WO2021184391A1

WO2021184391A1 - Method for earlier screening of novel coronavirus

Info

Publication number: WO2021184391A1
Application number: PCT/CN2020/080524
Authority: WO
Inventors: 金腾川; 姜德华; 马欢; 常志远; 曾威红; 易如婷; 黄文喜
Original assignee: 广州市康润生物科技有限公司; 生物岛实验室
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2021-09-23

Abstract

Provided are a kit for screening for novel coronavirus infections and a detection reagent thereof. The kit contains a SARS-CoV-2 magnetic bead coating material serving as R1 and an anti-human IgA antibody acridinium ester marker serving as R2, wherein the SARS-CoV-2 magnetic bead coating material contains a magnetic bead coating material trihydroxymethyl aminomethane buffer solution (with a pH value of 7.1-7.4) coated with a SARS-CoV-2 recombinant antigen, and the anti-human IgA antibody acridinium ester marker is a 2-(N-morpholino) ethanesulfonic acid buffer solution containing an acridinium ester labeled mouse anti-human IgA monoclonal antibody.

Description

Early screening methods for the new coronavirus

Technical field

The present invention relates to the technical field of immunoassays, in particular to an earlier screening method, reagents and kits for SARS-CoV-2 IgA antibodies of novel coronavirus infection pneumonia (new coronary pneumonia).

Background technique

Since December 2019, the new type of coronavirus pneumonia (new crown pneumonia) has been raging. The cumulative number of people infected in China has exceeded 80,000, and the mortality rate has reached 1.4-3.4%. The Class B infectious diseases stipulated in the Law shall be managed as Class A infectious diseases. New coronary pneumonia usually develops 3-7 days after being infected with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). The symptoms are mainly fever, dry cough, fatigue, and can also be accompanied by nasal congestion, runny nose, sore throat, myalgia and diarrhea, etc. . Mild patients have no symptoms of pneumonia. Severe patients often develop dyspnea and/or hypoxemia one week after the onset of onset. In severe cases, they can rapidly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis, and coagulation. Dysfunction and multiple organ failure, etc. It is worth noting that severe and critically ill patients may have moderate to low fever during the course of their illness, or even no obvious fever. Therefore, it is of great significance to efficiently detect patients with new coronary pneumonia and predict patients with severe pneumonia.

The virus nucleic acid detection reagent was first used for the diagnosis of new coronary pneumonia. Because of its low detection rate, many companies have begun to develop rapid detection kits for SARS-CoV-2IgG and IgM, including colloidal gold method and chemiluminescence immunoassay. The seventh edition of the new coronary pneumonia diagnosis and treatment plan incorporates IgM and IgG serological testing as laboratory diagnostic methods. Currently, the registered products include 11 nucleic acid detection products and 6 antibody detection products. The latter include: colloidal gold method new coronavirus (2019-nCoV) antibody detection kit, colloidal gold method new coronavirus (2019-nCoV) IgM Antibody detection kit, magnetic particle chemiluminescence method new coronavirus (2019-nCoV) IgM antibody detection kit, magnetic particle chemiluminescence method new coronavirus (2019-nCoV) IgG antibody detection kit, new type of scientific luminescent particle immunoassay method Coronavirus (2019-nCoV) antibody detection kit.

Because SARS-CoV-2 infects the lower respiratory tract, the patient produces less sputum, and there is a high probability that the sample collection will fail with a throat swab. The detection rate of nucleic acid detection reagents for new coronary pneumonia is low, and the sensitivity is only 30-50%, which cannot meet the needs of screening all patients.

Measuring the total antibody improves the sensitivity but cannot effectively distinguish which stage of the patient is in the course of the disease and whether it is still infectious. The colloidal gold method is simple and quick to detect, but the accuracy is not enough. The sensitivity and specificity of chemiluminescence detection of IgM and IgG antibodies are high, but the window period of the two is long. IgM needs to be detected 3-5 days after the onset of onset, and the detection value of IgG needs to increase by more than 4 times during the recovery period to be diagnosed.

