CN111638332B - Novel coronavirus IgA/IgM/IgG antibody ELISA detection kit - Google Patents

Abstract

The invention discloses a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit, which belongs to the technical field of biological detection, and comprises an ELISA plate, substrate liquid, sample diluent, positive control liquid, negative control liquid, concentrated washing liquid, enzyme conjugate and stop solution, wherein the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein, the enzyme conjugate is mouse anti-human IgA/IgM/IgG-HRP antibody diluent marked by horseradish peroxidase, and the detection is carried out through immune specificity reaction of antigen antibody so as to qualitatively judge whether novel coronavirus antibody exists in a test sample, and the kit has high sensitivity and high specificity; the kit detects through the immunospecific reaction of the antigen and the antibody, has the characteristics of high specificity and high accuracy, is simple and convenient in step, reasonable in design and suitable for popularization.

Description

Novel coronavirus IgA/IgM/IgG antibody ELISA detection kit

Technical Field

The invention relates to the technical field of biological detection, in particular to a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit.

Background

Common signs of novel coronaviruses (2019-nCoV) are fever, hypodynamia, dry cough and dyspnea gradually, and partial patients have slight symptoms and even have clinical symptom-free patients, and the novel coronaviruses have the characteristics of people, the common incubation period is 1-14 days, the incubation period is infectious, asymptomatic infectious people can become an infectious source, and the novel coronaviruses are mainly transmitted through respiratory tract droplets and close contact infection, so that the population is generally susceptible.

Currently, 2019-nCoV nucleic acid detection is a routine detection method and diagnostic basis for diagnosing COVID-19. However, the detection of nucleic acids has a false negative problem due to the influence of various factors such as sample collection and storage, virus-infected sites, RNA extraction methods, and quality problems of nucleic acid detection kits. With the research and development of 2019-nCoV IgM and IgG antibody immunodetection kit, the antibody detection can effectively make up the risk of missed detection of nucleic acid detection, and plays a significant role in diagnosis, treatment, prevention and control of COVID-19 in time.

At present, a method and a corresponding kit for detecting 2019-nCoV IgM/IgG antibodies have certain researches and reports, and certain products are marketed. The existing methods for detecting 2019-nCoV antibodies comprise a common ELISA method, a fluorescence immunoassay method, a radioimmunoassay method, a chemiluminescence method and the like, and have certain defects. The common ELISA kit uses prokaryotic expression recombinant protein or cultured whole virus as a coating antigen, but has lower sensitivity and specificity. Therefore, due to the special properties of 2019-nCov-SARS, development of a novel coronavirus ELISA detection kit with good specificity and high sensitivity is needed.

Disclosure of Invention

The invention aims to provide a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit, which solves the problems in the prior art, adopts His+N protein gene+glycine flexible peptide+S protein gene to be inserted into PET-28a carrier to construct prokaryotic expression carrier to obtain recombinant protein, coats a microplate and forms a kit of 2019-nCoV IgA/IgM/IgG antibody, besides the advantages of the common ELISA detection kit, the kit also overcomes the defect of low sensitivity caused by recombinant antigen, and improves the detection sensitivity and specificity.

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

the invention provides a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit, which comprises an ELISA plate, substrate liquid, sample diluent, positive control liquid, negative control liquid, concentrated washing liquid, enzyme conjugate and stop solution, wherein the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein;

the enzyme conjugate is a murine anti-human IgA/IgM/IgG working solution prepared by enzyme-labeled diluent according to working concentration.

Further, the enzyme conjugate is a horseradish peroxidase-labeled murine anti-human IgA/IgM/IgG-HRP antibody dilution.

Further, the novel coronavirus is coated after being blocked by bovine serum albumin with the mass concentration of 1-10%.

Further, the novel coronavirus was coated in an amount of 0.1. Mu.g/well.

Further, the sample diluent is phosphate buffer solution with pH of 7.2-7.4 containing 1-10% of bovine serum albumin by mass fraction.

Further, the concentrated washing solution is phosphate buffer solution with pH of 7.2-7.4 and containing 0.05% Tween-20 by volume fraction, and the stopping solution is sulfuric acid solution with concentration of 2 mol/L.

Further, the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1:30-1:100, and the absorbance value of the positive control solution is greater than 1.0;

the negative control serum is prepared by adding negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control serum is less than 0.1.

