CN111638332A - 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, 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, wherein the ELISA plate is pre-coated with novel coronavirus S + N recombinant protein, the enzyme conjugate is a horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent, detection is carried out through immune specific reaction of an antigen antibody, so that whether the novel coronavirus antibody exists in a test sample is qualitatively determined, and the kit has high sensitivity and high specificity; the kit provided by the invention is used for detecting through the immune specificity reaction of the antigen antibody, has the characteristics of high specificity and strong accuracy, is simple and convenient in steps, is reasonable in design, and is 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

The common signs of the novel coronavirus (2019-nCoV) are fever, hypodynamia, dry cough and dyspnea gradually, part of patients have slight symptoms, even patients without clinical symptoms exist, the novel coronavirus has the characteristic of people transmission, the incubation period is generally 1-14 days, the incubation period is infectious, asymptomatic infectors can also become infection sources, the novel coronavirus is mainly transmitted through respiratory droplets and close contact infection, and the population is generally susceptible.

At present, the 2019-nCoV nucleic acid detection is a conventional detection method and diagnosis basis for diagnosing COVID-19. However, the nucleic acid detection has a problem of false negative 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 the 2019-nCoV IgM and IgG antibody immunodetection kit, the antibody detection can effectively make up the risk of nucleic acid detection omission, and plays an important role in the timely diagnosis, control and prevention of COVID-19 in laboratory diagnosis.

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

Disclosure of Invention

The invention aims to provide a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit, which is used for solving the problems in the prior art, and adopts the technical scheme that His + N protein gene + glycine flexible peptide + S protein gene are inserted into a PET-28a carrier to construct a prokaryotic expression carrier to obtain recombinant protein, and the prokaryotic expression carrier is coated on a microporous plate to form a 2019-nCoVIgA/IgM/IgG antibody kit.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a novel coronavirus IgA/IgM/IgG antibody ELISA detection kit, which 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, wherein the ELISA plate is pre-coated with novel coronavirus S + N recombinant protein;

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

Further, the enzyme conjugate is horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent.

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

Further, the coating amount of the novel coronavirus was 0.1. mu.g/well.

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

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

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

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

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

(1) designing specific primers according to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, and respectively amplifying the N and S protein genes; inserting an N protein gene + glycine flexible peptide + S protein gene + 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 total length of the N protein gene is 1257bp, 419 amino acids are coded, the total length of the S protein gene is 1479bp, and 493 amino acids are coded;

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

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

(3) and (4) analyzing by SDS/PAGE to determine the distribution of the target protein in the eluent, and collecting the target protein.

The invention discloses the following technical effects:

the novel coronavirus IgA/IgM/IgG antibody ELISA detection kit provided by the invention is characterized in that His + N protein gene + glycine flexible peptide + S protein gene is inserted into a PET-28a vector to construct a prokaryotic expression vector to obtain recombinant protein, and a microporous plate is coated on the prokaryotic expression vector to form the 2019-nCoV IgA/IgM/IgG antibody kit.

The kit comprises an enzyme label 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, wherein the enzyme label plate is pre-coated with novel coronavirus S + N recombinant protein, the enzyme conjugate is horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent, detection is performed through immune specific reaction of an antigen antibody so as to qualitatively judge whether the novel coronavirus antibody exists in a test sample, and the kit has high sensitivity and high specificity.

The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit provided by the invention detects through the immune specificity reaction of the antigen antibody, has the characteristics of high specificity and strong 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 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 it is obvious for those skilled in the art to obtain other drawings without creative efforts.

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

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

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

(1) designing specific primers according to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, and respectively amplifying the N and S protein genes; inserting an N protein gene + glycine flexible peptide + S protein gene + 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 total length of the N protein gene is 1257bp, 419 amino acids are coded, the total length of the S protein gene is 1479bp, and 493 amino acids are coded;

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

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

(3) SDS/PAGE analysis is carried out to determine the distribution of the target protein in the eluent, and the target protein is collected as shown in figure 1.

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, 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, wherein the ELISA plate is pre-coated with novel coronavirus S + N recombinant protein.

In this example, the enzyme conjugate was a horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent.

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

In this example, the coating amount of the novel coronavirus was 0.1. mu.g/well.

In this example, the sample diluent is a phosphate buffer solution with a mass fraction of 5% bovine serum albumin and a ph of 7.2-7.4.

In this embodiment, the concentrated washing solution is a phosphate buffer solution with a volume fraction of 0.05% tween-20 and a ph of 7.2-7.4, and the stop solution is a 2mol/L sulfuric acid solution.

In this example, the substrate solution contained 0.06 wt% of TMB hydrochloride in a citric acid buffer, 0.001 wt% of sodium thiosulfate, and 0.02 wt% of 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 the negative serum into a sample diluent according to the proportion of 5 wt%, and the absorbance value of the negative control liquid is less than 0.1.

Example 3

A 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, wherein the ELISA plate is pre-coated with novel coronavirus S + N recombinant protein.

In this example, the enzyme conjugate was a horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent.

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

In this example, the coating amount of the novel coronavirus was 0.1. mu.g/well.

In this embodiment, the sample diluent is a phosphate buffer solution with a mass fraction of 10% bovine serum albumin and a ph of 7.2-7.4.

