阅读说明：本技术 一种SARS-CoV-2感染细胞免疫检测的抗原多肽组合物及其应用、试剂盒 (Antigen polypeptide composition for immune detection of SARS-CoV-2 infected cell, application and kit thereof ) 是由 王子腾 王新明 吴文娟 贺卓 赵兵杰 晋高伟 于 2021-09-03 设计创作，主要内容包括：本发明涉及生物医药技术领域,尤其涉及一种SARS-CoV-2感染细胞免疫检测的抗原多肽组合物及其应用、试剂盒。本发明抗原多肽组合物能够特异性地刺激CD4～(+)、CD8～(+)细胞分泌IFN-γ,用于SARS-CoV-2检测具有较高的灵敏度和特异性,且CD4～(+)、CD8～(+)联合检测可更为准确的反映被检测者对SARS-CoV-2抗原的细胞免疫状态,进而可配合抗体及核酸结果对患者感染SARS-CoV-2后患病的严重程度、治疗效果以及疫苗接种后的保护效应做出评估。(The invention relates to the technical field of biological medicine, in particular to an antigen polypeptide composition for immune detection of SARS-CoV-2 infected cells, and application and a kit thereof. The antigen polypeptide composition of the invention can specifically stimulate CD4 + 、CD8 + IFN-gamma is secreted by cells, has higher sensitivity and specificity when being used for SARS-CoV-2 detection, and CD4 + 、CD8 + The combined detection can more accurately reflect the cell immune state of a detected person to SARS-CoV-2 antigen, and further can be matched with antibody and nucleic acid results to make evaluation on the severity of the patient infected with SARS-CoV-2, the treatment effect and the protective effect after vaccination.)

1. An antigenic polypeptide composition for immunological detection of SARS-CoV-2 infected cells, comprising at least one of I) to IV):

I) the amino acid sequence of the NP epitope peptide is shown as SEQID.No. 1;

and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 2;

II) and NP epitope peptide with the amino acid sequence shown as SEQID.No. 3-5;

III) and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 6-9.

2. Use of the antigenic polypeptide composition of claim 1 in the preparation of a kit for the in vitro detection of a SARS-CoV-2 specific cellular immune response.

3. A kit for detecting SARS-CoV-2 specific cell immune reaction is characterized by comprising a cell culture reagent and an IFN-gamma antigen detection reagent;

the cell culture reagent comprising the antigenic polypeptide composition of claim 1 and a buffer.

4. The kit according to claim 3, wherein the cell culture reagent further comprises a negative control culture solution, the negative control culture solution comprises a buffer solution, glucose with a mass fraction of 2-7%, glycerol with a volume fraction of 1-5%, and pH 7.35.

5. The kit of claim 3, wherein the cell culture reagents further comprise a positive control culture fluid; the positive control culture solution comprises a buffer solution and phytohemagglutinin of 0.8-4.8 mg/ml.

6. The kit according to any one of claims 3 to 5, wherein the cell culture reagent contains a culture medium which is PBS buffer and/or HEPES buffer.

7. The kit of claim 3, wherein the IFN- γ antigen detection reagent comprises a magnetic microparticle suspension coupled with an IFN- γ coated antibody, an enzyme conjugate with horseradish peroxidase (HRP) -labeled IFN- γ labeled antibody as a tracer; the calibrator, chemiluminescence substrate solution and bar code card.

8. The kit of claim 7, wherein the magnetic particles have a diameter of 0.5-4 um, the anti-IFN- γ antibody is covalently coupled to the magnetic particles, and the working concentration of the anti-IFN- γ antibody is 0.5-1.5 ug/ml.

9. The kit according to claim 7, wherein the chemiluminescent substrate solution comprises a substrate solution A and a substrate solution B, wherein the substrate solution A is isoluminol derivative, and the substrate solution B is hydrogen peroxide.

10. The kit of claim 3, wherein the calibrator is IFN- γ antigen high value plasma.

11. The kit according to any one of claims 3 to 10, further comprising a cell culture well plate and a cell culture tube, wherein the tube body of the cell culture tube is provided with an identification bar code.

