CN111122864A - Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof - Google Patents
Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses a novel coronavirus IgG antibody enzyme-linked immunoassay kit and a preparation method thereof, and relates to the field of biological medicine, wherein the kit comprises an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate A solution, a substrate B solution, a concentrated washing solution, a stop solution and a coating plate pre-coated with a novel coronavirus antigen, the enzyme conjugate is a goat anti-human IgG-HRP working solution prepared by using an enzyme-labeled diluent according to working concentration, detection is carried out through the immune specific reaction of an antigen antibody so as to judge whether the novel coronavirus IgG antibody exists in a test sample qualitatively, and the kit has the characteristics of high specificity and strong anti-interference performance The method has the characteristics of high accuracy, simple steps, reasonable design and suitability for popularization.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to a novel coronavirus IgG antibody enzyme-linked immunoassay kit and a detection method thereof, and particularly relates to a novel coronavirus IgG antibody in a serum or plasma sample by qualitative detection of an enzyme-linked immunoassay method based on an indirect method principle.
Background
Since 12 months in 2019, a novel coronavirus (2019-nCoV) epidemic situation appears. The common signs of the cough are fever, hypodynamia, dry cough and dyspnea gradually, while some patients have slight disease symptoms, even asymptomatic patients without clinical symptoms. The human-transmissible agent has the characteristic of human transmissible, the incubation period is 1-14 days generally, the incubation period is infectious, asymptomatic infected persons can also become an infection source, and the infection is transmitted mainly through respiratory droplets and close contact, so that the human is generally susceptible.
The epidemic situation develops to the present, a plurality of suspected cases are not yet diagnosed due to reasons such as insufficient detection efficiency, but the existing detection reagent has the defects of low sensitivity and weak anti-interference capability, so that the problems of false negative and false positive of reagent diagnosis often appear, which not only troubles the diagnosis link, but also exists in the rehabilitation link, and accurate treatment is difficult to achieve without accurate diagnosis, so that how to accurately screen the suspected cases in batches for clinicians to accurately judge whether the suspected cases are novel coronavirus pneumonia, so as to be convenient for quick isolation and avoid infection to more people, and the problem to be solved is urgent.
Disclosure of Invention
The invention aims to provide a novel coronavirus IgG antibody enzyme-linked immunoassay kit which is high in sensitivity, strong in specificity and good in accuracy.
Another purpose of the invention is to provide a test method of the novel coronavirus IgG antibody enzyme-linked immunoassay kit.
The technical scheme provided by the invention is as follows: comprises an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate pre-coated with a new coronavirus antigen;
the enzyme conjugate is goat anti-human IgG-HRP working solution prepared by using enzyme-labeled diluent according to working concentration;
the preparation method of the coating plate for pre-coating the new coronavirus antigen comprises the following steps:
(1) coating: diluting a new coronavirus antigen by using a coating buffer solution, coating the diluted antigen into the holes of a coating plate according to 100 mu L/hole, and adsorbing the antigen for 21-24 hours at the temperature of 2-8 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate with 10mmol/L concentrated washing solution according to 300 mu L/hole, and sealing with 150 mu L/hole sealing solution at 2-8 ℃ for 16-18 hours;
(3) drying and storing: removing the confining liquid, drying, sealing, and storing at 2-8 deg.C.
As a further improvement of the invention, the new coronavirus antigen is a new coronavirus S2 protein which is expressed by genetic engineering recombination.
As a further improvement of the invention, the coating buffer solution is 0.05mol/L carbonate buffer solution with 0.02wt% of sodium azide and pH9.6, and the neocoronavirus antigen is diluted according to the volume ratio of 1: 500-1: 1000.
As a further improvement of the invention, the blocking solution is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.25wt% of sodium caseinate and 0.1wt% of ProClin 300.
As a further improvement of the invention, the sample diluent is 10mmol/L pH7.4PBS containing 20wt% goat serum, 0.2wt% sodium caseinate, 5wt% PEG6000, 4wt% sucrose, 0.1wt% ProClin 300.
As a further improvement of the invention, the enzyme-labeled diluent is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.1wt% of aminopyrine, 1wt% of glycerol, 20wt% of calf serum and 0.1wt% of ProClin 300.