Therefore, the currently disclosed technology lags behind the detection of the new coronavirus, and the detection results need to be improved. There is no report on the use of IgA in screening tests for new coronary pneumonia, and there is no report on the successful development of IgA test kits.

Summary of the invention

The purpose of the present invention is to avoid the shortcomings of the prior art and provide a kit and reagents for screening new coronavirus infections that can detect SARS-CoV-2 IgA antibodies earlier.

The purpose of the present invention is achieved by the following technical measures.

Provide a kit for detecting SARS-CoV-2 IgA antibody for screening new coronavirus infection.

Preferably, the above-mentioned kit for screening for novel coronavirus infection is used for early screening of SARS-CoV-2 IgA antibodies.

Preferably, the above kit for screening for novel coronavirus infection is used as the SARS-CoV-2 magnetic bead coating of R1: a magnetic bead coating containing SARS-CoV-2 recombinant antigen-coated trimethylol Aminomethane buffer, pH value is 7.1-7.4;

As R2 anti-human IgA antibody acridinium ester label: 2-(N-morpholine) ethanesulfonic acid buffer containing acridinium ester-labeled mouse anti-human IgA monoclonal antibody.

Preferably, in the above kit for screening new coronavirus infections, the SARS-CoV-2 recombinant antigen is a spike protein expressed in a suspension cultured human embryonic kidney cell line HEK293F, referred to as S protein (Spike protein, S protein). ), the amino acid sequence at positions Gln321-Ser591 of the RBD domain was truncated for protein expression;

The protein expression process is: the SARS-CoV-2 S protein RBD gene is connected to human IgG1 Fc through a TEV restriction site sequence at the C-terminus, and then it is cloned into the mammalian expression vector pTT5 and Kozak is added to the translation initiation region The sequence was then transfected into suspension culture cells HEK293F, using interferon α-1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column for two purifications; the first purification To use Protein A column to obtain RBD-TEV-Fc fusion protein from the culture supernatant; the second purification is to use TEV enzyme with 6His tag to digest the fusion protein, and then pass the digested products through Protein A column in turn And nickel column to remove Fc, TEV enzymes, and the impurity protein bound to the Protein A column during the first purification, and finally obtain pure RBD protein in the flow-through.

In another preferred embodiment, in the above kit for screening new coronavirus infections, the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as N protein, and N protein is a cut of Met1-Ala419 The amino acid sequence at position is expressed;

The protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria. Add 0.5-2.0M sodium chloride to the solution to reduce non-specific binding of protein and nucleic acid, and then pass the lysate through a nickel column. After elution, add ammonium sulfate with a final concentration of 0.25-1.0M and stir at room temperature for 10-20 minutes to expose the protein Its hydrophobic core is further purified by a hydrophobic column, and then superdex-200 molecular sieve is used to remove polymers and other impurity proteins, and finally a high-purity N protein with uniform conformation is obtained.

Preferably, in the above kit for screening new coronavirus infection, the tris buffer contains 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin ( w/v), 0.25-1% Proclin300(v/v);

2-(N-morpholine) ethanesulfonic acid buffer contains 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin 300 (v/v) ).

Preferably, the above kit for screening for novel coronavirus infection also contains:

As the reaction buffer of R3: phosphate buffer containing goat anti-human IgG antibody, containing 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25- 1% Proclin300(v/v);

SARS-CoV-2 IgA negative control: SARS-CoV-2 IgA negative serum;

SARS-CoV-2 IgA positive control: human-derived purified SARS-CoV-2 IgM antibody;

Negative and positive control information card: contains the luminescence value of the negative and positive control, which is used for correction during screening.

In another preferred embodiment, the above-mentioned kit for screening for novel coronavirus infection contains:

Test card: It is composed of SARS-CoV-2 IgA test strip and plastic box; the test strip contains nitrocellulose membrane, sample pad, bonding pad, absorbent paper, and PVC board support;

The nitrocellulose membrane is coated with anti-human-IgA antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled SARS-CoV-2 antigen.

