CN112213497A - polypeptide-ELISA kit for detecting novel coronavirus S protein idiotypic antibody - Google Patents
polypeptide-ELISA kit for detecting novel coronavirus S protein idiotypic antibody Download PDFInfo
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Abstract
The invention discloses a polypeptide-enzyme linked immunosorbent kit for detecting a unique specific antibody of novel coronavirus surface spinous process protein S. The kit comprises an ELISA plate coated with unique specific dominant linear B cell antigen polypeptide of SARS-CoV2 surface spinous protein S, sample diluent, negative control serum, positive control serum, horseradish peroxidase labeled antibody, concentrated washing liquid, enzyme substrate solution and stop solution. The kit has high specificity and good repeatability, can be simply and conveniently used for detecting the unique antibody of the novel coronavirus, reduces false positive, and can be used for large-scale serological detection and epidemiological investigation to evaluate the infection condition of the novel coronavirus.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a polypeptide-enzyme-linked immunosorbent assay (ELISA) kit for detecting a unique specific antibody of a novel coronavirus (SARS-CoV2) surface spike protein S.
Background
Coronaviruses are a single-stranded positive-strand RNA virus with a linear envelope genome, and a novel coronavirus SARS-CoV2 is the 7 th coronavirus which is known to infect humans and cause related diseases, and is currently in a global pandemic state. The first 6 coronaviruses causing human diseases include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, of which the first 4 cause mild self-limiting diseases with local epidemics mainly in the global range, accounting for 10% to 30% of upper respiratory infections in adults, and the last two cause severe respiratory syndrome with fatality rates of 10% and 36%, respectively. The surface spinous process protein (S) of coronaviruses is the most important surface structural protein in relation to the infectivity of the virus. The S protein comprises two functional subunits, S1 and S2, S1 comprises a receptor binding Region (RBD) responsible for recognizing and binding to cellular receptors, and S2 contains the essential elements required for the membrane fusion process, responsible for anchoring the S protein to the viral membrane and mediating fusion of the viral membrane to the host cell membrane. The S protein is therefore the major protein mediating coronavirus entry into the host cell and is also the major target for the host immune system to produce antibodies, including neutralizing antibodies, following infection.
The surface spinous process protein S (containing 1273 amino acids) of the novel coronavirus SARS-CoV2 is located on the surface of the virus particle in the form of trimer, and contains linear and space conformational B cell epitope sites/epitopes inducing the production of specific antibodies. The linear B cell antigenic determinant consists of continuous amino acid sequences and can be combined with a specific antibody induced by a complete antigen, so that the specific antibody can be captured by utilizing the antigenic epitopes to realize the detection purpose. Sequence alignment shows that SARS-CoV2 surface spinous process protein S has 30% to 76% identity and 46% to 86% similarity with the above 6 other coronavirus surface spinous process proteins S, respectively, so that cross-immune reaction may occur to affect its specificity when serological antibody detection using the S protein or its subunit such as Receptor Binding Domain (RBD) as a capture antigen. For example, the cross-immune response of antibody production induced by S protein or RBD between SARS-CoV2 and SARS-CoV with the closest homology has been demonstrated. Furthermore, predictive analysis of B-cell epitopes or T-cell epitopes also showed that most epitopes overlap or are similar between SARS-CoV2 and SARS-CoV. Therefore, the method and the kit for determining the unique specific B cell antigenic determinant of SARS-CoV2 and establishing the corresponding antibody detection method and kit to eliminate the interference of other coronavirus infection induced antibodies have important practical significance, namely, the new coronavirus antibody is distinguished from other coronavirus antibodies. The polypeptide-ELISA method and the kit can be used for serological screening of convalescent period (including recessive infection cases) and general population to confirm the existence and corresponding level of the unique antibody of the novel coronavirus, provide an effective means for epidemiological investigation of SARS-CoV2 serum antibody, and contribute to the investigation and prevention and control of the new crown epidemic situation which is widely spread at present.