As a further improvement of the invention, the substrate A solution is a 10mmol/L citric acid-sodium acetate buffer solution containing 0.06wt% of pH 5.2-5.3;
the substrate B solution contains 0.06wt% of TMB hydrochloride in citric acid buffer, 0.001wt% of sodium thiosulfate and 0.02wt% of sodium carbonate.
As a further improvement of the invention, the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1: 30-1: 100, and the absorbance value of the positive control solution is more than 1.0;
the negative control serum is prepared by adding the negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control liquid is less than 0.1. As a further improvement of the invention, the component of the stop solution is 1mol/L sulfuric acid.
As a further improvement of the invention, the concentrated washing solution comprises the components of 0.25mol/L pH6.0-6.5 PB containing 20wt% NaCl, 0.5wt% KCl and 3wt% Tween-20.
A novel coronavirus IgG antibody enzyme-linked immunoassay method comprises the novel coronavirus IgG antibody enzyme-linked immunoassay kit, and comprises the following steps:
s1, sample dilution: diluting a blood sample to be detected by sample diluent according to a ratio of 1: 100;
s2, antigen-antibody reaction: adding the diluted blood sample to be detected, the negative control solution and the positive control into different holes of a coating plate pre-coated with the new coronavirus antigen respectively according to the amount of 100 mu L/hole, and incubating for 60 minutes at 37 ℃;
s3, reacting the antigen-antibody complex with an enzyme-labeled antibody: discarding the reaction solution, washing the coated plate with concentrated washing solution to remove unbound components, adding 100. mu.L/well of the enzyme conjugate, and incubating at 37 ℃ for 30 minutes;
s4, color development: discarding reaction liquid, washing the coated plate by using concentrated washing liquid to remove unbound components, adding 50 mu L of substrate A liquid and substrate B liquid into each hole of the coated plate respectively, and developing for 15 minutes at 37 ℃ in a dark place;
s5, terminating the reaction: adding 50 mu L of stop solution into each hole of the coated plate to stop the reaction;
s6, absorbance measurement: sequentially measuring the absorbance value of each hole of the coated plate at the wavelength of 450nm/630nm by using an enzyme-labeling instrument;
s7, analyzing and judging results: the test effectiveness is verified through an S6 absorbance determination result, and if the absorbance average value of the positive control solution/the absorbance average value of the negative control solution is more than or equal to 2.1, the test is established; and (3) judging the detection result through a critical value, wherein the critical value =0.15+ the absorbance average value of the negative control solution (when the absorbance average value of the negative control solution is less than 0.05, the calculation is carried out according to 0.05), if the absorbance value of the blood sample to be detected is greater than the critical value, the blood sample to be detected is a positive result, and if the absorbance value of the blood sample to be detected is less than the critical value, the blood sample to be detected is a negative result.
The detection principle of the application is as follows: coating a new coronavirus antigen on a coating plate to form a coating plate pre-coated with the new coronavirus antigen, diluting a blood sample to be detected, dripping the diluted blood sample onto the coating plate pre-coated with the new coronavirus antigen, combining the novel coronavirus IgG antibody with a solid phase novel coronavirus S2 antigen on the coating plate pre-coated with the new coronavirus antigen to form an antigen-antibody complex if the specific novel coronavirus IgG antibody exists in the blood sample to be detected, adding an enzyme conjugate (enzyme-labeled anti-human IgG, which is sheep anti-human IgG-HRP working solution), combining an IgG component in the antigen-antibody complex with the enzyme-labeled anti-human IgG to form a solid phase complex, reacting horseradish peroxidase (HRP) in the solid phase complex with a substrate A, B solution to generate a color (blue) when a color developing agent is added, and adding a stop solution to stop the reaction (the color is changed from blue to yellow), and finally, reading the absorbance (A) value by an enzyme-labeling instrument, and comparing the absorbance (A) value with the critical value of the absorbance of the normal human negative control solution, thereby qualitatively judging whether the novel coronavirus IgG antibody exists in the sample.
Meanwhile, the control group is arranged, the absorbance of the negative control liquid and the positive control liquid and the blood sample to be detected are measured together to verify the effectiveness of the test, and if the absorbance average value of the positive control liquid/the absorbance average value of the negative control liquid is more than or equal to 2.1, the test is established, so that the detection accuracy is further improved.