Technical Field

The invention relates to the technical field of new coronavirus detection, in particular to an antigen polypeptide composition for immune detection of SARS-CoV-2 infected cells, and application and a kit thereof.

Background

The novel coronavirus pneumonia is an acute infectious disease caused by the novel coronavirus, patients and asymptomatic infected persons are main infectious sources, and the main transmission routes are respiratory transmission and close contact transmission. The humoral and cellular immunity of the host to SARS-CoV-2 infection is a key factor in determining the prognosis of a patient.

The SARS-CoV-2 infection diagnostic reagent kit developed at present can be divided into two types according to the detection target: one is nucleic acid for detecting viruses, against pathogens; one is to detect the corresponding antibody, directed against humoral immunity. Neither of these can make a corresponding determination of the cellular immune response of the patient.

The gamma-interferon in vitro release test can reflect the cellular immunity level to a certain degree, at present, antigens are mainly used for stimulating CD4+ cells in T cells to generate gamma interferon on an immune platform, but only the CD4+ cellular immunity level can be used for judging whether a patient is infected with a certain pathogen, and the severity, the treatment effect, the protection effect of related vaccines and the like of the patient infected with the certain pathogen are difficult to accurately evaluate.

Disclosure of Invention

In view of the above, the invention provides an antigen polypeptide composition for SARS-CoV-2 infected cell immunoassay, and applications and kits thereof. The antigen polypeptide composition can specifically stimulate CD4⁺、CD8⁺IFN-gamma is secreted by cells, has higher sensitivity and specificity when being used for SARS-CoV-2 detection, and CD4⁺、CD8⁺The combined detection can more accurately reflect the cell immune state of a detected person to SARS-CoV-2 antigen.

In order to achieve the above object, the present invention provides the following technical solutions:

1. an antigenic polypeptide composition for immunological detection of SARS-CoV-2 infected cells, comprising at least one of I) to IV):

I) the amino acid sequence of the NP epitope peptide is shown as SEQID.No. 1;

and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 2;

II) and NP epitope peptide with the amino acid sequence shown as SEQID.No. 3-5;

III) and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 6-9.

Specifically, the antigen polypeptide of I), namely group 1, is a virus antigen polypeptide group capable of combining with HLA class I molecules and HLA class II molecules, and mainly acts on CD8⁺、CD4⁺A cell comprising a total of two polypeptides, numbered S-1 and N-2, respectively, wherein the S-1 polypeptide is derived from an S protein and has the sequence: NNCTFEYVSQPFLMDLEGK (SEQ ID No. 1); the N-2 polypeptide is derived from N protein and has the sequence: KMKDLSPRWYFYYLGTGPEAGL (SEQ ID No. 2);

II) the antigenic polypeptides of group 2, are groups of viral antigenic polypeptides binding to HLA class I molecules, acting mainly on CD8⁺A cell comprising a total of three polypeptides, numbered N-1, N-3, ORF-1, wherein the N-1 polypeptide is derived from an N protein and has the sequence: MSDNGPQNQRNAPRITFGGP (SEQ ID No. 3); the N-3 polypeptide is derived from N protein and has the sequence: DQVILLNKHIDAYKTFPPTEPKK (SEQ ID No. 4); the ORF-1 polypeptide is derived from ORF protein and has the sequence: LHSYFTSDYYQLYS (SEQ ID No. 5);

III) the antigenic polypeptides of group 2, are groups of viral antigenic polypeptides binding to HLA class II molecules, acting mainly on CD4⁺The cell comprises four polypeptides which are respectively numbered as S-2, S-3, M-1 and M-2, wherein the S-2 polypeptide is derived from S protein and has the sequence: CSNLLLQYGSFCTQLNRA (SEQ ID No. 6); the S-3 polypeptide is derived from S protein and has the sequence: LTDEMIAQYTSALLAGTIT (SEQ ID No. 7); the M-1 polypeptide is derived from an M protein and has the sequence: LVIGAVILRGHLRIAGHHLGRC (SEQ ID No. 8); the M-2 polypeptide is derived from an M protein and has the sequence: VATSRTLSYYKLGASQRVAGDS (SEQ ID No. 9).