Disclosure of Invention
The invention aims to provide a polypeptide-enzyme linked immunosorbent kit for detecting a unique specific antibody of novel coronavirus surface spinous process protein S aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme: a polypeptide-enzyme-linked immunosorbent (ELISA) kit for detecting a unique specific antibody of novel coronavirus surface spinous process protein S comprises an ELISA plate coated with a unique specific dominant linear B cell antigen polypeptide of SARS-CoV2 surface spinous process protein S, a sample diluent, a negative control serum, a positive control serum, a horseradish peroxidase-labeled antibody, a concentrated washing solution, an enzyme substrate solution and a stop solution. The main technical scheme is as follows: artificially synthesizing four sections of unique specific polypeptide sequences SQCVNLTTRTQLPPAYTNSFT, HVSGTNGTKRFD, LGVYYHKNNKSWMESEFRVYSS, ALHRSYLTPGDSSSGWTAG containing dominant linear B cell antigenic determinant sites in SARS-CoV2 surface spinous protein S, and diluting the polypeptide to 10 microgram/ml by using coating buffer solution; taking 1 ml of each polypeptide solution, uniformly mixing, coating the polypeptide solution in a 96-hole enzyme label plate in an amount of 100 microliter/hole, reacting at 4 ℃ overnight, and washing the plate for 3 times; adding 200 microliters of phosphate buffer solution containing bovine serum albumin with the mass concentration of 1% into each hole for sealing, incubating at 37 ℃ for 1 hour, washing the plate for 3 times, and airing for later use.
The plate was incubated with the serum sample to be tested for 1 hour at 37 ℃ to bind the specific antibodies in the serum sample. Negative control wells, positive control wells and blank wells were set simultaneously. Washing for 3 times, adding an HRP enzyme-labeled antibody diluted by 5000 times, incubating for 30 minutes at 37 ℃ after washing the plate for 3 times, adding an enzyme substrate o-phenylenediamine (OPD) for color development, patting and uniformly mixing, and incubating for 10 minutes at 37 ℃. Adding 50 microliters of stop solution into each hole, shaking for 10 seconds, uniformly mixing, and measuring the absorbance value of 450 nanometers by using an enzyme-labeling instrument. And judging the result by a P/N ratio method, wherein the sample absorbance value/negative control absorbance value is more than or equal to 2.1, and the sample absorbance value/negative control absorbance value is positive, otherwise, the sample absorbance value/negative control absorbance value is negative.
The polypeptide-ELISA kit for detecting the unique specific antibody of the novel coronavirus surface spinous process protein S can be used for detecting the specific antibody of the novel coronavirus.
The polypeptide-ELISA kit for detecting the unique specific antibody of the novel coronavirus surface spinous process protein S can be used for evaluating the infection condition of the novel coronavirus, and is applied to serology, epidemiology research and the like.
The invention has the beneficial effects that the invention can be used for detecting the specific antibody of the novel coronavirus and evaluating the infection condition of the novel coronavirus. The invention uses an artificial synthesis method to prepare the polypeptide containing the unique specific dominant linear B cell antigenic determinant site of the spinous process protein S on the surface of SARS-CoV2, has high purity and good specificity, directly aims at the corresponding antibody, avoids non-specific reaction caused by other substances, reduces false positive and improves the detection accuracy; the method has the advantages of high sensitivity, low cost, simple and convenient operation, high speed, strong specificity and the like, and can be widely used by basic units.
Drawings
FIG. 1 is a flow chart of the preparation process of the kit of the present invention;
FIG. 2 is a graph showing the prediction results of the linear epitope in S1A region of surface spinous process protein of novel coronavirus SARS-CoV2 (4 unique loci are pointed by arrows);
FIG. 3 is a diagram showing the result of polypeptide-ELISA for detecting the S-specific antibody against the surface spike protein S of the novel coronavirus SARS-CoV2 in serum by using the kit of the present invention.
Detailed Description
As shown in the attached figure 1, the polypeptide-ELISA kit for detecting the unique specific antibody of the novel coronavirus surface spinous process protein S is prepared by the following method:
(1) according to the characteristics of strong antigenicity, good surface property and strong hydrophilicity of the linear B cell antigenic determinant, a bioinformatics software is used for analyzing the amino acid sequence of the novel coronavirus surface spinous process protein S, and the dominant linear B cell antigenic determinant site (shown in figure 2) in the novel coronavirus surface spinous process protein S is predicted and determined; then comparing the new type coronavirus SARS-CoV2 with other 6 kinds of coronavirus known to infect human and cause related diseases, especially with the surface spinous process protein S amino acid sequence of SARS-CoV with the most similar homology, defining the unique specificity of dominant linear B cell epitope.