Compared with the prior art, the invention has the following advantages:
the invention relates to a novel coronavirus IgG antibody enzyme-linked immunoassay kit, which comprises an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate A solution, a substrate B solution, a concentrated washing solution, a stop solution and a coating plate which pre-coats a new coronavirus antigen, wherein the enzyme conjugate is a goat anti-human IgG-HRP working solution prepared by using an enzyme-labeled diluent according to working concentration, detection is carried out through the immune specific reaction of an antigen antibody so as to qualitatively judge whether the novel coronavirus IgG antibody exists in a test sample, the detection is carried out through the immune specific reaction of the antigen antibody, the kit has high accuracy and high specificity, the CV values of the test positive serum sample are all within the range of 5-10%, and the result shows that the product has strong precision and good stability, and simultaneously the kit can be used for novel H1N1 influenza A virus, respiratory syncytial virus, EB virus, The serum of infectors such as measles virus, human cytomegalovirus, mumps virus, varicella-zoster virus and the like has no cross reaction, and does not interfere the detection of the product for common endogenous substances (mucin, bilirubin, triglyceride, hemoglobin, biotin, rheumatoid factor and 2-mercaptoethanol) and exogenous substances (gentamicin and dexamethasone), and meanwhile, the application has the advantages of simple structure, low detection cost and good accuracy.
The novel coronavirus IgG antibody enzyme-linked immunoassay method 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 step, is reasonable in design, and is suitable for popularization.
Detailed Description
The following describes a specific embodiment of the present invention with reference to specific embodiments.
Embodiment 1, a novel coronavirus IgG antibody enzyme-linked immunoassay kit, comprising an enzyme conjugate, a sample diluent, an enzyme-labeled diluent, a negative control solution, a positive control solution, a substrate a solution, a substrate B solution, a stop solution, a concentrated washing solution, and a pre-coated plate pre-coated with a novel coronavirus antigen; the enzyme conjugate is goat anti-human IgG-HRP working solution prepared by using enzyme-labeled diluent according to the working concentration of 1: 25000;
the preparation method of the coating plate for pre-coating the new coronavirus antigen comprises the following steps:
(1) coating: diluting the new coronavirus antigen with coating buffer solution (0.05 mol/L, pH9.6 carbonate buffer solution containing 0.02wt% sodium azide) at volume ratio of 1:500, coating the diluted antigen into the pores of the coating plate at volume ratio of 100 μ L/pore, and adsorbing at 2 deg.C for 21 hr;
(2) and (3) sealing: discarding the coating solution, washing the plate with 10mmol/L concentrated washing solution at 300. mu.L/well, and sealing with 150. mu.L/well of sealing solution (0.07 mol/L boric acid-borax buffer solution containing 5wt% sucrose, 2wt% liquid gelatin, 0.25wt% sodium caseinate, 0.1wt% ProClin 300) at 6 deg.C for 16 hr;
(3) drying and storing: discarding the sealing solution, drying, sealing, and storing at 6 deg.C.
The sample diluent is 10mmol/L PBS (pH7.4PBS) containing 20wt% goat serum, 0.2wt% casein sodium, 5wt% PEG6000, 4wt% sucrose and 0.1wt% ProClin 300.
The enzyme-labeled diluent is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.1wt% of aminopyrine, 1wt% of glycerol, 20wt% of calf serum and 0.1wt% of ProClin 300; the substrate A solution is 10mmol/L citric acid-sodium acetate buffer solution containing 0.06wt% pH5.3; the substrate B solution contains 0.06wt% of TMB hydrochloride in a citric acid buffer solution, 0.001wt% of sodium thiosulfate and 0.02wt% of sodium carbonate; the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 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 5wt%, and the absorbance value of the negative control liquid is less than 0.1; the component of the stop solution is 1mol/L sulfuric acid. The concentrated washing solution comprises the components of 0.25mol/L pH6.5 PB containing 20wt% NaCl, 0.5wt% KCl and 3wt% Tween-20.
