Example 1). Detect the IFN-γ using a fully automated chemiluminescence immunoassay analyzer. The results are shown in Table 2. The meanings of N, P, and T values ​​are the same as in Table 1. No CpG ODN was added to the control; the remaining reagents were the same as those in the corresponding N, P, and T tubes.

[0187] Table 2. IFN-γ levels (μmol / mL) at different concentrations of CpG ODN oligonucleotides.

[0188]

[0189]

[0190] Table 2 shows that TN and (TN) / (PN) can be detected in Examples 4 and 5 compared to the control group. When the concentration of CpGODN is 10 μM, the TN and (TN) / (PN) values ​​of the positive sample are the highest, which can be used to make a more accurate judgment on the detection results.

[0191] Test Example 3

[0192] This test example provides the test results of the kits used in Examples 1 and 4.

[0193] The negative control reagent, positive control reagent, and test culture reagent from Examples 1 and 4 were each placed into 4 mL culture tubes, designated as negative control tubes (N), positive control tubes (P), and test culture tubes (T), respectively, with 50 μL dispensed into each tube. Different colored screw caps were used for each culture tube to distinguish the different components. Specifically, the screw cap for the negative control tube (N) was white; the screw cap for the positive control tube (P) was red; and the screw cap for the test culture tube (T) was blue.

[0194] Take 9 mL each of negative samples (blood from subjects who have not been vaccinated against COVID-19 and are not infected with COVID-19) and positive samples (blood from vaccinated subjects), and aliquot 1 mL into N, P, and T tubes respectively. Place the blood culture tubes vertically in an incubator at 37°C and incubate for 22 hours. Remove the culture tubes, centrifuge at 25°C and 5000 rpm for 10 minutes, and transfer 50 μL of the supernatant to an IFN-γ detection kit (same as in Example 1). Detect the IFN-γ using a fully automated chemiluminescence immunoassay analyzer. The results are shown in Table 3. The meanings of N, P, and T values ​​are the same as in Table 1. No agonist was added to the control group; the remaining reagents were the same as those in the corresponding N, P, and T tubes.

[0195] Table 3. IFN-γ levels (μmol / mL) at different agonist concentrations

[0196]

[0197]

[0198] Table 3 shows that the detection effect is better when the agonist is 0.1 μg / mL imidazoquinoline compound R848, which neither increases the background concentration of N value nor stimulates T cells to secrete IFN-γ.

[0199] Test Example 4

[0200] This test example provides the test results of the kits used in Examples 1, 6, and 7.

[0201] The negative control reagent, positive control reagent, and test culture reagent from Examples 1, 6, and 7 were each placed into 4 mL culture tubes, designated as negative control tubes (N), positive control tubes (P), and test culture tubes (T), respectively, with 50 μL dispensed into each tube. Different colored screw caps were used for each culture tube to distinguish the different components. Specifically, the negative control tube (N) had a white screw cap; the positive control tube (P) had a red screw cap; and the test culture tube (T) had a blue screw cap.

[0202] Take 12 mL each of negative samples (blood from subjects who have not been vaccinated against COVID-19 and are not infected with COVID-19) and positive samples (blood from vaccinated subjects), and aliquot 1 mL into N, P, and T tubes respectively. Place the blood culture tubes vertically in an incubator at 37°C and incubate for 22 hours. Remove the culture tubes, centrifuge at 25°C and 5000 rpm for 10 minutes, and take 50 μL of the supernatant into an IFN-γ detection kit (same as in Example 1). Detect the results on a fully automated chemiluminescence immunoassay analyzer. The results are shown in Table 4. The meanings of N, P, and T values ​​are the same as in Table 1. No antibody was added to the control; the remaining reagents were the same as those in the corresponding N, P, and T tubes.

[0203] Table 4. IFN-γ levels (μmol / mL) at different inhibitor concentrations

[0204]

[0205]

[0206] Table 4 shows that the combination of (CD25+CD127) antibodies yields the best results.

[0207] Test Example 5

[0208] This test case provides the performance of the IFN-γ detection kit in Example 1.

[0209] 1. Blank limit

[0210] The zero-value calibrator without the analyte was repeatedly tested 20 times. The average relative light intensity (RLU) (M) and standard deviation (SD) of the 20 measurements were obtained. The RLU value corresponding to M+2SD was then calculated. Substituting the relative light intensity corresponding to M+2SD into the master curve equation under the conditions of the reagent system, the corresponding concentration value was obtained, which is the blank limit. The blank limit of this IFN-γ detection kit was found to be ≤5.69 pg / mL.