(2) Four segments of the novel coronavirus surface spinous process protein S are artificially synthesized to contain unique specific polypeptide sequences (SEQ ID NO. 1-SEQ ID NO.4) SQCVNLTTRTQLPPAYTNSFT, HVSGTNGTKRFD, LGVYYHKNNKSWMESEFRVYSS, ALHRSYLTPGDSSSGWTAG of dominant linear B cell antigenic determinant sites, and the purity is identified. Diluting each polypeptide to 10 microgram/ml, coating the polypeptide in 96-well enzyme label plate by using 100 microliter/well as capture antigen, reacting with recovery phase serum of patient infected with SARS-CoV2, and checking and determining antigenicity/immunogenicity and specificity of each polypeptide fragment by using normal human serum as control.
(3) The synthesized polypeptide is used as a capture antigen, the capture antigen is respectively diluted to 10 micrograms/ml by using a coating buffer solution, 1 ml of polypeptide solution is respectively mixed, the mixture is uniformly mixed and coated in a 96-hole enzyme label plate by the amount of 100 microliters/hole, and the plate is washed for 3 times at 4 ℃ after overnight. Adding 200 microliters of phosphate buffer solution containing bovine serum albumin with the mass concentration of 1% into each hole for sealing, incubating at 37 ℃ for 1 hour, washing the plate for 3 times, drying in the air, packaging the ELISA plate by using a sealing bag, and storing at 4 ℃ to obtain the ELISA plate coated with the antigen in advance.
(4) The negative control serum contained in the kit is a normal serum sample, and the positive control serum is a serum sample of a patient who is definitely infected with the virus and is verified by experiments.
(5) The preparation method of other reagents related to the kit comprises the following steps:
coating buffer (0.05 mol/l carbonate buffer, pH 9.6): 1000 ml of distilled water was added with 1.59 g of sodium carbonate (Na)2CO3) And 2.93 grams of sodium bicarbonate (NaHCO)3) And dissolving and mixing uniformly.
Phosphate buffer (0.01 mol/l PBS, pH 7.4): 1000 ml of distilled water was added with 8.0 g of sodium chloride (NaCl), 0.2 g of potassium dihydrogen phosphate (KH)2PO4) 2.9 g disodium hydrogen phosphate (Na)2HPO4·12H2O) and 0.2 g of potassium chloride (KCl) are dissolved and mixed evenly.
Sealing liquid: 1 g bovine serum albumin was added to 100 ml PBS wash, and the mixture was dissolved and mixed.
Sample diluent: bovine serum albumin and tween-20 were added to PBS until the mass concentration of bovine serum albumin was 1%, the volume concentration of tween-20 was 0.1%, and ph 7.4.
Horseradish peroxidase-labeled antibody: HRP-labeled antibody, commercially available, was used by diluting 5000-fold with sample dilution.
Concentrating the washing solution: PBS containing 1% Tween-20 by volume, pH7.4, was used by diluting 10-fold with PBS at the time of washing.
Enzyme substrate a solution: 5.1 g citric acid (C)6H8O7·H2O), 18.4 g disodium hydrogen phosphate (Na)2HPO4·12H2O), adding water to reach the volume of 1000 ml, and mixing uniformly.
Enzyme substrate B solution: 30% (volume concentration) hydrogen peroxide (H)2O2)1 ml.
Enzyme substrate C solution: 1 g of o-phenylenediamine (OPD) powder.
Stopping liquid: 2 mol/l sulfuric acid solution, 108.7 ml of 98% concentrated sulfuric acid by volume is added into 891.3 ml, mixed evenly and cooled.
