CN111337673A - Synthetic polypeptide composition for novel coronavirus immunodetection and application - Google Patents
Synthetic polypeptide composition for novel coronavirus immunodetection and application Download PDFInfo
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Abstract
The invention provides a synthetic polypeptide composition for immunoassay of novel coronavirus, which comprises four synthetic polypeptides; the synthetic polypeptide can react with an antibody IgG/IgM of a novel coronavirus (2019-nCoV), does not react with other lung infectious coronaviruses and the like, and does not react with normal human serum; the synthetic polypeptide provided by the invention is used for detecting a new coronavirus (2019-nCoV) IgM/IgG antibody in serum, the sensitivity can reach more than 90%, and the specificity can reach more than 99%.
Description
Technical Field
The invention belongs to the technical field of immunoassay detection, and particularly relates to a synthetic polypeptide composition for immunoassay of novel coronavirus and application thereof.
Background
The novel coronavirus (SARS-CoV-2) is a new strain of coronavirus which is found in human body in 2019, the coronavirus (Coronavirus, CoV) belongs to the order of nidoviridae, the family of coronaviridae, which is divided into α, β and gamma, α and β, the gamma is only pathogenic to mammals, the gamma is mainly caused to cause bird infection, and the CoV is mainly transmitted by directly contacting with secretion or by aerosol and spray, and has also evidence to show that the coronavirus can be transmitted by a fecal oral route.
To date, the human coronavirus (HCoV) responsible for human respiratory disease has reached 7 species: HCoV-229E, HCoV-OC 43, SARS-CoV, HCoV-NL 63, HCoV-HKU 1, MERS-CoV, and novel coronaviruses (2019), are important pathogens of human respiratory infections. Among them, the new type of coronavirus (2019-nCoV) is clinically manifested as fever, hypodynamia and other general symptoms accompanied by dry cough, dyspnea, etc., and can rapidly develop severe pneumonia, respiratory failure, acute respiratory distress syndrome, toxic shock, multiple organ failure, severe acid-base metabolic disorder and the like, even endanger life.
At present, the detection method of a clinical laboratory mainly depends on nucleic acid detection, which is capable of finding the virus evidence of infection, but the nucleic acid detection needs to be carried out in a laboratory with conditions and qualification, has the defects of long detection time, multiple steps, high requirements on fields, equipment and professional detection personnel and the like, is difficult to carry out on a large scale, and has higher cost.
Once a human body is infected with a virus, the immune system within the body immediately begins to react, producing antibodies. Generally, the IgM antibody index rapidly increases 3 to 7 days in the early stage of infection, gradually decreases after reaching a peak, and then IgG antibodies exist in the human body for a long period.
Due to methodological limitations, false positives and false negatives can occur in nucleic acid detection, resulting in misdiagnosis and missed diagnosis. When the IgM antibody is analyzed by adopting a chemiluminescence immunoassay method, a mild patient, a suspected patient and a person in close contact can be rapidly examined in a large scale in a short time, and the cost is reduced by about two thirds. Once a human body is infected with a virus, the immune system within the body immediately begins to react, producing antibodies. Generally, the IgM antibody index rapidly increases in3 to 7 days from the initial stage of infection, and gradually decreases after reaching a peak.
The antibody determination can also be used for epidemiological investigation to know how many individuals in the population have been exposed to the corresponding novel virus, which is helpful for the overall judgment and control of epidemic situations.
In the antibody detection, how to select an antigen which reacts with an antibody IgG/IgM of a novel coronavirus (2019-nCoV), does not react with other lung infectious coronaviruses and the like, does not react with normal human serum, and has high specificity is a problem to be solved urgently.
Disclosure of Invention
In view of the above, the present invention is directed to a synthetic polypeptide composition for immunoassay of novel coronavirus and its application, wherein the synthetic polypeptide composition can satisfy the specificity and sensitivity of antibody detection, and has simple synthesis, only needs to use the chemical synthesis technology in the prior art, does not need to use biological recombinant expression for preparation, and has low cost.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a synthetic polypeptide composition for immunoassay of a novel coronavirus, comprising four synthetic polypeptides, the amino acid sequences of the four synthetic polypeptides are shown in Table 1:
TABLE 1 amino acid sequence of the synthetic polypeptides
The invention also provides an application of the synthetic polypeptide composition in a novel coronavirus (2019-nCoV) immunodetection reagent.