Embodiment 2, a novel coronavirus IgG antibody enzyme-linked immunoassay kit, comprising an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate a solution, a substrate B solution, a stop solution, a concentrated washing solution, and a pre-coated plate pre-coated with a new coronavirus antigen; the enzyme conjugate is goat anti-human IgG-HRP working solution prepared by using enzyme-labeled diluent according to the working concentration of 1: 18000;
the preparation method of the coating plate for pre-coating the new coronavirus antigen comprises the following steps:
(1) coating: diluting new coronavirus antigen with coating buffer solution (0.05 mol/L, pH9.6 carbonate buffer solution containing 0.02wt% sodium azide) at volume ratio of 1:1000, coating the diluted solution into the pores of the coating plate at volume ratio of 100 μ L/pore, and adsorbing at 8 deg.C for 22 hr;
(2) and (3) sealing: discarding the coating solution, washing the plate with 10mmol/L concentrated washing solution at 300. mu.L/well, and sealing with 150. mu.L/well of sealing solution (0.07 mol/L boric acid-borax buffer solution containing 5wt% sucrose, 2wt% liquid gelatin, 0.25wt% sodium caseinate, 0.1wt% ProClin 300) at 8 deg.C for 17 hr;
(3) drying and storing: discarding the sealing solution, drying, sealing, and storing at 8 deg.C.
The sample diluent is 10mmol/L PBS (pH7.4PBS) containing 20wt% goat serum, 0.2wt% casein sodium, 5wt% PEG6000, 4wt% sucrose and 0.1wt% ProClin 300.
The enzyme-labeled diluent is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.1wt% of aminopyrine, 1wt% of glycerol, 20wt% of calf serum and 0.1wt% of ProClin 300; the substrate A solution is 10mmol/L citric acid-sodium acetate buffer solution containing 0.06wt% pH5.2; the substrate B solution contains 0.06wt% of TMB hydrochloride in a citric acid buffer solution, 0.001wt% of sodium thiosulfate and 0.02wt% of sodium carbonate; the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1:30, 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 5wt%, and the absorbance value of the negative control liquid is less than 0.1; the component of the stop solution is 1mol/L sulfuric acid. The concentrated washing solution comprises the components of 0.25mol/L pH6.0 PB containing 20wt% NaCl, 0.5wt% KCl and 3wt% Tween-20.
Example 3, a novel coronavirus IgG antibody enzyme-linked immunoassay kit, comprising an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate a solution, a substrate B solution, a stop solution, a concentrated washing solution, and a pre-coated plate pre-coated with a new coronavirus antigen; the enzyme conjugate is goat anti-human IgG-HRP working solution prepared by using enzyme-labeled diluent according to the working concentration of 1: 25000;
the preparation method of the coating plate for pre-coating the new coronavirus antigen comprises the following steps:
(1) coating: diluting the new coronavirus antigen with coating buffer solution (0.05 mol/L, pH9.6 carbonate buffer solution containing 0.02wt% sodium azide) at volume ratio of 1:700, coating the diluted antigen into the pores of the coating plate at volume ratio of 100 μ L/pore, and adsorbing at 6 deg.C for 24 hr;
(2) and (3) sealing: discarding the coating solution, washing the plate with 10mmol/L concentrated washing solution (Tween phosphate buffer) at 300. mu.L/well, and sealing with 150. mu.L/well of sealing solution (0.07 mol/L boric acid-borax buffer solution containing 5wt% sucrose, 2wt% liquid gelatin, 0.25wt% sodium caseinate, 0.1wt% ProClin 300) at 2 deg.C for 18 hours;
(3) drying and storing: discarding the confining liquid, drying, sealing, and storing at 2 deg.C.
The sample diluent is 10mmol/L PBS (pH7.4PBS) containing 20wt% goat serum, 0.2wt% casein sodium, 5wt% PEG6000, 4wt% sucrose and 0.1wt% ProClin 300.
The enzyme-labeled diluent is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.1wt% of aminopyrine, 1wt% of glycerol, 20wt% of calf serum and 0.1wt% of ProClin 300; the substrate A solution is 10mmol/L citric acid-sodium acetate buffer solution containing 0.06wt% pH5.3; the substrate B solution contains 0.06wt% of TMB hydrochloride in a citric acid buffer solution, 0.001wt% of sodium thiosulfate and 0.02wt% of sodium carbonate; the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1:60, 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 5wt%, and the absorbance value of the negative control liquid is less than 0.1; the component of the stop solution is 1mol/L sulfuric acid. The concentrated washing solution comprises the components of 0.25mol/L pH6.0 PB containing 20wt% NaCl, 0.5wt% KCl and 3wt% Tween-20.