(6) The specific method for detecting the sample by using the kit of the invention is as follows:
and taking out the enzyme label plate which is already coated with the linear B cell antigen polypeptide with unique specificity and superiority of the novel coronavirus surface spinous process protein S in the kit. Diluting each serum sample by 100 times with a sample diluent, adding into a prepared well of an enzyme-labeled plate with each well being 100 microliter, simultaneously setting a negative control well, a positive control well and a blank well, incubating for 1 hour at 37 ℃, and washing the plate for 3 times by using a washing solution. Diluting the enzyme-labeled antibody by 5000 times with a sample diluent, adding the diluted enzyme-labeled antibody into the wells, incubating for 30 minutes at 37 ℃ and washing the plate for 3 times. Preparing OPD color developing solution, weighing 20 mg of enzyme substrate C, dissolving in 50 ml of enzyme substrate A, adding 20 microliters of enzyme substrate B, uniformly mixing, adding 100 microliters of enzyme substrate B into each hole of an ELISA plate, standing in the dark for 10 minutes, adding 2 mol/L sulfuric acid into 50 microliters of each hole to terminate the reaction, adjusting the blank hole to zero, and measuring the absorbance value at 450 nm by using an ELISA reader. And judging the result by using a P/N ratio method, wherein the sample serum absorbance value/negative control absorbance value is positive when being more than or equal to 2.1, and the sample serum absorbance value/negative control absorbance value is negative when not being more than or equal to 2.1. The identification results are exemplified as follows: sample 1 was negative and sample 2 was positive (see figure 3).
The following examples are intended to illustrate the present invention, but not to limit the present invention, and any modifications and changes made within the spirit of the present invention and the scope of the claims fall within the scope of the present invention.
Example 1: prediction and determination of unique dominant linear B cell epitope in novel coronavirus surface spinous process protein S
According to the characteristics of strong antigenicity, good surface property and strong hydrophilicity of the linear B cell antigenic determinant, bioinformatics software is used for analyzing the amino acid sequence of the novel coronavirus surface spinous process protein S, and the dominant linear B cell antigenic determinant site in the novel coronavirus surface spinous process protein S is predicted and determined (figure 2). The unique specificity of the dominant linear B cell epitope is clarified by comparing the amino acid sequence of the novel coronavirus SARS-CoV2 with the amino acid sequence of other 6 kinds of coronavirus known to infect human and cause related diseases, especially the surface spinous process protein S of SARS-CoV with the closest homology. Artificially synthesizing each polypeptide, then respectively diluting the polypeptides to 10 micrograms/milliliter, coating the polypeptides in a 96-well enzyme label plate by taking the amount of 100 microliters/well as a capture antigen, reacting with recovery-phase serum of a patient clinically infected with SARS-CoV2, and verifying and determining the antigenicity/immunogenicity and specificity of each polypeptide fragment by taking normal human serum as a control.
Example 2: preparation of linear B cell antigen polypeptide with unique advantage of novel coronavirus surface spinous process protein S
Artificially synthesizing four sections of polypeptides (SEQ ID NO. 1-SEQ ID NO.4) containing unique dominant linear B cell antigenic determinant sites in novel coronavirus surface spinous process protein S: SQCVNLTTRTQLPPAYTNSFT, HVSGTNGTKRFD, LGVYYHKNNKSWMESEFRVYSS, ALHRSYLTPGDSSSGWTAG, the purity after purification is more than 95%.
Example 3: the kit is used for detecting the specific antibody of the surface spinous process protein S of the novel coronavirus in a sample
Sample incubation: taking out the ELISA plate coated with the unique specific dominant linear B cell antigen polypeptide of the SARS-CoV2 surface spinous process protein S in the kit. Diluting each sample by 100 times with a sample diluent, adding the diluted sample into a prepared well of an enzyme-labeled plate with each well being 100 microliters, simultaneously setting a negative control well, a positive control well and a blank well, incubating for 1 hour at 37 ℃, and washing the plate for 3 times by using a washing solution.
And (3) secondary antibody incubation: diluting the enzyme-labeled antibody by 5000 times with a sample diluent, adding the diluted enzyme-labeled antibody into the wells, incubating for 30 minutes at 37 ℃ and washing the plate for 3 times.