Preferably, in the synthetic polypeptide composition, the mass ratio of the four synthetic polypeptides is (0.5-2): (0.5-2): (0.5-2): (0.5-2).
Preferably, in the synthetic polypeptide composition, the mass ratio of the four synthetic polypeptides is 1:1:1: 1.
Preferably, the four synthetic polypeptides are biotinylated or FITC-linked Spike polypeptide, nucleocapsid polypeptide, respectively.
Preferably, the immunoassay comprises one of chemiluminescence immunoassay and enzyme-linked immunoassay.
Preferably, the detection is carried out by combining total antibodies or IgM or IgG antibodies or IgM and IgG antibodies of the novel coronavirus (2019-nCoV).
The kit is prepared by optimally combining biotinylated or FITC-modified Spike polypeptide (structural protein surface glycoprotein polypeptide), nucleocapsid polypeptide (structural protein nucleocapsid phosphoprotein polypeptide), streptavidin coated plate/magnetic particle or anti-FITC antibody coated plate/magnetic particle, labeled mouse anti-human IgM/IgG monoclonal antibody, chemiluminescence method substrate liquid and the like, and can detect novel coronavirus (2019-nCoV) IgM/IgG antibody in serum.
Compared with the prior art, the synthetic polypeptide composition for the novel coronavirus immunodetection and the application thereof have the following advantages:
the synthetic polypeptide can react with an antibody IgG/IgM of a novel coronavirus (2019-nCoV), does not react with other lung infectious coronaviruses and the like, and does not react with normal human serum; the synthetic polypeptide provided by the invention is used for detecting a new coronavirus (2019-nCoV) IgM/IgG antibody in serum, the sensitivity can reach more than 90%, and the specificity can reach more than 99%.
Drawings
FIG. 1 is a graph of ROC curve analysis of the first embodiment;
FIG. 2 is a graph of ROC curve analysis for example two;
FIG. 3 is a ROC curve analysis chart of comparative example one.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
Example one
Preparing magnetic particles-anti-FITC antibody comprises the following steps of taking 200 mL0.2M hydroxyethyl piperazine ethiosulfonic acid (HEPES) buffer solution, adding 60mg of magnetic particles with amino or carboxyl attached to the surface, stirring at room temperature for 30min, then adding 21mg of anti-FITC antibody, then adding EDC with the concentration of 10mg/mL, reacting at room temperature in a dark place for 1h, washing with 0.01M PBS buffer solution for 3 times, and finally dissolving with 0.01M PBS to 1L; the magnetic particles are ferroferric oxide with amino or carboxyl active groups wrapped on the surfaces, and the particle size is 1-2 mu m.
The preparation of the FITC labeled novel coronavirus antigen comprises the following steps,
1) mixing FITC and the novel coronavirus synthetic polypeptide according to a molar ratio of 1:1, and reacting for 12 hours at 37 ℃ in the absence of light.
2) Dialyzing the labeling solution obtained in step 1) with 0.01M PBS at 2-8 deg.C for 24h, adding equal volume of glycerol, and storing at-20 deg.C.
Wherein, the novel coronavirus antigen is formed by mixing 4 synthetic polypeptides according to the same mass ratio; the amino acid sequences of the 4 synthetic polypeptides are shown in table 1.
It should be noted that the FITC-labeled novel coronavirus synthetic polypeptide used in the present application may be synthesized by the above method, or may be synthesized by other conventional methods, as long as it is possible to link FITC to the novel coronavirus synthetic polypeptide.
The 20-time concentrated washing solution comprises 58g/L of disodium hydrogen phosphate, 5.92g/L of sodium dihydrogen phosphate, 180g/L of NaCl, 10mL/LTween-20 and 2% of Proclin300 by mass concentration.
Negative control and positive control reagent, wherein the negative control is prepared by the following steps: adding buffer solution containing bovine serum albumin into ProClin with the volume concentration of 1%TM300. Subpackaging, labeling and storing at 2-8 ℃. The formula of the buffer solution is as follows: 10g/L BSA, 0.02mol/L PBS (pH 7-8, expiration date: 14 months)
Positive control, preparation method: mixing 5 parts of positive human serum, performing heat inactivation at 56 ℃ for 45 minutes, diluting the mixture to a working concentration by using a buffer solution containing bovine serum albumin, adding ProClin TM300, subpackaging, labeling and storing at 2-8 ℃. The formula of the buffer solution is as follows: 10g/L BSA, 0.02mol/L PBS (pH 7-8, effective period: 14 months).