Preferably, the above-mentioned kit for screening for novel coronavirus infection also contains Tris hydrochloric acid buffer as a sample diluent, the pH of the Tris hydrochloric acid buffer is 7.1-7.4, and it contains 0.5-2% bovine serum white Protein (w/v), 0.05-0.2% Tween 20 (v/v), 0.25-1% Proclin 300 (v/v).

The present invention also provides a use of a reagent for detecting SARS-CoV-2 IgA antibody in preparing the above reagent or kit for screening novel coronavirus infection.

The kit used for screening the novel coronavirus infection and its detection reagent of the present invention can detect the novel coronavirus. The applicant found that patients with SARS-CoV-2 IgA antibodies appeared earlier than IgM antibodies; and SARS-CoV-2 IgA antibodies lasted longer in humans than IgM antibodies. Therefore, the kit of the present invention and the SARS-CoV-2 IgA antibody immunoassay reagent can be used to supplement the diagnosis of new coronary pneumonia at an earlier stage, and have the characteristics of accurate detection results.

Attached drawings

The present invention is further explained by using the accompanying drawings, but the content in the accompanying drawings does not constitute any limitation to the present invention.

Figure 1 is a schematic diagram of the results of serum SARS-CoV-2 IgA antibody levels in patients with new coronary pneumonia and a control group in an experimental example of the present invention.

Fig. 2 is a graph showing the change trend of SARS-CoV-2 IgA and IgM antibodies with the course of the disease in an experimental example of the present invention.

Detailed ways

The present invention will be further illustrated in combination with the following examples.

Example 1.

A kit for the detection of SARS-CoV-2 IgA antibodies for screening new coronavirus infections. Especially for early screening of SARS-CoV-2 IgA antibodies.

This kit for screening for novel coronavirus infection includes:

1. SARS-CoV-2 magnetic bead coating (R1): Tris buffer containing magnetic bead coating of SARS-CoV-2 recombinant antigen, pH 7.1-7.4, containing 0.05 -0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin 300 (v/v); magnetic beads are used as the reaction solid phase carrier, and the antigen is used for binding The target antibody in the test substance.

2. Anti-human IgA antibody acridinium ester marker (R2): 2-(N-morpholine) ethanesulfonic acid buffer of acridinium ester-labeled mouse anti-human IgA monoclonal antibody, containing 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin300 (v/v). Combining with the antigen-antibody complex, under alkaline conditions, the acridinium ester can emit light after being excited.

3. Reaction buffer (R3): phosphate buffer containing goat anti-human IgG antibody, containing 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin300 (v/v). Used to adsorb IgG in the sample to be tested to prevent interference.

4. SARS-CoV-2 IgA negative control: SARS-CoV-2 IgA negative serum.

5. SARS-CoV-2 IgA positive control: It is a human-derived purified SARS-CoV-2 IgM antibody.

6. Negative and positive control information card: contains the luminescence value of the negative and positive control, which is used for correction during screening.

In addition, the experiment requires the substrate solution (aqueous solution containing NaOH) and the cleaning solution (phosphate buffer containing surfactant) as general reagents, which are not provided in this kit.

Through clinical research, some patients' SARS-CoV-2 IgA turns positive earlier than IgM antibodies, 1-4 days earlier on average. Therefore, the clinical detection of SARS-CoV-2 IgA antibodies can be carried out earlier, and some patients with new coronary pneumonia can be screened and dealt with early.

The experiment found that the sensitivity and specificity of the test results of this kit for diagnosing pneumonia reached 99% and 96%, respectively, and it is an excellent test kit.

Example 2.