Color reading: preparing OPD color developing solution, weighing 20 mg of enzyme substrate C, dissolving in 50 ml of enzyme substrate A, adding 20 microliters of enzyme substrate B, uniformly mixing, adding 100 microliters of enzyme substrate B into each hole of an ELISA plate, standing in the dark for 10 minutes, adding 2 mol/L sulfuric acid into 50 microliters of each hole to terminate the reaction, adjusting the blank hole to zero, and measuring the absorbance value at 450 nm by using an ELISA reader.
And (4) judging a result: and (3) by a P/N ratio method, the sample serum absorbance value/negative control absorbance value is more than or equal to 2.1, the sample serum absorbance value/negative control absorbance value is positive, otherwise, the sample serum absorbance value/negative control absorbance value is negative. For example, the ELISA results in FIG. 3: sample 1 was negative and sample 2 was positive.
Sequence listing
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<120> polypeptide-ELISA kit for detecting novel coronavirus S protein idiotypic antibody
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Claims (6)
1. A polypeptide-enzyme-linked immunosorbent assay kit for detecting unique specific antibodies of novel coronavirus surface spinous process protein S is characterized by comprising an enzyme label plate coated with unique specific dominant linear B cell antigen polypeptide of the novel coronavirus surface spinous process protein S, a sample diluent, a negative control serum, a positive control serum, an antibody marked by horseradish peroxidase, a concentrated washing solution, an enzyme substrate solution and a stop solution; the ELISA plate coated with the linear B cell antigen polypeptide with unique specificity and superiority of the novel coronavirus surface spinous process protein S is prepared by the following method: artificially synthesizing four sections of unique specific polypeptide sequences containing dominant linear B cell antigenic determinant sites in SARS-CoV2 surface spinous process protein S, diluting each polypeptide to 10 microgram/ml by using coating buffer solution, wherein the sequences of the unique specific polypeptide sequences containing the dominant linear B cell antigenic determinant sites are shown in SEQ ID NO. 1-SEQ ID NO. 4; taking 1 ml of each polypeptide solution, uniformly mixing, coating the polypeptide solution in a 96-hole enzyme label plate in an amount of 100 microliter/hole, reacting at 4 ℃ overnight, and washing the plate for 3 times; adding 200 microliters of phosphate buffer solution containing bovine serum albumin with the mass concentration of 1% into each hole for sealing, incubating at 37 ℃ for 1 hour, washing the plate for 3 times, and airing for later use.
2. The kit of claim 1, wherein the sample diluent is prepared by adding bovine serum albumin and tween-20 to PBS, wherein the final concentration of bovine serum albumin is 1% by mass, the final concentration of tween-20 is 0.1% by volume, and ph 7.4.
3. The kit of claim 1, wherein the concentrated wash solution is a PBS solution containing tween-20 at a concentration of 1% by volume, ph 7.4.
4. The kit of claim 1, wherein the enzyme substrate solution comprises an enzyme substrate a solution, an enzyme substrate B solution, and an enzyme substrate C solution; the enzyme substrate A solution is prepared by adding water into 5.1 g of citric acid and 18.4 g of disodium hydrogen phosphate to reach the constant volume of 1000 ml and then uniformly mixing. The enzyme substrate B solution is hydrogen peroxide with the volume concentration of 30 percent. The enzyme substrate C solution is o-phenylenediamine.
5. The kit according to claim 1, wherein the stop solution is a sulfuric acid solution having a concentration of 2 mol/l.
6. The kit according to claim 1, wherein the phosphate buffer is prepared by adding 8.0 g of sodium chloride, 0.2 g of potassium dihydrogen phosphate, 2.9 g of disodium hydrogen phosphate and 0.2 g of potassium chloride to 1000 ml of distilled water, dissolving and mixing them, and adjusting the pH to 7.4.
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| CN113092780A (en) * | 2021-03-31 | 2021-07-09 | 武汉大学中南医院 | Marker for identifying neocoronary pneumonia and application thereof |
| CN115541880A (en) * | 2022-10-08 | 2022-12-30 | 云南大学 | Method, material and application for detecting new coronavirus antigen based on copper metal organic framework nano-enzyme laccase |
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