The preparation method of the alkaline phosphatase marked mouse anti-human IgM monoclonal antibody comprises the following steps:
A. alkaline phosphatase (ALP) activation
1) Preparing 10mg/mL ALP solution;
2) preparing 12.8 mg/mL sodium periodate NaIO4A solution;
3) preparing a solution prepared in the step 1) and the step 2) according to a volume ratio of 1:1, uniformly mixing, and reacting for 30min at 2-8 ℃ in a dark place;
4) preparing a 20 mu L/mL ethylene glycol aqueous solution, mixing the ethylene glycol aqueous solution with the solution obtained in the step 3) in the same volume, reacting at normal temperature in a dark place for 30min, completing activation, and storing at-20 ℃ (the storage time is not more than 3 months);
B. alkaline phosphatase labeled mouse anti-human IgM monoclonal antibody
1) Putting the raw materials to be marked into a dialysis bag, and dialyzing for 30min by using 0.05M carbonate buffer solution with pH9.6;
2) mixing the labeled raw material and activated ALP at a mass ratio of 1:3, and dialyzing with 0.05M carbonate buffer solution at 2-8 deg.C for 24h, wherein the solution is changed for 2-3 times;
3) NaBH with the concentration of 2mg/mL is prepared4Aqueous solution of NaBH prepared by adding 80 μ L to 1mgALP4Mixing the aqueous solutions in proportion, and reacting for 2 hours at 2-8 ℃ in a dark place;
4) dialyzing the labeling solution obtained in step 3) with 0.01 PBS at 2-8 deg.C for 24h, adding equal volume of glycerol, and storing at-20 deg.C.
The diluent is prepared by adding 6g Tris, 1.36g ZnCl into 1L process water2，9.6gMgCl2Stirring until the mixture is completely dissolved; then adding 2.1g of polymerized BSA, and stirring until the polymerized BSA is completely dissolved; 1.1mL of ProClin was addedTM300, stirring for 30 minutes. Adjusting the pH value to be within the range of 8.0 +/-0.2 by using 6M HCl or 2M NaOH.
The chemiluminescence substrate is prepared by the following steps of measuring 900mL of purified water; 0.25g of AMPPD and 0.05g of Na were added to the reaction mixture, respectively2SO35g of SDS (sodium dodecyl sulfate) and 6g of Tris, and stirring until complete dissolution; then 0.05mL of tween-20 and 1mL of Proclin were addedTM300, constant volume to 1L. Adjusting the pH value to 9.0, and storing at 2-8 ℃.
Diluting a FITC-labeled novel coronavirus recombinant antigen and an alkaline phosphatase-labeled mouse anti-human IgM monoclonal antibody with a diluent until the working concentration is 0.2 mu g/mL; or the related reagent can be directly diluted to working concentration after being prepared, and can be directly used without dilution during operation.
And (3) sample analysis process:
1. and (3) balancing the kit to be detected for 30 minutes at room temperature (18-25 ℃).
2. Preparing a washing liquid: the concentrated washings were diluted 1:20 with distilled water (1 mL washings plus 19mL distilled water). If the concentrated washing solution has crystals, the concentrated washing solution can be placed at room temperature or 37 ℃ for dilution after the crystals are dissolved.
According to the above research, the reaction steps of the kit are determined as follows:
1. sample treatment: and adding 20 mu L of sample into 1mL of physiological saline, uniformly mixing for 5 seconds by using a vortex mixer, and standing for 15 minutes to start the experiment.
2. And taking out a proper amount of reaction tubes according to the experimental requirements. Set up 2 tubes for negative and positive controls. Each tube was first filled with 50. mu.L of LFITC-labeled novel coronavirus antigen (synthetic polypeptide), 75. mu.L of the treated sample or negative and positive controls, and then 50. mu.L of anti-FITC antibody-labeled magnetic particles were added and reacted at 37 ℃ for 20 minutes.
3. And (5) magnetic separation and cleaning.
4. 75 μ L of alkaline phosphatase-labeled mouse anti-human IgM monoclonal antibody was added to each tube.