Clinical test experiments were conducted on the kits prepared in examples 1 to 3 above
The detection steps are as follows:
s1, sample dilution: diluting a blood sample to be detected by sample diluent according to a ratio of 1: 100;
s2, antigen-antibody reaction: adding the diluted blood sample to be detected, the negative control solution and the positive control into different holes of a coating plate pre-coated with the new coronavirus antigen respectively according to the amount of 100 mu L/hole, and incubating for 60 minutes at 37 ℃;
s3, reacting the antigen-antibody complex with an enzyme-labeled antibody: discarding the reaction solution, washing the coated plate with concentrated washing solution to remove unbound components, adding 100. mu.L/well of the enzyme conjugate, and incubating at 37 ℃ for 30 minutes;
s4, color development: discarding reaction liquid, washing the coated plate by using concentrated washing liquid to remove unbound components, adding 50 mu L of substrate A liquid and substrate B liquid into each hole of the coated plate respectively, and developing for 15 minutes at 37 ℃ in a dark place;
s5, terminating the reaction: adding 50 mu L of stop solution into each hole of the coated plate to stop the reaction;
s6, absorbance measurement: sequentially measuring the absorbance value of each hole of the coated plate at the wavelength of 450nm/630nm by using an enzyme-labeling instrument;
s7, analyzing and judging results: the test effectiveness is verified through an S6 absorbance determination result, and if the absorbance average value of the positive control solution/the absorbance average value of the negative control solution is more than or equal to 2.1, the test is established; and judging the detection result through a critical value, wherein the critical value (cut-off) =0.15+ the absorbance average value of the negative control solution (when the absorbance average value of the negative control solution is less than 0.05, the calculation is carried out according to 0.05), if the absorbance value of the blood sample to be detected is greater than the critical value, the blood sample to be detected is a positive result, and if the absorbance value of the blood sample to be detected is less than the critical value, the blood sample to be detected is a negative result.
1. And (3) performance testing: 9 normal human serum samples, 6 weak positive sera and 6 strong positive sera (samples provided by the Guangdong province disease control center and IgG antibody detection amount marked on the sample information respectively) were divided into three groups, and the test was performed 10 times respectively by the kits prepared in examples 1 to 3 according to the above steps, with the test results shown in tables 1 to 3:
table 1: example 1 test results
The data in table 1 can be obtained, the test result in example 1, in which the absorbance average value of the positive control solution 1/the absorbance average value of the negative control solution 1 is greater than or equal to 2.1, indicates that the test data in example 1 is valid, the cut-off value (cut-off) =0.15+ the absorbance average value of the negative control solution 1 =0.17, and the absorbance (a) values of the tested normal human serum samples (normal 1, normal 2, normal 3) are all less than 0.17, which indicates that the novel coronavirus IgG antibody exists in the normal human serum sample, the absorbance (a) values of the weak positive serum samples (weak positive 1, weak positive 2), and strong positive serum samples (strong positive 1, strong positive 2) are all greater than 0.17, indicates that the novel coronavirus IgG antibody exists in the weak positive serum sample and the strong positive serum sample, and the results of each test are correct for 10 times, which indicates that the present application has high accuracy and high specificity, and the CV values of the tested positive serum samples are all in the range of 5% -10%, the product has strong precision and good stability, and the content of the antibody in the sample can be reflected by the value A.
Table 2: example 2 test results
The data in table 2 can be obtained, the absorbance average value of the positive control solution 2/the absorbance average value of the negative control solution 2 in the test result of example 2 is greater than or equal to 2.1, which indicates that the test data of example 2 is valid, the critical value (cut-off) =0.15+ absorbance average value of the negative control solution 2 =0.169, and the absorbance (a) values of the tested normal human serum samples (normal 4, normal 5, normal 6) are all less than 0.169, which indicates that the novel coronavirus IgG antibody exists in the normal human serum sample, the absorbance (a) values of the weak positive serum samples (weak positive 3, weak positive 4), and strong positive serum samples (strong positive 3, strong positive 4) are all greater than 0.169, which indicates that the novel coronavirus IgG antibody exists in the tested weak positive serum and strong positive serum samples, and the results of each 10 tests are correct in groups, which indicates that the application has high accuracy and high specificity, and the CV values of the tested positive serum samples are all in the range of 5% -10%, the product has strong precision and good stability, and the content of the antibody in the sample can be reflected by the value A.