Further, the preparation method of the novel coronavirus S+N recombinant protein comprises the following steps:

(1) According to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, specific primers are designed for the amplification of the N and S protein genes respectively; inserting an N protein gene, glycine flexible peptide, an S protein gene and a His gene into a PET-28a vector through target fragment amplification, enzyme digestion, connection and transformation to obtain a plasmid containing N and S protein gene sequences, wherein the sequences are shown as SEQ ID No.1, the full length of the N protein gene is 1257bp, 419 amino acids are encoded, the full length of the S protein gene is 1479bp, and 493 amino acids are encoded;

(2) Carrying out protein expression on plasmids containing N and S protein gene sequences by using escherichia coli, carrying out ultrasound on thalli, dissolving sediment by using 50mM Tris+2M urea pH=12 overnight, centrifuging, and dialyzing supernatant to a Tris solution with pH=9.5 to obtain protein;

the dialysis procedure was as follows: loading NTA resin into a chromatographic column, adding target protein into the NTA chromatographic column, washing with 10 times of NTA volume of NTA-0Buffer at a flow rate of 15mL/h, collecting the penetrating part, eluting with 5 times of NTA volumes of NTA-20, NTA-40, NTA-60, NTA-100 and NTA-500 respectively at a flow rate of 15mL/h, and collecting the eluent;

(3) SDS/PAGE analysis is carried out to determine the distribution of target proteins in the eluent, and the target proteins are collected.

The invention discloses the following technical effects:

the novel coronavirus IgA/IgM/IgG antibody ELISA detection kit disclosed by the invention adopts His+N protein gene+glycine flexible peptide+S protein gene to be inserted into a PET-28a carrier to construct a prokaryotic expression carrier to obtain recombinant protein, and the prokaryotic expression carrier is coated with a micropore plate to form a 2019-nCoV IgA/IgM/IgG antibody kit.

The kit comprises an ELISA plate, substrate liquid, sample diluent, positive control liquid, negative control liquid, concentrated washing liquid, enzyme conjugate and stop solution, wherein the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein, the enzyme conjugate is mouse anti-human IgA/IgM/IgG-HRP antibody diluent marked by horseradish peroxidase, and the detection is carried out through the immunospecific reaction of antigen antibody so as to qualitatively judge whether the novel coronavirus antibody exists in a test sample, thereby having high sensitivity and high specificity.

The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit detects through the immunospecific reaction of antigen and antibody, has the characteristics of high specificity and high accuracy, is simple and convenient in step, reasonable in design and suitable for popularization.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an SDS/PAGE electrophoresis of the recombinant protein of example 1.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1

The preparation method of the novel coronavirus S+N recombinant protein comprises the following steps:

(1) According to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, specific primers are designed for the amplification of the N and S protein genes respectively; inserting an N protein gene, glycine flexible peptide, an S protein gene and a His gene into a PET-28a vector through target fragment amplification, enzyme digestion, connection and transformation to obtain a plasmid containing N and S protein gene sequences, wherein the sequences are shown as SEQ ID No.1, the full length of the N protein gene is 1257bp, 419 amino acids are encoded, the full length of the S protein gene is 1479bp, and 493 amino acids are encoded;

(2) Carrying out protein expression on plasmids containing N and S protein gene sequences by using escherichia coli, carrying out ultrasound on thalli, dissolving sediment by using 50mM Tris+2M urea pH=12 overnight, centrifuging, and dialyzing supernatant to a Tris solution with pH=9.5 to obtain protein;

the dialysis procedure was as follows: loading NTA resin into a chromatographic column, adding target protein into the NTA chromatographic column, washing with 10 times of NTA volume of NTA-0Buffer at a flow rate of 15mL/h, collecting the penetrating part, eluting with 5 times of NTA volumes of NTA-20, NTA-40, NTA-60, NTA-100 and NTA-500 respectively at a flow rate of 15mL/h, and collecting the eluent;

(3) SDS/PAGE analysis was performed to determine the distribution of the target protein in the eluate, as shown in FIG. 1, and the target protein was collected.

NAT buffer：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；

NAT buffer-20：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；20mmol/L Imidazole；

NAT buffer-40：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；40mmol/L Imidazole；

NAT buffer-60：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；60mmol/L Imidazole；

NAT buffer-100：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；100mmol/L Imidazole；

NAT buffer-500：20mmol/L Tris-Hcl，PH7.9，0.5mol/L NaCl，10％Glyerol；500mmol/L Imidazole。

Example 2

A novel coronavirus IgA/IgM/IgG antibody ELISA detection kit comprises an ELISA plate, substrate liquid, sample diluent, positive control liquid, negative control liquid, concentrated washing liquid, enzyme conjugate and stop solution, wherein the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein.