5. The reaction was carried out at 37 ℃ for 15 minutes.
6. And (5) magnetic separation and cleaning.
7. Add chemiluminescent substrate 200. mu.L per tube, detect in dark and read 5 seconds for reaction.
The test effect of the kit prepared in this example was evaluated as follows:
the clinical test of the product takes the definite disease diagnosis/elimination standard of 'novel coronavirus pneumonia diagnosis and treatment plan' as comparison, 684 cases are selected, wherein 282 cases are diagnosed, and 402 cases are eliminated.
The following instruments were used for the tests: an immunoassay analyzer axced 260 by chemiluminescence; product registration number-jin machinery standard 20182400046;
1. stability of
1.1 design requirements: the kit is placed at 37 +/-1 ℃ for 7 days, and the appearance, the negative reference product compliance rate, the positive reference product compliance rate, the lowest detection limit and the precision detection result all meet the design requirements.
1.2 test methods: the kit is stored at 37 ℃ for 7 days and then taken out, and a reference substance is detected.
Table 2 example a stability test result
Table 3 example a stability specific assay data
2. Precision degree
2.1 design requirements
2.1.1 in-batch precision: the detection of 3 different levels of precision reference products in the reference products should meet the following requirements
2.1.1.1 precision reference N: negative detection rate should be 100% (n = 20);
2.1.1.2 precision reference L: the positive detection rate is more than or equal to 90 percent (n = 20);
2.1.1.3 precision reference CV: the positive detection rate is 100 percent, and CV is less than or equal to 10 percent (n = 20).
2.1.2 batch-to-batch precision: and detecting the positive quality control product with the precision in the reference product, wherein the positive detection rate is 100 percent, and the CV is less than or equal to 15 percent.
2.2 test methods
2.2.1 in-batch precision: detecting the precision reference substance, repeating the detection for 20 times at each level, and calculating the negative detection rate and the positive detection rate. And (3) calculating a Coefficient of Variation (CV) according to the formula (2) according to the average value () and the Standard Deviation (SD) of the positive quality control product determination result (S/CO), wherein the result meets the requirement of 2.1.1.
Coefficient of variation (CV%) ═ SD/mean × 100% … … … … equation (2)
2.2.2 batch to batch precision: and (3) detecting the precision reference product CV in the reference product by using three batches of kits, testing 20 tubes in each batch, and calculating the positive detection rate. And simultaneously calculating the average value and the Standard Deviation (SD) of 60 detection results, and calculating the Coefficient of Variation (CV) according to a formula (2), wherein the result meets the requirement of 2.1.2.
2.3 results of measurement of precision reference
Table 4 example a precision test data
3. Sensitivity and specificity
TABLE 5 example partial test results excluding 2019-nCoV infection
TABLE 6 example-partial test results for confirmed 2019-nCoV infection
The results of the ROC curve analysis using the SPSS software are shown in the following table, and the ROC curve analysis is shown in fig. 1.
TABLE 7 area under the curve of the example
The S/CO of the sample is more than or equal to 1, and the detection result is judged to be positive; the sample S/CO is less than 1, and the detection result is judged to be negative. Wherein the S/CO value is the luminescence value/cutoff value of the detection sample. And determining the sensitivity and specificity of the kit under the condition of different cutoff values through an ROC curve, and screening out the optimal cutoff value.
The ROC curve analysis result shows that the area under the curve is 0.963, which indicates that the kit has higher accuracy in clinical diagnosis.
Example two
Preparing magnetic particles-anti-FITC antibody comprises the following steps of taking 100 mL0.1M morpholine ethanesulfonic acid (MES) buffer solution, adding 20mg of magnetic particles with amino or carboxyl groups attached to the surfaces, stirring at room temperature for 40min, then adding 7mg of anti-FITC antibody, then adding EDC with the concentration of 8mg/mL, reacting at 2-8 ℃ for 1h, washing with 0.01M PBS buffer solution for 3 times, and finally dissolving with 0.01 MPBS to 1L; the magnetic particles are ferroferric oxide with amino or carboxyl active groups wrapped on the surfaces, and the particle size is 1-2 mu m.
The preparation of the FITC labeled novel coronavirus antigen comprises the following steps,
1) mixing FITC and the novel coronavirus synthetic polypeptide according to a molar ratio of 1:1, and reacting for 12 hours at 37 ℃ in the absence of light.