[0211] 2. Negative compliance rate

[0212] Whole blood was drawn from 10 volunteers (healthy individuals who had not been vaccinated against COVID-19 and were not infected with COVID-19) using vacuum blood collection tubes containing heparin (lithium heparin or sodium heparin). Each tube contained at least 4.0 mL of blood, which was inverted 8-10 times to ensure thorough mixing with the anticoagulant. Within 4 hours of blood collection, 1.0 mL of blood sample was added to negative control tubes, positive control tubes, and test culture tubes containing the blood stimulation reagent from Example 1. Each culture tube was inverted 8-10 times to ensure thorough mixing; air bubbles generated during this stage did not affect subsequent detection. The blood culture tubes were placed vertically in a 37°C incubator for 22 hours. The culture tubes were then removed, centrifuged at 25°C and 5000 rpm for 10 minutes, and 50 μL of the supernatant was transferred to an IFN-γ detection kit for analysis using a fully automated chemiluminescence immunoassay analyzer. The negative concordance rate of this IFN-γ detection kit was no less than 90%.

[0213] 3. Positive compliance rate

[0214] Ten volunteers who tested positive for COVID-19 IgM / IgG / neutralizing antibodies (COVID-19 IgM / IgG / neutralizing antibodies) using a rapid COVID-19 test kit (colloidal gold method) (Tianjin Hongyutai) were selected (patients who had been vaccinated against COVID-19 or had recovered from COVID-19). Whole venous blood was drawn from the positive volunteers using vacuum blood collection tubes containing heparin (lithium heparin or sodium heparin), at a rate of at least 4.0 mL per tube. The tubes were inverted 8-10 times to ensure thorough mixing with the anticoagulant. Within 4 hours of blood collection, 1.0 mL of blood sample was added to negative control tubes, positive control tubes, and test culture tubes containing the blood stimulation reagent from Example 1. Each culture tube was inverted 8-10 times to ensure thorough mixing with the contents of the culture tube. The blood culture tubes were then placed vertically in a 37°C incubator for 22 hours. Remove the culture tubes, centrifuge at 25°C and 5000 rpm for 10 minutes, and transfer 50 μL of the supernatant to the IFN-γ detection kit for detection on a fully automated chemiluminescence immunoassay analyzer. The positive concordance rate of this IFN-γ detection kit meets the requirement of 90%.

[0215] 4. Accuracy

[0216] Within the measurement range specified by the kit, using the PB buffer system, two accuracy samples of γ-interferon biological reference standard (No. 87 / 586) were prepared at concentration points: 50 pg / mL and 500 pg / mL. Each point was measured in triplicate, and the average value M was taken. The ratio of the measured value to the labeled value was calculated. The results are detailed in the table below. Table 5 shows that the accuracy of this IFN-γ detection kit is within the range of 0.900–1.100.

[0217] Table 5 Accuracy Test Results

[0218]

[0219]

[0220] 5. Precision

[0221] Six serum samples stimulated with specific antigens were collected, combined, and mixed to serve as a precision reference. For each sample, the same batch of reagents was used to perform 10 replicates, with intra-batch variation (CV) ≤ 8.0%; for each sample, three batches of reagents were used to perform 10 replicates, with inter-batch variation (CV) ≤ 10.0%. See Table 6 for details.

[0222] Table 6. Results of reagent kit precision testing

[0223]

[0224] 6. Linearity Detection

[0225] Eight concentrations of gamma-interferon biological reference material (number 87 / 586) were prepared at 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, 15.6 pg / mL, and 0 pg / mL. Each concentration was measured twice. The relative light unit (RLU) value for each concentration was calculated and fitted with the corresponding concentration value. The linear regression equation was Y = 296.19X + 1402.4, with a correlation coefficient r ≥ 0.9900. The results are as follows. Figure 1 As shown.