In some preferred embodiments, the antigenic polypeptide composition comprises composition 1 and composition 2, wherein composition 1 is an antigenic epitope polypeptide represented by I) to III). Wherein, the antigen epitope polypeptides are mixed in equal proportion; the concentration of each epitope polypeptide solution is 0.02-0.1mg/ml, specifically 0.02mg/ml, 0.04mg/ml or 0.1 mg/ml. The composition 2 is the epitope polypeptide shown in II), wherein the epitope polypeptides are mixed in equal proportion; the concentration of each epitope polypeptide solution is 0.02-0.1mg/ml, specifically 0.02mg/ml, 0.04mg/ml or 0.1 mg/ml.

Experiments show that the antigenic polypeptide composition can specifically stimulate CD4⁺、CD8⁺IFN-gamma is secreted by cells, has higher sensitivity and specificity when being used for SARS-CoV-2 detection, and CD4⁺、CD8⁺The combined detection can more accurately reflect the cell immune state of a detected person to SARS-CoV-2 antigen.

Based on the above results, the present invention also provides the application of the antigen polypeptide composition in the preparation of a kit for in vitro detection of SARS-CoV-2 specific cellular immune response.

The invention also provides a kit for detecting SARS-CoV-2 specific cellular immune reaction, which comprises a cell culture reagent and an IFN-gamma antigen detection reagent;

the cell culture reagent comprising the antigenic polypeptide composition of claim 1 and a buffer.

In some embodiments, the cell culture reagent further comprises a negative control culture fluid comprising a buffer, 2-7 wt% glucose, a volume fraction of 1-5 vol% glycerol, and a pH of 7.35.

In some embodiments, the cell culture reagent further comprises a positive control medium; the positive control culture solution comprises a buffer solution and 0.8-4.8mg/ml of phytohemagglutinin and is used for nonspecific stimulation of IFN-gamma secretion of cells.

In the cell culture reagent, the buffer solution is PBS buffer solution and/or HEPES buffer solution. In some embodiments specifically PBS buffer.

In some embodiments, the IFN- γ antigen detection reagent comprises a magnetic microparticle suspension coupled with an IFN- γ coated antibody, an enzyme conjugate with horseradish peroxidase (HRP) -labeled IFN- γ labeled antibody as a tracer; the calibrator, chemiluminescence substrate solution and bar code card. The IFN-gamma coating antibody and the IFN-gamma marking antibody recognize different antigen epitopes on the IFN-gamma antigen to form a double sandwich structure.

Wherein, the diameter of the magnetic particle is 0.5-4 um, the anti-IFN-gamma antibody is covalently coupled on the magnetic particle, and the working concentration of the anti-IFN-gamma antibody is 0.5-1.5 ug/ml.

The anti-IFN-gamma antibody is a murine monoclonal antibody. The anti-IFN-gamma antibody can be combined with gamma-interferon (IFN-gamma) in human plasma to form an antigen-antibody immune complex.

The bar code card can be identified by the matched software of instruments such as a full-automatic chemiluminescence instrument AutoLumo A2000 and the like, and is used for calibrating a calibration curve.

The chemiluminescence substrate liquid comprises a substrate A liquid and a substrate B liquid, wherein the substrate A liquid is isoluminol derivative, and the substrate B liquid is hydrogen peroxide.

The calibrator is mixed IFN-gamma antigen high-value plasma and is obtained by the following method: mixing fresh human peripheral blood, adding positive control culture solution (P) at a ratio of 10:1, and culturing for 16-24 h; and centrifugally extracting the upper plasma after culturing to obtain the mixed IFN-gamma antigen high-value plasma. The concentration of the calibrator is 0:0 IU/ml; 1: 0.15 IU/ml; 2:0.5 IU/ml; 3:1 IU/ml; 4:3 IU/ml; the kit is characterized by comprising the following components of 5:10IU/ml, wherein the components are numbered as 0, 1, 2, 3, 4 and 5 in sequence, are used for quantitatively determining the concentration of gamma-interferon (IFN-gamma) antigen, and can be matched with a bar code card to be used for the identification of matched software of instruments such as a full-automatic chemiluminescence instrument AutoLumoA2000 and the like of an applicant company.