In this example, the enzyme conjugate is a horseradish peroxidase-labeled murine anti-human IgA/IgM/IgG-HRP antibody dilution.

In this example, the novel coronavirus was encapsulated with bovine serum albumin at a mass concentration of 5%.

In this example, the novel coronavirus was coated in an amount of 0.1. Mu.g/well.

In this example, the sample diluent is phosphate buffer at pH7.2-7.4 containing 5% by mass of bovine serum albumin.

In this example, the concentrated washing solution was phosphate buffer of pH7.2-7.4 containing 0.05% Tween-20 by volume fraction, and the stop solution was 2mol/L sulfuric acid solution.

In this example, the substrate solution contained 0.06wt% TMB hydrochloride in citric acid buffer, 0.001wt% sodium thiosulfate and 0.02wt% sodium carbonate.

In this embodiment, the positive control solution is prepared by adding positive serum into a sample diluent according to a ratio of 1:70, and the absorbance value of the positive control solution is greater than 1.0;

the negative control serum is prepared by adding negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control serum is less than 0.1.

Example 3

A novel coronavirus IgA/IgM/IgG antibody ELISA detection kit comprises an ELISA plate, substrate liquid, sample diluent, positive control liquid, negative control liquid, concentrated washing liquid, enzyme conjugate and stop solution, wherein the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein.

In this example, the enzyme conjugate is a horseradish peroxidase-labeled murine anti-human IgA/IgM/IgG-HRP antibody dilution.

In this example, the novel coronavirus was encapsulated with bovine serum albumin at a mass concentration of 1%.

In this example, the novel coronavirus was coated in an amount of 0.1. Mu.g/well.

In this example, the sample diluent is phosphate buffer at pH7.2-7.4 containing 10% by mass of bovine serum albumin.

In this example, the concentrated washing solution was phosphate buffer of pH7.2-7.4 containing 0.05% Tween-20 by volume fraction, and the stop solution was 2mol/L sulfuric acid solution.

In the embodiment, the positive control solution is prepared by adding positive serum into a sample diluent according to the ratio of 1:30, and the absorbance value of the positive control solution is greater than 1.0;

the negative control serum is prepared by adding negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control serum is less than 0.1.

Test example 1 detection test was performed using the kit of example 1

1. Reagent and sample preparation:

the reagent is rewarmed before use;

dilution of concentrated washing solution: 25mL of the concentrated washing solution is taken, 475mL of purified water is added for dilution, and shaking and mixing are carried out.

Enzyme conjugate dilution: 1mL of the sample dilution was taken, 10. Mu.L of the enzyme conjugate was added, and mixed by shaking.

Sample to be measured: the sample to be tested is subjected to 100-fold pre-dilution. And taking 2 mu L of a sample to be tested, adding 198 mu L of sample diluent, and shaking and uniformly mixing.

2. Detection method

The desired ELISA plate was removed, allowed to return to room temperature, 300. Mu.L of diluted wash solution was added to each well, and allowed to stand for 30s. Negative control 3 wells and positive control 2 wells were added, 100 μl per well. The diluted sample to be tested was added at 100. Mu.L per well.

And (3) sealing the plates by using sealing plates, and incubating for 30min at 37 ℃. The liquid was discarded, 300 μl of wash solution (1×) was added to each well using an automatic plate washer, repeated 5 times, and finally patted dry on a clean paper towel. All wells were added with 100. Mu.L of diluted murine anti-human IgA/IgM/IgG-HRP antibody. And (3) sealing the plates by using sealing plates, and incubating for 30min at 37 ℃. After 3 washes, 100. Mu.L of substrate solution was added to each well and incubated at 37℃for 10min. 100. Mu.L of stop solution was added to each well. In half an hour, dual wavelength detection is performed on an enzyme-labeled instrument, OD450 is read, and OD570 or OD630 is used as a reference wavelength. OD450-OD570 (or OD 630) is the calibration value.