A kit for detecting SARS-CoV-2 IgA antibody for screening new coronavirus infection. Other features are the same as Example 1, except that it also has the following features: SARS-CoV-2 recombinant antigen is in Spike protein (S protein) expressed in suspension cultured human embryonic kidney cell line HEK293F, referred to as S protein, was extracted from the Gln321-Ser591 amino acid sequence of the RBD domain for protein expression. The protein expression process is: the SARS-CoV-2 S protein RBD gene is connected to human IgG1 Fc through a TEV restriction site sequence at the C-terminus, and then it is cloned into the mammalian expression vector pTT5 and Kozak is added to the translation initiation region The sequence was then transfected into suspension culture cells HEK293F, using interferon α-1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column for two purifications; the first purification To obtain the RBD-TEV-Fc fusion protein from the culture supernatant using the Protein A column; the second purification is to digest the fusion protein with the TEV enzyme with 6His tag, and then flow the digested products through the Protein A column in turn And nickel column to remove Fc, TEV enzymes, and the impurity protein bound to the Protein A column during the first purification, and finally obtain pure RBD protein in the flow-through.

The SARS-COV-2 spike RBD protein expressed by this method is intercepted from the Gln321-Ser591 amino acid sequence of the SARS-COV-2 spike protein. The coding gene sequence is shown in the following sequence 1, and its amino acid sequence is shown in the following sequence 2.

Sequence 1 The gene sequence encoding SARS-COV-2 spike RBD in this patent

Sequence 2 The amino acid sequence of SARS-COV-2 spike RBD in this patent

The SARS-COV-2 spike RBD protein of this method is secreted and expressed, and the signal peptide used is the IFNA1 (Interferon α-1) signal peptide, and the Kozak sequence is added to the translation initiation region. The C-terminus of the SARS-COV-2 spike RBD protein of this patent is connected to the human IgG1 Fc sequence through a TEV restriction site sequence. The expressed fusion protein is named SARS-COV-2-RBD-TEV-Fc, and its gene sequence (including The signal peptide) is shown in the following sequence 3, and the amino acid sequence is shown in the following sequence 4.

Sequence 3 The gene sequence of the fusion protein SARS-COV-2-RBD-TEV-Fc

Sequence 4 Amino acid sequence of fusion protein SARS-COV-2-RBD-TEV-Fc

This patent obtains high-purity SARS-COV-2 spike RBD protein through two purifications. For the first purification, the protein A column was used to obtain the SARS-COV-2-RBD-TEV-Fc fusion protein from the culture supernatant. The fusion protein was digested with TEV enzyme with 6His tag, and the digested products were passed through the protein A column and the nickel column in turn. At this time, the Fc, TEV enzyme, and the protein bound to the Protein A column during the first purification were all removed. After removal, the purified SARS-COV-2 spike RBD protein is finally obtained in the flow through.

As another implementation method, the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as the N protein, and the N protein is expressed by truncating the amino acid sequence at position Met1-Ala 419. The protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria. Add 0.5-2.0M sodium chloride to the solution, preferably 1.0M sodium chloride to reduce non-specific binding of protein and nucleic acid, then pass the lysate through a nickel column, and add ammonium sulfate with a final concentration of 0.25-1.0M at room temperature after elution. Stir for 10-20min, preferably after elution, add ammonium sulfate with a final concentration of 0.5M and stir for 15min at room temperature to expose the hydrophobic core of the protein, and then use a hydrophobic column to further purify the protein, and then use superdex-200 molecular sieve to remove polymers and others Hybrid protein, and finally obtain high-purity N protein with uniform conformation.

The SARS-COV-2 N protein expressed in this patent is truncated from the SARS-COV-2 N protein Met1-Ala 419 amino acid sequence. The coding gene sequence is shown in the following sequence 5, and its amino acid sequence is shown in the following sequence 6.

Sequence 5 SARS-COV-2 N protein gene sequence expressed in this patent

Sequence 6 SARS-COV-2 N protein amino acid sequence expressed in this patent

The SARS-COV-2 N protein of this patent is soluble expression in bacteria, with 6His tag connected to its N end, and a nickel column is used to purify the soluble SARS-COV-2 N from the supernatant of the bacterial fragments. At this time, it is purified. A large amount of nucleic acid is bound to the N protein. In order to remove the nucleic acid, ammonium sulfate is added and stirred at room temperature to expose the hydrophobic core of the protein, and then a hydrophobic column is used to further purify the protein. At this time, the nucleic acid is removed. In order to further remove polymers and other impurity proteins, the superdex200 molecular sieve was used to further purify the protein, and finally a high-purity SARS-COV-2 N protein with uniform conformation was obtained.