In some embodiments, the kit of the invention further comprises a cell culture well plate and a cell culture tube, wherein the tube body of the cell culture tube is provided with an identification bar code. The cell culture pore plate is made of a Polypropylene (PS) material; the cell culture tube is made of polyethylene terephthalate (PET).

The antigen composition provided by the invention is an antigen polypeptide composition for immune detection of SARS-CoV-2 infected cells, which is characterized by comprising at least one of I) to IV): I) the amino acid sequence of the NP epitope peptide is shown as SEQID.No. 1; and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 2; II) and NP epitope peptide with the amino acid sequence shown as SEQID.No. 3-5; III) and S1 epitope peptide with the amino acid sequence shown as SEQ ID No. 6-9. The invention also provides the application of the antigen polypeptide composition in preparing the detection reagent for in vitro detecting SARS-CoV-2 cell immune reaction, and the detection of SARS-CoV-2 specific cell immunityThe kit adopts an IGRA method in cell culture and a double-antibody sandwich method in IFN-gamma antigen detection, and experiments show that the antigen polypeptide composition can specifically stimulate CD4+ and CD8+ cells to secrete IFN-gamma, has higher sensitivity and specificity when being used for SARS-CoV-2 detection, and CD4⁺、CD8⁺The combined detection can more accurately reflect the cell immune state of a detected person to SARS-CoV-2 antigen, and further can be matched with antibody and nucleic acid results to make evaluation on the severity of the patient infected with SARS-CoV-2, the treatment effect and the protective effect after vaccination.

Detailed Description

The invention provides an antigen polypeptide composition for immune detection of SARS-CoV-2 infected cells, and application and a kit thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 the kit and the detection method of the present invention

The kit comprises the following components:

TABLE 1

Negative control culture (N): glucose and glycerol were added to PBS buffer at pH 7.35 to prepare a buffer containing 2 to 7% glucose and 1 to 5% glycerol.

Positive control culture (P): PBS buffer containing Phytohemagglutinin (PHA) at 0.8-4.8 mg/ml.

Test medium (T1): the test culture medium for the antigen polypeptides of groups 1 to 3 of SARS-CoV-2-specific CD4+ and CD8+ was added to the PBS buffer. The antigens are mixed in equal proportion, and the concentration range is 0.02-0.1 mg/ml.

Test medium (T2): test medium for the group 2 antigen polypeptide of SARS-CoV-2 specific CD8+ was added to PBS buffer. Mixing the antigens at equal ratio, wherein the concentration of each antigen is 0.02-0.1 mg/ml.

Wherein the group 1 antigen polypeptide is a virus antigen polypeptide group capable of combining with HLA class I molecule and HLA class II molecule, and mainly acts on CD8⁺、CD4⁺A cell comprising a total of two polypeptides, numbered S-1 and N-2, respectively, wherein the S-1 polypeptide is derived from an S protein and has the sequence: NNCTFEYVSQPFLMDLEGK (SEQ ID No. 1); the N-2 polypeptide is derived from N protein and has the sequence: KMKDLSPRWYFYYLGTGPEAGL (SEQ ID No. 2);

the group 2 antigen polypeptide is a virus antigen polypeptide group combined with HLA class I molecules, and mainly acts on CD8⁺A cell comprising a total of three polypeptides, numbered N-1, N-3, ORF-1, wherein the N-1 polypeptide is derived from an N protein and has the sequence: MSDNGPQNQRNAPRITFGGP (SEQ ID No. 3); the N-3 polypeptide is derived from N protein and has the sequence: DQVILLNKHIDAYKTFPPTEPKK (SEQ ID No. 4); the ORF-1 polypeptide is derived from ORF protein and has the sequence: LHSYFTSDYYQLYS (SEQ ID No. 5);