2) Dialyzing the labeling solution obtained in step 1) with 0.01M PBS at 2-8 deg.C for 24h, adding equal volume of glycerol, and storing at-20 deg.C.
Wherein, the novel coronavirus antigen is formed by mixing 4 synthetic polypeptides according to the same mass ratio; the amino acid sequences of the 4 synthetic polypeptides are shown in table 1.
It should be noted that the FITC-labeled novel coronavirus synthetic polypeptide used in the present application may be synthesized by the above method, or may be synthesized by other conventional methods, as long as it is possible to link FITC to the novel coronavirus synthetic polypeptide.
The 20-time concentrated washing solution comprises 58g/L of disodium hydrogen phosphate, 5.92g/L of sodium dihydrogen phosphate, 180g/L of NaCl, 10mL/LTween-20 and 2% of Proclin300 by mass concentration.
Negative control and positive control reagent, wherein the negative control is prepared by the following steps: adding bovine serum albuminAdding ProClin with the volume concentration of 1% into the flushing liquidTM300. Subpackaging, labeling and storing at 2-8 ℃. The formula of the buffer solution is as follows: 10g/L BSA, 0.02mol/L PBS (pH 7-8, expiration date: 14 months)
Positive control, preparation method: mixing 5 parts of positive human serum, performing heat inactivation at 56 ℃ for 45 minutes, diluting the mixture to a working concentration by using a buffer solution containing bovine serum albumin, adding ProClin TM300, subpackaging, labeling and storing at 2-8 ℃. The formula of the buffer solution is as follows: 10g/L BSA, 0.02mol/L PBS (pH 7-8, effective period: 14 months).
The preparation method of the alkaline phosphatase labeled mouse anti-human IgG monoclonal antibody comprises the following steps:
A. alkaline phosphatase (ALP) activation
1) Preparing 10mg/mL ALP solution;
2) 12.8 mg/mL sodium periodate NaIO is prepared4A solution;
3) preparing a solution prepared in the step 1) and the step 2) according to a volume ratio of 1:1, uniformly mixing, and reacting for 30min at 2-8 ℃ in a dark place;
4) preparing a 20 mu L/mL ethylene glycol aqueous solution, mixing the ethylene glycol aqueous solution with the solution obtained in the step 3) in the same volume, reacting at normal temperature in the dark for 20min, completing activation, and storing at-20 ℃ (the storage time is not more than 3 months);
B. alkaline phosphatase-labeled mouse anti-human IgG monoclonal antibody
1) Putting the raw materials to be marked into a dialysis bag, and dialyzing for 30min by using 0.05M carbonate buffer solution with pH9.6;
2) mixing the labeled raw material and activated ALP at a mass ratio of 1:3, and dialyzing with 0.05M carbonate buffer solution at 2-8 deg.C for 24h, wherein the solution is changed for 2-3 times;
3) NaBH with the concentration of 2mg/mL is prepared4Aqueous solution of NaBH prepared by adding 80 μ L to 1mgALP4Mixing the aqueous solutions in proportion, and reacting for 2 hours at 2-8 ℃ in a dark place;
4) dialyzing the labeling solution obtained in step 3) with 0.01 PBS at 2-8 deg.C for 24h, adding equal volume of glycerol, and storing at-20 deg.C.
The diluent is prepared by adding 12.684g Tris, 18.396g NaCl into 1.6L process water, and stirring until the solution is completely dissolved; then adding 2.1g of CaseinNa and stirring until the CaseinNa is completely dissolved; 0.2331g of MgCl2 is added and stirred until the mixture is completely dissolved; then, 2.1mL ProClin TM300 was added and the mixture was stirred for 30 minutes. The volume is adjusted to 2L by purified water, the pH value is measured by a pH meter, and the pH value is adjusted to be within 7.4 +/-0.2 by 6M HCl or 2M NaOH.
The chemiluminescent substrate comprises 0.25g/L AMPPD, 0.05g/L Na2SO35g/L SDS (sodium dodecyl sulfate), 6g/L Tris; 0.05mL/L Tween-20 and 1mL ProclinTM300, pH 9.0.
Diluting a FITC-labeled novel coronavirus recombinant antigen and an alkaline phosphatase-labeled mouse anti-human IgG monoclonal antibody to a working concentration of 0.2 mu g/mL by using a diluent; or the related reagent can be directly diluted to working concentration after being prepared, and can be directly used without dilution during operation.