The kit of the present invention can detect the SARS-CoV-2 IgA antibody through the clinic earlier, and screen and deal with some patients with new coronary pneumonia early. The sensitivity and specificity of the test results of the kit for diagnosing pneumonia reached 99% and 96%, respectively, and it is an excellent test kit.

Example 3.

A kit for the detection of SARS-CoV-2 IgA antibodies for screening new coronavirus infections, especially for early screening of SARS-CoV-2 IgA antibodies.

This kit for screening for novel coronavirus infection includes:

1. SARS-CoV-2 magnetic bead coating (R1): Tris buffer containing magnetic bead coating of SARS-CoV-2 recombinant antigen, pH 7.2, containing 0.1% spit Temperature 20 (v/v), 5% bovine serum albumin (w/v), 0.5% Proclin300 (v/v); magnetic beads are used as the reaction solid phase carrier, and the antigen is used to bind the target antibody in the test object.

2. Anti-human IgA antibody acridinium ester marker (R2): 2-(N-morpholine) ethanesulfonic acid buffer solution of acridinium ester-labeled mouse anti-human IgA monoclonal antibody, containing 0.1% Tween 20 (v /v), 5% bovine serum albumin (w/v), 0.5% Proclin300 (v/v). Combining with the antigen-antibody complex, under alkaline conditions, the acridinium ester can emit light after being excited.

3. Reaction buffer (R3): phosphate buffer containing goat anti-human IgG antibody, containing 0.1% Tween 20 (v/v), 5% bovine serum albumin (w/v), 0.5% Proclin 300 ( v/v). Used to adsorb IgG in the sample to be tested to prevent interference.

4. SARS-CoV-2 IgA negative control: SARS-CoV-2 IgA negative serum.

The experiment also requires the substrate solution (aqueous solution containing NaOH) and the cleaning solution (phosphate buffer containing surfactant) as general reagents, which are not provided in this kit.

Example 4.

The RBD protein was used as a magnetic bead-coated antigen kit for detection, and clinical studies were conducted on 174 patients and 202 control populations, and the serum SARS-CoV-2 IgA antibody levels of patients with new coronary pneumonia and the control group were obtained. The results are shown in Figure 1. . As can be seen in Figure 1, the IgA level of patients with new coronary pneumonia is significantly higher than that of the control group. The trend of SARS-CoV-2 IgA and IgM antibodies with the course of the disease is shown in Figure 2. In Figure 2, it can be seen that the patient’s SARS-CoV-2 IgA turns positive earlier than IgM antibodies, on average 2(1-4) )sky. Therefore, clinical testing of SARS-CoV-2 and IgA antibodies at the time of patient consultation can diagnose some patients with new coronary pneumonia earlier.

The sensitivity and specificity of the test results of this kit for diagnosing new coronary pneumonia reached 99.43% and 96.04%, respectively. It is a test kit with excellent performance.

Example 5.

The N protein was used as a magnetic bead-coated antigen kit for testing. A clinical study was conducted on 100 patients with new coronary pneumonia and 100 controls. The test results showed that the positive rates of SARS-CoV-2 IgA in patients and controls were respectively 98.9% and 4.2%, there is a significant difference between the two groups.

The sensitivity and specificity of the test results of this kit for diagnosing new coronary pneumonia reached 98.9% and 95.8%, respectively. It is a test kit with excellent performance.

Example 6.

The kit described in any one of Examples 1 to 5 was used to detect 10 normal human sera and 10 new coronary pneumonia patients' sera by chemiluminescence immunoassay.

1. Before installing the machine, the magnetic bead coating (R1) needs to be gently turned upside down about 30 times to make the magnetic bead particles evenly dispersed. There is no need to continue mixing after loading the magnetic bead coating (R1) for the first time.