group 3 is a group of viral antigen polypeptides that bind to HLA class II molecules and act primarily on CD4⁺The cell comprises four polypeptides which are respectively numbered as S-2, S-3, M-1 and M-2, wherein the S-2 polypeptide is derived from S protein and has the sequence: CSNLLLQYGSFCTQLNRA (SEQ ID No. 5); the S-3 polypeptide is derived from S protein and has the sequence: LTDEMIAQYTSALLAGTIT (SEQ ID No. 6); the M-1 polypeptide is derived from an M protein and has the sequence: LVIGAVILRGHLRIAGHHLGRC (SEQ ID No. 7); the M-2 polypeptide is derived from an M protein and has the sequence: VATSRTLSYYKLGASQRVAGDS (SEQ ID No. 8).

Preparation of a calibrator: the calibrator is high-value plasma containing mixed IFN-gamma antigens, and is obtained by the following method: mixing fresh human peripheral blood, adding positive control culture solution (P) at a ratio of 10:1, and culturing for 16-24 h; and centrifugally extracting the upper plasma after culturing to obtain the mixed IFN-gamma antigen high-value plasma.

The detection method comprises the following steps:

respectively culturing human fresh peripheral vein anticoagulation by using the four culture solutions, and determining the IFN-gamma concentrations stimulated by the four culture solutions to judge the specific cellular immune response of the test culture solution (T1) and the test culture solution (T2).

(II) in vitro release of IFN-gamma

1) The collection adopts venipuncture, and a heparin sodium or heparin lithium anticoagulation vacuum blood collection tube treated by an aseptic technique is used for collecting a whole blood sample, wherein the collection amount is not less than 5 mL.

2) Subpackaging the culture solution:

cell culture well plate: adding a negative control culture solution (N), a test culture solution (T1), a test culture solution (T2) and a positive control culture solution (P) into corresponding holes of a cell culture plate according to a handle mark in a biosafety cabinet, wherein each hole is 100 mu L;

cell culture tube: this step may be omitted.

3) Subpackaging whole blood:

cell culture well plate: after whole blood is collected, the whole blood is gently inverted and uniformly mixed in a blood collection tube for 3-5 times, and the whole blood is subpackaged in corresponding holes (containing 100 mu L of culture solution of N, P, T1 and T2 respectively) of a cell culture plate in a biosafety cabinet within 8 hours, wherein each hole is 1.0 mL;

cell culture tube: after whole blood is collected, the whole blood is gently inverted and uniformly mixed in a blood collection tube for 3-5 times, and the whole blood is subpackaged to a group of corresponding cell culture tubes with 1.0 mL/hole in a biosafety cabinet within 8 hours.

4) Culturing: covering the upper cover of the cell culture plate tightly/screwing the cover of the cell culture tube tightly, quickly putting the cell culture plate into an electric heating constant-temperature incubator at 37 ℃ for culturing for 16-24 hours, and keeping the cell culture plate flat during the culture process without being turned upside down.

5) Collecting:

cell culture well plate: after the culture is finished, the cell culture plate is placed on a flat desktop, the cell culture plate is placed still for 1 minute, the cover is opened carefully, and supernatant is sucked from each cell culture plate hole to carry out IFN-gamma detection. Note that the cell layer is not absorbed, preventing hemolysis.

Cell culture tube: after the culture is finished, centrifuging the cell culture tubes for 10 minutes at 3000-. Note that the cell layer is not absorbed, preventing hemolysis. The post-centrifugation step can also be performed automatically by an automated chemiluminescence apparatus from the applicant company, such as AutoLumo a 2000.

Detection of IFN-gamma

6) A calibrator (for calibration) and the supernatant after the culture were sequentially added to a reaction vessel (hereinafter referred to as "well"), each at a loading of 100. mu.l/well.

7) mu.L of magnetic particle suspension was added to each well.

8) 50 μ L of enzyme conjugate was added to each well.

9) After mixing, incubation was carried out at 37 ℃ for 34 minutes.

10) Washing with the cleaning solution was performed 5 times.

11) Each well was filled with 50. mu.L each of the luminescent substrate A solution and the luminescent substrate B solution.