[0226] 7. Establishment of positive judgment value

[0227] Eighty whole blood samples were collected using vacuum blood collection tubes containing heparin (lithium heparin or sodium heparin). Sixty samples were negative (from healthy individuals who had not been vaccinated against COVID-19 and were not infected with COVID-19), and 20 samples were from volunteers who tested positive for COVID-19 IgM / IgG / neutralizing antibodies (who had been vaccinated against COVID-19). Each tube contained at least 4.0 mL of blood, which was inverted 8-10 times to ensure thorough mixing with the anticoagulant. Within 4 hours of blood collection, 1.0 mL of blood sample was added to negative control tubes, positive control tubes, and test culture tubes containing the blood stimulation reagent from Example 1. Each culture tube was inverted 8-10 times to ensure thorough mixing with the culture medium. The blood culture tubes were placed vertically in an incubator at 37°C for 22 hours. The culture tubes were then removed, centrifuged at 25°C and 5000 rpm for 10 minutes, and 50 μL of the supernatant was transferred to an IFN-γ detection kit for analysis using a fully automated chemiluminescence immunoassay analyzer. Based on the statistical analysis of negative samples, positive samples, and the total sample, the criteria for determining positive reference values ​​were derived, as shown in Table 7:

[0228] Table 7. Result Interpretation Criteria for In Vitro Detection Methods of COVID-19 Gamma Interferon

[0229]

[0230] 8. Evaluation of reagent kit equivalence

[0231] Of the 60 clinical trial samples, 8 were from healthy individuals who had not received the COVID-19 vaccine, 7 from individuals who had received one dose of the COVID-19 vaccine, 25 from individuals who had received two doses of the COVID-19 vaccine, and 20 from individuals who had received three doses of the COVID-19 vaccine. Using the COVID-19 (IgM / IgG / neutralizing antibody) rapid test kit (colloidal gold method) produced by Tianjin Hongyutai Biotechnology Co., Ltd. as a control reagent, the positive detection rate of the test kit after COVID-19 vaccination was 98.07%, and the negative detection rate was 100.0%. The results are shown in Table 8.

[0232] Table 8 Summary of positive detection rates for γ-interferon

[0233]

[0234]

[0235] As can be seen from Table 8 above, compared with the currently available COVID-19 IgG / IgM / neutralizing antibody detection kits and ELISA kits, the kit of this invention has the advantages of high accuracy, high sensitivity, high specificity, good stability, and fully automated detection.

[0236] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for quantitative detection of COVID-19 IFN-γ using peripheral blood, characterized in that, The kit consists of a blood stimulation reagent and an IFN-γ detection kit. The blood stimulation reagent consists of a negative control reagent, a positive control reagent, and a test culture reagent. Each of the blood stimulation reagents includes an activator that enhances the immune stimulation of T cells and an inhibitor that inhibits the negative regulatory effect of Treg cells. The positive control reagent also includes phytohemagglutinin, and the test culture reagent also includes T cell epitope peptides. The T-cell epitope peptide is a COVID-19 nucleocapsid protein T-cell epitope peptide fragment with the amino acid sequence LSPRWYFYY and a COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment with the amino acid sequence CVADYSVLY; the activator that enhances T-cell immune stimulation is an imidazoquinone compound R848 and an oligonucleotide fragment of unmethylated CpG, wherein the sequence of the unmethylated CpG oligonucleotide fragment is 5'-ggGGGACGATCGTCggggg-3'; the concentration of the imidazoquinone compound R848 is 0.05~0.2 μg / mL, and the concentration of the unmethylated CpG oligonucleotide fragment is 1~10 μmol / mL; the inhibitor that inhibits the negative regulatory effect of Treg cells is a combination of CD25 antibody and CD127 antibody.

2. The kit for quantitative detection of COVID-19 IFN-γ using peripheral blood according to claim 1, characterized in that, The concentration of the COVID-19 nucleocapsid protein T-cell epitope peptide fragment is 0.6~0.8 µg / mL, and the concentration of the COVID-19 S protein receptor-binding domain antigen T-cell epitope peptide fragment is 0.9~1.2 µg / mL.

3. The kit for quantitative detection of COVID-19 IFN-γ using peripheral blood according to claim 1, characterized in that, The concentration of the phytohemagglutinin is 10 µg / mL.

4. The kit for quantitative detection of COVID-19 IFN-γ using peripheral blood according to any one of claims 1 to 3, characterized in that, The reagents in the IFN-γ detection kit include magnetic bead coating working solution, enzyme-labeled antibody working solution, luminescent colorimetric solution, and washing solution.

5. The application of the kit for quantitative detection of COVID-19 IFN-γ in peripheral blood according to any one of claims 1 to 4 in the quantitative detection of COVID-19 IFN-γ in peripheral blood for non-diagnostic purposes: After co-culturing the peripheral blood sample to be tested with each of the blood stimulating reagents, centrifuge, take the supernatant, and use the IFN-γ detection kit to quantitatively detect IFN-γ in the supernatant.

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