And (3) sample analysis process:
1. and (3) balancing the kit to be detected for 30 minutes at room temperature (18-25 ℃).
2. Preparing a washing liquid: the 20-fold concentrated washings were diluted 1:20 with distilled water (1 mL of washings plus 19mL of distilled water). If the concentrated washing solution has crystals, the concentrated washing solution can be placed at room temperature or 37 ℃ for dilution after the crystals are dissolved.
According to the above research, the reaction steps of the kit are determined as follows:
1. sample treatment: and adding 20 mu L of sample into 1mL of physiological saline, uniformly mixing for 5 seconds by using a vortex mixer, and standing for 15 minutes to start the experiment.
2. And taking out a proper amount of reaction tubes according to the experimental requirements. Set up 2 tubes for negative and positive controls. Each tube was first filled with 50. mu.L of LFITC-labeled novel coronavirus antigen (synthetic polypeptide), 75. mu.L of the treated sample or negative and positive controls, and then 50. mu.L of anti-FITC antibody-labeled magnetic particles were added and reacted at 37 ℃ for 20 minutes.
3. And (5) magnetic separation and cleaning.
4. 75 μ L of alkaline phosphatase-labeled mouse anti-human IgG monoclonal antibody was added to each tube.
5. The reaction was carried out at 37 ℃ for 15 minutes.
6. And (5) magnetic separation and cleaning.
7. Add chemiluminescent substrate solution 200. mu.L per tube, detect in dark, and read 5 seconds after reaction.
The test effect of the kit prepared in this example was evaluated as follows:
the clinical test of the product takes the definite disease diagnosis/elimination standard of 'novel coronavirus pneumonia diagnosis and treatment plan' as comparison, 684 cases are selected, wherein 282 cases are diagnosed, and 402 cases are eliminated.
The test effect of the kit prepared in this example was evaluated as follows:
the following instruments were used for the tests: an immunoassay analyzer axced 260 by chemiluminescence; product registration number-jin machinery standard 20182400046;
1. stability of
1.1 design requirements: the kit is placed at 37 +/-1 ℃ for 7 days, and the appearance, the negative reference product compliance rate, the positive reference product compliance rate, the lowest detection limit and the precision detection result all meet the design requirements.
1.2 test methods: the kit is stored at 37 ℃ for 7 days and then taken out, and a reference substance is detected.
1.3 test results
Table 8 example two stability test results
TABLE 9 example two stability specific assay data
2. Precision degree
2.1 design requirements
2.1.1 in-batch precision: the detection of 3 different levels of precision reference products in the reference products should meet the following requirements
2.1.1.1 precision reference N: negative detection rate should be 100% (n = 20);
2.1.1.2 precision reference L: the positive detection rate is more than or equal to 90 percent (n = 20);
2.1.1.3 precision reference CV: the positive detection rate is 100 percent, and CV is less than or equal to 10 percent (n = 20).
2.1.2 batch-to-batch precision: and detecting the CV of the precision reference product in the reference product, wherein the positive detection rate is 100 percent, and the CV is less than or equal to 15 percent.
2.2 test methods
2.2.1 in-batch precision: detecting the precision reference substance, repeating the detection for 20 times at each level, and calculating the negative detection rate and the positive detection rate. And (3) calculating the Coefficient of Variation (CV) according to the formula (2) according to the average value () and the Standard Deviation (SD) of the measurement result (S/CO), wherein the result meets the requirement of 2.1.1.
Coefficient of variation (CV%) ═ SD/mean × 100% … … … … equation (2)
2.2.2 batch-to-batch precision: and (3) detecting the precision reference product CV in the reference product by using three batches of kits, testing 20 tubes in each batch, and calculating the positive detection rate. And simultaneously calculating the average value and the Standard Deviation (SD) of 60 detection results, and calculating the Coefficient of Variation (CV) according to a formula (2), wherein the result meets the requirement of 2.1.2.
2.3 precision reference measurement results
TABLE 10 results of secondary precision measurements of examples
3. Sensitivity and specificity
TABLE 11 example two partial test results excluding 2019-nCoV infection
TABLE 12 partial test results for two confirmed diagnoses of 2019-nCoV infection in the examples
The results of the ROC curve analysis using the SPSS software are shown in the following table, and the ROC curve analysis is shown in fig. 2.