12) And detecting the luminous intensity 1-5 minutes after uniformly mixing.

13) The steps 6-12 can be performed automatically by a full-automatic chemiluminescence apparatus from the applicant company, such as AutoLumo a 2000.

Calculation of results

14) And selecting a proper curve fitting mode to calculate the result, preferably selecting a four-parameter fitting mode for the kit, and establishing a calibration curve by taking the concentration value of the calibrator as an x axis and taking the log value of the luminous value of the calibrator as a y axis. And (4) calculating a corresponding concentration value according to the luminous value of the sample to be detected. The results can be interpreted in two ways:

interpretation method 1:

(test culture broth (T1, T2) result-negative control culture broth (N) result)/(positive control culture broth (P) result-negative control culture broth (N) result);

(test culture solution (T2) result-negative control culture solution (N) result)/(test culture solution (T1) result-negative control culture solution (N) result)

Interpretation method 2:

test medium (T1, T2) results-negative control medium (N) results;

positive control culture (P) results-negative control culture (N) results.

Infection and non-infection differential detection/mild and severe disease prompt detection: the judgment method 1 is selected, in the following examples, (test culture solution (T1) result-negative control culture solution (N) result)/(positive control culture solution (P) result-negative control culture solution (N) result) or (test culture solution (T2) result-negative control culture solution (N) result)/(positive control culture solution (P) result-negative control culture solution (N) result) > 2% is positive for infection, and (test culture solution (T2) result-negative control culture solution (N) result)/(test culture solution (T1) result-negative control culture solution (N) result) < 30% is a severe indication. Detection of cellular immune response intensity of infected/vaccinated population: in the subsequent example of interpretation mode 2, the result of the test culture solution (T1) -the result of the negative control culture solution (N) or the result of the test culture solution (T2) -the result of the negative control culture solution (N) is more than 0.438IU/ml, which indicates that stronger cellular immune response exists against SARS-CoV-2 virus.

Example 2

In order to verify the effect of the kit for detecting SARS-CoV-2 specific cellular immune response in human fresh peripheral vein anticoagulation based on IFN-gamma release test in the aspect of SARS-CoV-2 infection detection, the present example uses clinically confirmed new coronary pneumonia patients as experimental group and healthy human as control group for verification.

In a hospital in Wuhan City, 97 confirmed (cured) patients fresh whole blood heparin samples were tested, the 97 confirmed (cured) samples contained 15 severe patients and 82 mild patients, and 40 healthy fresh whole blood heparin samples were tested as controls. The interpretation method 1 was selected, and the results are shown in tables 2 and 3, with 2% as the cutoff value:

TABLE 2

TABLE 3

The result shows that the sensitivity and the specificity of the kit are both over 90 percent, the ratio of the T2 detection result to the T1 detection result of a severe patient to a mild patient shows obvious difference, and the kit can be matched with other methodologies to make more comprehensive assessment on the infection severity.

Example 3

In this case, the application of the kit of example 1 of the present invention in SARS-CoV-2 vaccination was verified by using a healthy human vaccination as an experimental group and a healthy human non-vaccination as a control group.

In Zhengzhou city, 7 vaccinated healthy persons (3 days after the 2 nd needle vaccination) were tested for fresh whole blood heparin samples, and 19 unvaccinated fresh whole blood heparin samples of healthy persons. The result of using interpretation 2 at a cut-off of 0.1IU/ml is shown in Table 4, in which RBD-Ab is an antibody bound to the RBD region of the virus.

TABLE 4

Note: the numbers 1-7 are the specific cellular immune response and antibody results after the vaccination of healthy people (3 days after the 2 nd needle inoculation); no. 8-26 are healthy human non-vaccinated.

From the above results, the kit of the present invention showed a significant difference between the vaccinated (3 days after the 2 nd needle inoculation) and the unvaccinated human, and the cellular immune response appeared earlier than the antibody response in the individual (e.g. sample No. 6). The whole cellular immune response and the antibody response show positive correlation, and can be matched with antibody detection to provide more comprehensive evaluation on the vaccine protection effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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