2. Select the reagent position on the instrument operation interface, scan the QR code on the reagent rack, and put the reagent rack into the reagent compartment.

3. Prepare the sample diluent according to the sample diluent instructions.

4. Prepare the cleaning fluid according to the cleaning fluid instructions.

5. Prepare the substrate solution A and the substrate solution B according to the instructions for the substrate solution for the automatic immunoassay system.

6. Put the negative control and positive control on the sample rack and push them into the sample bin. In the "sample application" interface, set the sample type as negative control and positive control respectively, and select SARS-CoV-2 IgA project, negative control and The positive control should be made 2 replicates, and click "Run" after confirming.

7. Detection: Put the sample in the sample rack (the sample size should be greater than 300μL), push it into the sample rack, edit the sample information on the operation interface, select the SARS-CoV-2 IgA project, and click "Run" after confirming. The system will perform the following operations:

The total incubation time is 15 minutes.

●Transfer the test substance (negative and positive control, sample) to the suction point.

●Load the reaction cup into the running channel.

●Respectively draw 30μL of the test substance into the reaction cup.

●Transport the reaction cup to the reagent compartment and add 50μL of reagent R1.

●After shaking and mixing, transport the reaction cup to the incubator and incubate at 37°C for 10 minutes.

●Transport the reaction cup to the washing channel for magnetic separation, wash the reaction mixture with washing liquid, and repeat the magnetic separation-washing 3 times.

●Transport the reaction cup to the reagent compartment again, and add 50μL of reagent R2.

●After shaking and mixing, transport the reaction cup to the incubator and incubate at 37°C for 5 minutes.

●Transfer the reaction cup to the substrate channel, add 100μL of substrate solution A, shake and mix.

●Transport the reaction cup to the detection channel, grab the reaction cup to the detection chamber, add 100μL of substrate solution B, and immediately detect the luminescence signal, and calculate the COI value of IgA.

●Grab the reaction cup to the waste bin.

8. Result judgment

When COI<0.8, the SARS-CoV-2 IgA in the sample is non-reactive;

When 0.8≤COI<1.0, the SARS-CoV-2 IgA in the sample is uncertain;

When COI≥1.0, the SARS-CoV-2 IgA in the sample is reactive.

The principle of chemiluminescence immunoassay is: the new coronavirus (SARS-CoV-2) IgA antibody detection kit (chemiluminescence immunoassay) adopts two-step indirect immunoassay. The sample to be tested is incubated with the magnetic bead coating. After magnetic separation and washing of unbound substances, the acridinium ester label is added and incubated together. After washing again, the substrate solution is added, and then the luminescence reaction of the acridinium ester is detected. If there is a novel coronavirus IgA antibody in the sample, it can form a magnetic bead coating-new coronavirus IgA antibody-acridinium ester marker complex. The luminescence intensity of the acridinium ester is positively correlated with the content of the novel coronavirus IgA antibody Relationship, the test result is expressed by the critical value index (COI).

The test results of this embodiment are shown in Table 1.

Table I

It can be seen that the kit of the present invention can detect 100% of patients with new coronary pneumonia, with accurate detection results and high sensitivity.

Example 7.

A kit for screening for novel coronavirus infection, containing,

Test card: consists of SARS-CoV-2 IgA test strip and plastic box; the test strip contains nitrocellulose membrane, sample pad, binding pad, absorbent paper, PVC board and other supports; the nitrocellulose membrane is coated with anti-human -IgA antibody and anti-mouse IgG polyclonal antibody, the binding pad contains colloidal gold-labeled SARS-CoV-2 antigen, and the method of obtaining the antigen can be the method in Example 2.

Tris hydrochloric acid buffer of the sample diluent: The pH of the Tris hydrochloric acid buffer is 7.1-7.4, containing 0.5-2% bovine serum albumin (w/v), 0.05-0.2% Tween 20 (v/v) , 0.25-1% Proclin300 (v/v). Preferably, it contains 1% bovine serum albumin (w/v), 0.1% Tween 20 (v/v), and 0.5% Proclin 300 (v/v).