TABLE 13 area under the second curve of the example
The S/CO of the sample is more than or equal to 1, and the detection result is judged to be positive; the sample S/CO is less than 1, and the detection result is judged to be negative. Wherein the S/CO value is the luminescence value/cutoff value of the detection sample. And determining the sensitivity and specificity of the kit under the condition of different cutoff values through an ROC curve, and screening out the optimal cutoff value.
The ROC curve analysis result shows that the area under the curve is 0.984, which indicates that the kit has higher accuracy in clinical diagnosis.
Comparative example 1
Changing the novel coronavirus antigen to the novel coronavirus recombinant antigen in the first embodiment; the amino acid sequence of the recombinant antigen is shown in the following table
TABLE 14 amino acid sequence information of recombinant antigens
The preparation of the FITC labeled novel coronavirus antigen comprises the following steps,
1) putting the novel coronavirus recombinant antigen into a dialysis bag, and dialyzing with 0.02-0.1M carbonate buffer solution with pH of 8.5-10 for 0.5-2 h;
2) mixing FITC and novel coronavirus recombinant antigen at a molar ratio of 4:1, dialyzing with 0.02-0.1M carbonate buffer solution at 2-8 deg.C for 20-24h, and changing the solution for 2-3 times;
3) dialyzing the labeling solution obtained in step 2) with 0.01-0.05M PBS at 2-8 deg.C for 24h, adding equal volume of glycerol, and storing at-20 deg.C.
Sensitivity and specificity
TABLE 15 comparative example a partial test result excluding 2019-nCoV infection
TABLE 16 partial test results for the confirmed 2019-nCoV infection of comparative example one
The results of the ROC curve analysis using the SPSS software are shown in the following table, and the ROC curve analysis is shown in fig. 3.
TABLE 17 area under the curve of comparative example 1
The S/CO of the sample is more than or equal to 1, and the detection result is judged to be positive; the sample S/CO is less than 1, and the detection result is judged to be negative. Wherein the S/CO value is the luminescence value/cutoff value of the detection sample. And determining the sensitivity and specificity of the kit under the condition of different cutoff values through an ROC curve, and screening out the optimal cutoff value.
The ROC curve analysis result shows that the area under the curve is 0.960, which indicates that the kit has higher accuracy in clinical diagnosis.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Booskasei Biotechnology Ltd
<120> a synthetic polypeptide composition for novel coronavirus immunodetection and application thereof
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>29
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Gly Pro Val Pro Leu Ala Ile Ala Gly Thr Pro Leu Ile Thr Ser
1 5 10 15
Leu His Thr Pro Ile Ala Leu Val Ala Ala Leu Pro Gly
20 25
<210>2
<211>45
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro
1 5 10 15
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
20 25 30
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
<210>3
<211>27
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe Pro
1 5 10 15
Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser
20 25
<210>4
<211>40
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln
1 5 10 15
Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln
20 25 30
Lys Arg Thr Ala Thr Lys Ala Tyr
35 40
<210>5
<211>204
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly
195 200
<210>6
<211>244
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>6
Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg
1 5 10 15
Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro
20 25 30
Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu
35 40 45
Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln
50 55 60
Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser
65 70 75 80
Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr
85 90 95
Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly
100 105 110
Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln
115 120 125
Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg
130 135 140
Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Ala
145 150 155 160
Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile
165 170 175
Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu
180185 190
Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro
195 200 205
Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp
210 215 220
Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp
225 230 235 240
Ser Thr Gln Ala
<210>7
<211>325
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>7
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser
325
<210>8
<211>395
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>8
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
1 5 10 15
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
20 25 30
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
35 40 45
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
50 55 60
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
65 70 75 80
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
85 90 95
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
100 105 110
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
115 120 125
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
130 135 140
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
145 150 155 160
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
165 170 175
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
180 185 190
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
195 200 205
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
210 215 220
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
225 230 235 240
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
245 250 255
Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr
260 265 270
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
275 280 285
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
290 295 300
Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro
305 310 315 320
Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser
325 330 335
Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala
340 345 350
Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
355 360 365
Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg
370 375 380
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr
385 390 395
<210>9
<211>305
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>9
Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser
1 5 10 15
Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu
20 25 30
Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys
35 40 45
Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe
50 55 60
Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp
65 70 75 80
Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr
85 90 95
Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro
100105 110
Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe
115 120 125
Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp
130 135 140
Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe
145 150 155 160
Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala
165 170 175
Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr
180 185 190
Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala
195 200 205
Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn
210 215 220
Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln
225 230 235 240
Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val
245 250 255
Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser
260265 270
Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg
275 280 285
Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly
290 295 300
Arg
305
<210>10
<211>299
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser
1 5 10 15
Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu
20 25 30
Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser
35 40 45
Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val
50 55 60
Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr
65 70 75 80
Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
85 9095
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
100 105 110
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser
115 120 125
Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln
130 135 140
Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala
145 150 155 160
His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe
165 170 175
Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn
180 185 190
Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn
195 200 205
Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu
210 215 220
Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly
225 230 235 240
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile
245 250 255
Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp
260 265 270
Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
275 280 285
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala
290 295
Claims (7)
1. A synthetic polypeptide composition for immunodetection of novel coronaviruses, comprising the following four synthetic polypeptides, SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4.