In this embodiment, the concentrated washing solution is a phosphate buffer solution with a volume fraction of 0.05% tween-20 and a ph of 7.2-7.4, and the stop solution is a 2mol/L sulfuric acid solution.

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

Test example 1 detection test was carried out using the kit of example 1

1. Reagent and sample preparation:

rewarming the reagent before use;

diluting a concentrated washing solution: 25mL of the concentrated washing solution was diluted with 475mL of purified water, and the mixture was shaken and mixed.

Enzyme conjugate dilution: 1mL of sample diluent is added with 10 μ L of enzyme conjugate, and the mixture is shaken and mixed evenly.

A sample to be detected: the sample to be tested is pre-diluted 100 times. And adding 198 mul of sample diluent into 2 mul of sample to be detected, and shaking and mixing uniformly.

2. Detection method

The desired microplate was removed, returned to room temperature, and 300. mu.L of diluted washing solution was added to each well and allowed to stand for 30 seconds. Add negative control 3 wells and positive control 2 wells, 100. mu.L per well. The diluted test sample was added at 100. mu.L per well.

Incubate plate-sealing with plate-sealing membrane at 37 ℃ for 30 min. Discard the liquid, use an automatic plate washer, add 300 μ L of wash solution per well (1 ×), repeat 5 times, and finally pat dry on clean paper towels. mu.L of diluted murine anti-human IgA/IgM/IgG-HRP antibody was added to all wells. Incubate plate-sealing with plate-sealing membrane at 37 ℃ for 30 min. After washing 3 times, 100. mu.L of substrate solution was added to each well and incubated at 37 ℃ for 10 min. Add 100. mu.L of stop buffer to each well. Within half an hour, two-wavelength detection was performed on a microplate reader, reading OD450, with OD570 or OD630 as the reference wavelength. OD450-OD570 (or OD630) are calibration values.

3. Interpretation

The Cutoff value was determined as 2.1 times the average OD of the negative control, as calculated by 0.05 when the average OD of the negative control was less than 0.05. A value greater than or equal to the Cutoff value can be judged as positive, and a value less than the Cutoff value can be judged as negative.

Test example 2 determination of coating concentration of antigen at various concentrations

1. Coating quilt

Home-made S + N protein: 1350. mu.l PBS + 4. mu.l to 5. mu.g/ml; 400 μ l +600 μ l PBS was prepared to 2 μ g/ml; 50 μ l/well. Coating was carried out overnight at 4 ℃.

2.Blocking

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

3. A primary antibody

IgA/IgM/IgG antibodies were: anti-N and anti-S proteins: mu.l AB + 12.5. mu.l anti-N protein + 12.5. mu.l anti-S protein 10X, 25. mu.l + 225. mu.l AB 100X, 25. mu.l + 225. mu.l AB 1000X, 100. mu.l/well. Negtive was added at 100. mu. lAB.

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

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 amount of different antigens coated

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 percent (130/134); the total number of the positive specimens is 48, wherein the detection results of 35 positive specimens of patients with confirmed diagnosis and 13 positive specimens of patients with rehabilitation are 46 positive, and the coincidence rate is 95.8 percent (46/48), which indicates that the product has strong precision and good stability.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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Claims (8)

1. A 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 a mouse anti-human IgA/IgM/IgG working solution prepared by using an enzyme-labeled diluent according to working concentration.

2. The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit according to claim 1, wherein said enzyme conjugate is horse radish peroxidase-labeled mouse anti-human IgA/IgM/IgG-HRP antibody diluent.

3. The kit for detecting IgA/IgM/IgG antibody ELISA according to claim 1, wherein said novel coronavirus is coated after blocking with bovine serum albumin at a mass concentration of 1-10%.

4. The kit for ELISA detection of IgA/IgM/IgG antibodies of the novel coronavirus according to claim 3, wherein the coating amount of the novel coronavirus is 0.1 μ g/well.

5. The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit according to claim 1, wherein said sample diluent is a phosphate buffer solution with a pH of 7.2-7.4 containing bovine serum albumin in a mass fraction of 1-10%.

6. The novel ELISA detection kit for IgA/IgM/IgG antibodies of coronavirus according to claim 1, wherein said concentrated washing solution is a phosphate buffer solution with pH7.2-7.4 containing 0.05% Tween-20 by volume fraction, and said stop solution is a 2mol/L sulfuric acid solution.

7. The novel coronavirus IgA/IgM/IgG antibody ELISA detection kit according to claim 1, wherein the positive control solution is prepared by adding positive serum into a sample diluent according to a ratio 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 the negative serum into a sample diluent according to the proportion of 5 wt%, and the absorbance value of the negative control liquid is less than 0.1.

8. The novel ELISA detection kit for IgA/IgM/IgG antibodies of coronavirus according to claim 1, wherein said preparation method for S + N recombinant protein of coronavirus comprises the following steps:

(1) designing specific primers according to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, and respectively amplifying the N and S protein genes; inserting an N protein gene + glycine flexible peptide + S protein gene + 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 total length of the N protein gene is 1257bp, 419 amino acids are coded, the total length of the S protein gene is 1479bp, and 493 amino acids are coded;

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

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

(3) and (4) analyzing by SDS/PAGE to determine the distribution of the target protein in the eluent, and collecting the target protein.

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