The detection principle of the colloidal gold method is to use the principle of immunochromatographic technology to qualitatively detect SARS-CoV-2 IgA in human serum, plasma, and whole blood. When the sample contains SARS-CoV-2 IgA, it is combined with the SARS-CoV-2 CoV-2 antigen binds to form a reaction complex. Under the action of chromatography, the complex moves forward along the nitrocellulose membrane, and is captured by the anti-human-IgA antibody pre-coated on the detection zone (T) of the nitrocellulose membrane. The detection area (T) agglutinates to form a red reaction line visible to the naked eye. At this time, the result is positive; when the SARS-CoV-2 IgA in the sample is below the minimum detection limit, no red reaction line appears in the detection area (T). The result was negative.

The kit of Example 1 was used to detect 10 normal human sera and 10 new coronary pneumonia patients with the colloidal gold method.

1. Before testing, equilibrate the sample to be tested, testing reagents and other testing materials to room temperature, and testing should be performed at room temperature. Note: Room temperature refers to a temperature of 10°C to 30°C and a humidity of 45% to 75%.

2. Unscrew the dripper dripper, suck the sample to be tested with a pipette, add a drop of the sample to be tested to the dripper for dilution (ie 1:10 dilution), screw the dripper tightly, swing up and down, and mix.

3. Tear along the cut part of the aluminum foil bag, take out the test card and lay it flat on the table (note: do not touch the surface of the intermediate film of the test strip with your fingers).

4. Open the bottle cap and add 2 to 3 drops of the mixed sample on the test card.

5. Observe the display results of the two test strips for 15 minutes, and the display results after 30 minutes have no clinical significance.

6. Interpretation of test results

Positive: Two red lines, that is, one red reaction line appears in the detection area (T) and the quality control area (C).

Negative: A red line, that is, only a red reaction line appears in the quality control area (C).

Invalid: No red line appears in the quality control area (C).

The test results are shown in Table 2.

Table II

样本sample	SARS-CoV-2 IgASARS-CoV-2 IgA	样本sample	SARS-CoV-2 IgASARS-CoV-2 IgA
正常人1Normal person 1	阴性feminine	新冠肺炎1New Coronary Pneumonia 1	阳性positive
正常人2 Normal person 2	阴性feminine	新冠肺炎2 New Coronary Pneumonia 2	阳性positive
正常人3Normal person 3	阴性feminine	新冠肺炎3New Coronary Pneumonia 3	阳性positive
正常人4Normal person 4	阴性feminine	新冠肺炎4New Coronary Pneumonia 4	阳性positive
正常人5 Normal person 5	阴性feminine	新冠肺炎5 New Coronary Pneumonia 5	阳性positive
正常人6Normal person 6	阴性feminine	新冠肺炎6New Coronary Pneumonia 6	阳性positive
正常人7Normal 7	阴性feminine	新冠肺炎7New Coronary Pneumonia 7	阳性positive
正常人8Normal person 8	阴性feminine	新冠肺炎8New Coronary Pneumonia 8	阳性positive
正常人9Normal person 9	阴性feminine	新冠肺炎9New Coronary Pneumonia 9	阳性positive
正常人10Normal 10	阴性feminine	新冠肺炎10 New Coronary Pneumonia 10	阳性positive

Example 9.

The present invention also provides the use of a reagent for detecting SARS-CoV-2 IgA antibody in the preparation of the above-mentioned kit for screening new coronavirus infections and the preparation of the reagent for detecting SARS-CoV-2 IgA antibody for screening novel coronavirus infections. Use of coronavirus infection reagents. The invention can detect new coronaviruses, can be used to supplement the diagnosis of new coronary pneumonia at an earlier stage, and has the characteristics of accurate detection results.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that Modifications or equivalent replacements are made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

A kit for detecting SARS-CoV-2 IgA antibodies for screening new coronavirus infections.
The kit for screening for novel coronavirus infection according to claim 1, characterized in that it is used to screen for SARS-CoV-2 IgA antibody at an early stage.
The kit for screening for novel coronavirus infection according to claim 2, characterized in that it contains,