2. the synthetic polypeptide composition of claim 1, wherein the four synthetic polypeptides are biotinylated or FITC-linked Spike polypeptide, nucleocapsid polypeptide, respectively.
3. Use of the synthetic polypeptide composition according to claim 1 or 2 in novel coronavirus 2019-nCoV immunodetection reagents.
4. Use according to claim 3, characterized in that: in the synthetic polypeptide composition, the mass ratio of the four synthetic polypeptides is (0.5-2): (0.5-2): (0.5-2): (0.5-2).
5. Use according to claim 4, characterized in that: in the synthetic polypeptide composition, the mass ratio of the four synthetic polypeptides is 1:1:1: 1.
6. Use according to claim 3, characterized in that: the immunoassay comprises one of chemiluminescence immunoassay and enzyme-linked immunoassay.
7. Use according to claim 3, characterized in that: the detection is carried out by combining a novel coronavirus 2019-nCoV total antibody or an IgM or IgG antibody.
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| CN111349150A (en) * | 2020-03-24 | 2020-06-30 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting novel coronavirus and application thereof |
| CN111777684A (en) * | 2020-06-09 | 2020-10-16 | 重庆君同生物技术有限公司 | Preparation method and application of antibody induced by coronavirus tandem epitope protein |
| CN112379090A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus antibody detection kit and preparation method and application thereof |
| CN112858454A (en) * | 2020-10-16 | 2021-05-28 | 北京毅新博创生物科技有限公司 | Characteristic polypeptide composition for diagnosing new coronary pneumonia |
| WO2021232712A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯(重庆)生物科技有限公司 | Novel coronavirus igm/igg magnetic microparticle chemiluminescence immunoassay kit |
| WO2021232713A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯（天津）生物科技有限公司 | Enzyme-linked immunosorbent assay detection kit for novel coronavirus igg antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111349150A (en) * | 2020-03-24 | 2020-06-30 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting novel coronavirus and application thereof |
| WO2021232712A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯(重庆)生物科技有限公司 | Novel coronavirus igm/igg magnetic microparticle chemiluminescence immunoassay kit |
| WO2021232713A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯（天津）生物科技有限公司 | Enzyme-linked immunosorbent assay detection kit for novel coronavirus igg antibody |
| CN111777684A (en) * | 2020-06-09 | 2020-10-16 | 重庆君同生物技术有限公司 | Preparation method and application of antibody induced by coronavirus tandem epitope protein |
| WO2022006357A3 (en) * | 2020-06-30 | 2022-02-10 | Boost Biopharma, Inc. | Recombinant polypeptides containing at least one immunogenic fragment and antibody fc region and uses thereof |
| CN112858454A (en) * | 2020-10-16 | 2021-05-28 | 北京毅新博创生物科技有限公司 | Characteristic polypeptide composition for diagnosing new coronary pneumonia |
| CN112858454B (en) * | 2020-10-16 | 2022-09-30 | 北京毅新博创生物科技有限公司 | Characteristic polypeptide composition for diagnosing new coronary pneumonia |
| CN112379090A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus antibody detection kit and preparation method and application thereof |
| CN112379090B (en) * | 2021-01-07 | 2021-04-16 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus antibody detection kit and preparation method and application thereof |
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| CN111337673B (en) | 2020-08-11 |
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