Table 3: example 3 test results
The data in table 3 can be obtained, the absorbance average value of positive control solution 3/the absorbance average value of negative control solution 3 in the test result of example 3 is greater than or equal to 2.1, which indicates that the test data of example 3 is valid, the critical value (cut-off) =0.15+ absorbance average value of negative control solution 3 =0.166, and the absorbance (a) values of the tested normal human serum samples (normal 4, normal 5, normal 6) are all less than 0.166, which indicates that the novel coronavirus IgG antibody exists in the normal human serum sample, the absorbance (a) values of the weak positive serum samples (weak positive 5, weak positive 6), and strong positive serum samples (strong positive 5, strong positive 6) are all greater than 0.166, which indicates that the novel coronavirus IgG antibody exists in the tested weak positive serum and strong positive serum samples, and the results of each 10 tests are correct, which indicates that the present application has high accuracy and high specificity, and the CV values of the tested positive serum samples are all in the range of 5% -10%, the product has strong precision and good stability, and the content of the antibody in the sample can be reflected by the value A.
2. And (3) specificity verification: the serum of the infected persons with the novel influenza A H1N1 virus, the respiratory syncytial virus, the EB virus, the measles virus, the human cytomegalovirus, the mumps virus and the varicella-zoster virus are respectively used for detection by the kit products prepared in the examples 1 to 3, and the average value of absorbance is detected, and the results are shown in the table 4.
Table 4: results of absorbance test
As can be seen from the data in Table 4, the kit of the product detects the serum of a person infected with the novel H1N1 influenza A virus, the respiratory syncytial virus, the EB virus, the measles virus, the human cytomegalovirus, the mumps virus and the varicella-zoster virus, the detection results of the product of the application are negative, the detection results are not influenced by the viruses, and the kit has the characteristic of high specificity,
3. verification of interference performance of endogenous/exogenous substances:
(1) 5 parts of positive serum are treated by 2-mercaptoethanol (0.2 mol/L) in water bath at 37 ℃ for 60 minutes and then retested by the products of examples 1-3, and the results of the average value of the detected absorbance are shown in Table 5.
Table 5: results of absorbance test
As can be seen from the data in Table 5, the test of 5 parts of positive serum is positive before and after the positive serum is treated by 2-mercaptoethanol (0.2 mol/L) in water bath at 37 ℃ for 60 minutes, which shows that the product of the application is not interfered by the test result of 2-mercaptoethanol and has anti-interference performance.
(2) The following endogenous substances, mucin (10 g/L), bilirubin (574. mu. mol/L), triglyceride (30 g/L), hemoglobin (347. mu. mol/L), biotin (123 nmol/L) and rheumatoid factor (150 IU/ml), were added to the products of examples 1 to 3 at the labeled concentrations, and the average values of absorbance were measured, and the results are shown in Table 6.
Table 6: results of absorbance test
As can be seen from the test data in Table 6, the test results are negative after the endogenous substances such as mucin, bilirubin, triglyceride, hemoglobin, biotin and rheumatoid factor are added in the examples 1-3, which indicates that the product of the application is not interfered by the endogenous substances.
(3) The gentamicin concentration is 1 multiplied by 107U/L, the dexamethasone concentration is 500mg/L, the products of the embodiments 1 to 3 are respectively used for testing, and the detection results are shown in Table 7.
Table 7: results of absorbance test
As can be seen from the data in Table 7, when exogenous substances such as gentamicin and dexamethasone are added into the kit, the detection result is negative, and the product is not interfered by the exogenous substances.
According to the test results, the method performs detection through the immune specific reaction of the antigen and the antibody, has the characteristics of high specificity, high accuracy and high specificity, and the CV values of the positive serum samples are all in the range of 5-10%, which indicates that the product has strong precision and good stability, meanwhile, the kit has no cross reaction on the serum of infectors such as novel H1N1 influenza A virus, respiratory syncytial virus, EB virus, measles virus, human cytomegalovirus, mumps virus, varicella-zoster virus and the like, and does not interfere the detection of the product on common endogenous substances (mucin, bilirubin, triglyceride, hemoglobin, biotin, rheumatoid factors, 2-mercaptogentamicin) and exogenous substances (daptomycin and dexamethasone).