SARS-CoV-2 magnetic bead coating as R1: Tris buffer containing magnetic bead coating of SARS-CoV-2 recombinant antigen, pH 7.1-7.4;

As R2 anti-human IgA antibody acridinium ester label: 2-(N-morpholine) ethanesulfonic acid buffer containing acridinium ester-labeled mouse anti-human IgA monoclonal antibody.
The kit for screening new coronavirus infection according to claim 3, characterized in that: the SARS-CoV-2 recombinant antigen is a spike protein expressed in a suspension cultured human embryonic kidney cell line HEK293F, referred to as S For protein, the amino acid sequence of Gln321-Ser591 in the RBD domain was truncated for protein expression;

The protein expression process is: the SARS-CoV-2 S protein RBD gene is connected to human IgG1 Fc through a TEV restriction site sequence at the C-terminus, and then it is cloned into the mammalian expression vector pTT5 and Kozak is added to the translation initiation region The sequence was then transfected into suspension culture cells HEK293F, using interferon α-1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column for two purifications; the first purification To use Protein A column to obtain RBD-TEV-Fc fusion protein from the culture supernatant; the second purification is to use TEV enzyme with 6His tag to digest the fusion protein, and then pass the digested products through Protein A column in turn And nickel column to remove Fc, TEV enzymes, and the impurity protein bound to the Protein A column during the first purification, and finally obtain pure RBD protein in the flow-through.
The kit for screening novel coronavirus infection according to claim 3, characterized in that: the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as N protein, N protein The amino acid sequence at position 419 of Met1-Ala was truncated for expression;

The protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria. Add 0.5-2.0M sodium chloride to the solution to reduce non-specific binding of protein and nucleic acid, and then pass the lysate through a nickel column. After elution, add ammonium sulfate with a final concentration of 0.25-1.0M and stir at room temperature for 10-20 minutes to expose the protein Its hydrophobic core is further purified by a hydrophobic column, and then superdex-200 molecular sieve is used to remove polymers and other impurity proteins, and finally a high-purity N protein with uniform conformation is obtained.
The kit for screening new coronavirus infection according to any one of claims 3 to 5, characterized in that: the tris buffer contains 0.05-0.2% Tween 20 (v/ v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin300 (v/v);

2-(N-morpholine) ethanesulfonic acid buffer contains 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25-1% Proclin 300 (v/v) ).
The kit for screening for novel coronavirus infection according to claim 6, characterized in that: the kit further contains:

As the reaction buffer of R3: phosphate buffer containing goat anti-human IgG antibody, containing 0.05-0.2% Tween 20 (v/v), 2-8% bovine serum albumin (w/v), 0.25- 1% Proclin300(v/v);

SARS-CoV-2 IgA negative control: SARS-CoV-2 IgA negative serum;

SARS-CoV-2 IgA positive control: human-derived purified SARS-CoV-2 IgM antibody;

Negative and positive control information card: contains the luminescence value of the negative and positive control, which is used for correction during screening.
The kit for screening for novel coronavirus infection according to claim 1 or 2, characterized in that: the kit contains,

Test card: It is composed of SARS-CoV-2 IgA test strip and plastic box; the test strip contains nitrocellulose membrane, sample pad, bonding pad, absorbent paper, and PVC board support;

The nitrocellulose membrane is coated with anti-human-IgA antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled SARS-CoV-2 antigen.
The kit for screening novel coronavirus infection according to claim 7, characterized in that: the kit further contains Tris hydrochloric acid buffer as a sample diluent, and the pH of the Tris hydrochloric acid buffer is 7.1-7.4 , Containing 0.5-2% bovine serum albumin (w/v), 0.05-0.2% Tween 20 (v/v), 0.25-1% Proclin300 (v/v).
Use of a reagent for detecting SARS-CoV-2 IgA antibody in preparing the reagent or kit for screening new coronavirus infection according to any one of claims 1 to 9.