3. Interpretation of

The average OD of the negative control was 2.1 times as high as the Cutoff value, e.g., the average OD of the negative control was less than 0.05 calculated as 0.05. A value greater than or equal to Cutoff may be judged positive, and a value less than Cutoff may be judged negative.

Test example 2 determination of the coating concentrations of antigen at different concentrations

1. Coating

Homemade s+n protein: 1350. Mu.l PBS+4. Mu.l to 5. Mu.g/ml; 400 μl+600 μl PBS was used to make 2 μg/ml;50 μl/well. Coating overnight at 4 ℃.

2.Blocking

Mu.l Blocking was added to each well and incubated with shaking at room temperature for 2h.

3. First antibody

IgA/IgM/IgG antibodies were: anti-N protein and anti-S protein: 225. Mu.l of AB+12.5. Mu.l of anti-N protein+12.5. Mu.l of anti-S protein were prepared 10X, 25. Mu.l+225. Mu.l of AB were prepared 100X, 25. Mu.l+225. Mu.l of AB were prepared 1000X, 100. Mu.l/well. Negtive was added to 100 μ lAB.

4. Secondary antibody (HRP mouse anti-human IgA/IgM/IgG)

850. Mu.l+0.34. Mu.l anti-human IgA+0.34. Mu.l anti-human IgM+0.34. Mu.l anti-human IgG.

The results of the detection are shown in Table 1.

TABLE 1 comparison of the coating amounts of different antigens

Test example 3 Performance test

182 clinical serum samples are detected together, wherein 134 negative samples are detected, the detection result is 130 negative samples, and the coincidence rate is 97% (130/134); the total number of positive samples is 48, wherein 35 positive samples of the diagnosed patient and 13 positive samples of the recovered patient are detected to be 46 minutes positive, and the coincidence rate is 95.8 percent (46/48), so that the product has strong precision and good stability.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Sequence listing

<110> Zhejiang province people Hospital

<120> novel coronavirus IgA/IgM/IgG antibody ELISA detection kit

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Claims (1)

1. The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit comprises an ELISA plate, a substrate solution, a sample diluent, a positive control solution, a negative control solution, a concentrated washing solution, an enzyme conjugate and a stop solution, and is characterized in that the ELISA plate is pre-coated with novel coronavirus S+N recombinant protein;

the enzyme conjugate is horseradish peroxidase marked mouse anti-human IgA/IgM/IgG-HRP antibody diluent; the novel coronavirus is coated after being sealed by bovine serum albumin with the mass concentration of 1-10%; the coating amount of the novel coronavirus is 0.1 mug/well; the sample diluent is phosphate buffer solution with the pH value of 7.2-7.4 and containing 1-10% of bovine serum albumin by mass fraction; the concentrated washing liquid is phosphate buffer solution with the pH value of 7.2-7.4 and containing 0.05% Tween-20 by volume fraction, and the stop solution is sulfuric acid solution with the concentration of 2 mol/L; the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1:30-1:100, and the absorbance value of the positive control solution is greater than 1.0; the negative control serum is prepared by adding negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control serum is less than 0.1;

the preparation method of the novel coronavirus S+N recombinant protein comprises the following steps:

(1) Designing specific primers for amplifying two genes of N and S proteins respectively; inserting an N protein gene, glycine flexible peptide, S protein gene and His gene into a PET-28a vector through target fragment amplification, enzyme digestion, connection and transformation to obtain a plasmid containing N and S protein gene sequences, wherein the sequences are shown as SEQ ID No.1, the full length of the N protein gene is 1257bp, 419 amino acids are encoded, the full length of the S protein gene is 1479bp, and 493 amino acids are encoded;

(2) Carrying out protein expression on plasmids containing N and S protein gene sequences by using escherichia coli, carrying out ultrasound on thalli, dissolving sediment by using 50mM Tris+2M urea pH=12 overnight, centrifuging, and dialyzing supernatant to a Tris solution with pH=9.5 to obtain protein;

the dialysis procedure was as follows: loading NTA resin into a chromatographic column, adding target protein into the NTA chromatographic column, washing with 10 times of NTA volume of NTA-0Buffer at a flow rate of 15mL/h, collecting the penetrating part, eluting with 5 times of NTA volumes of NTA-20, NTA-40, NTA-60, NTA-100 and NTA-500 respectively at a flow rate of 15mL/h, and collecting the eluent;

(3) SDS/PAGE analysis is carried out to determine the distribution of target proteins in the eluent, and the target proteins are collected.