Claims (10)
1. A novel coronavirus IgG antibody enzyme-linked immunoassay kit is characterized by comprising an enzyme conjugate, a sample diluent, a negative control solution, a positive control solution, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate pre-coated with a novel coronavirus antigen;
the enzyme conjugate is goat anti-human IgG-HRP working solution prepared by using enzyme-labeled diluent according to working concentration;
the preparation method of the coating plate for pre-coating the new coronavirus antigen comprises the following steps:
(1) coating: diluting a new coronavirus antigen by using a coating buffer solution, coating the diluted antigen into the holes of a coating plate according to 100 mu L/hole, and adsorbing the antigen for 21-24 hours at the temperature of 2-8 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate with 10mmol/L concentrated washing solution according to 300 mu L/hole, and sealing with 150 mu L/hole sealing solution at 2-8 ℃ for 16-18 hours;
(3) drying and storing: removing the confining liquid, drying, sealing, and storing at 2-8 deg.C.
2. The novel coronavirus IgG antibody ELISA kit of claim 1, wherein the novel coronavirus antigen is a genetically engineered recombinant expressed novel coronavirus S2 protein.
3. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the coating buffer solution is 0.05mol/L carbonate buffer solution with 0.02wt% of sodium azide and pH9.6, and the new coronavirus antigen is diluted according to the volume ratio of 1: 500-1: 1000.
4. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the confining liquid is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.25wt% of casein sodium and 0.1wt% of ProClin 300.
5. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the sample diluent is 10mmol/L PBS (pH7.4PBS) containing 20wt% goat serum, 0.2wt% casein sodium, 5wt% PEG6000, 4wt% sucrose and 0.1wt% ProClin 300.
6. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein said: the enzyme-labeled diluent is 0.07mol/L boric acid-borax buffer solution containing 5wt% of sucrose, 2wt% of liquid gelatin, 0.1wt% of aminopyrine, 1wt% of glycerol, 20wt% of calf serum and 0.1wt% of ProClin 300.
7. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the substrate A solution is a 10mmol/L citric acid-sodium acetate buffer solution containing 0.06wt% of pH 5.2-5.3;
the substrate B solution contains 0.06wt% of TMB hydrochloride in citric acid buffer, 0.001wt% of sodium thiosulfate and 0.02wt% of sodium carbonate.
8. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the positive control solution is prepared by adding positive serum into a sample diluent according to the proportion of 1: 30-1: 100, and the absorbance value of the positive control solution is more than 1.0;
the negative control serum is prepared by adding the negative serum into a sample diluent according to the proportion of 5wt%, and the absorbance value of the negative control liquid is less than 0.1.
9. The novel coronavirus IgG antibody enzyme-linked immunoassay kit according to claim 1, wherein: the component of the stop solution is 1mol/L sulfuric acid;
the concentrated washing solution comprises the components of 0.25mol/L pH6.0-6.5 PB containing 20wt% NaCl, 0.5wt% KCl and 3wt% Tween-20.
10. A novel coronavirus IgG antibody enzyme-linked immunoassay method, which is characterized by comprising the novel coronavirus IgG antibody enzyme-linked immunoassay kit of any one of claims 1 to 9, wherein the detection method comprises the following steps:
s1, sample dilution: diluting a blood sample to be detected by sample diluent according to a ratio of 1: 100;
s2, antigen-antibody reaction: adding the diluted blood sample to be detected, the negative control solution and the positive control into different holes of a coating plate pre-coated with the new coronavirus antigen respectively according to the amount of 100 mu L/hole, and incubating for 60 minutes at 37 ℃;
s3, reacting the antigen-antibody complex with an enzyme-labeled antibody: discarding the reaction solution, washing the coated plate with concentrated washing solution to remove unbound components, adding 100. mu.L/well of the enzyme conjugate, and incubating at 37 ℃ for 30 minutes;
s4, color development: discarding reaction liquid, washing the coated plate by using concentrated washing liquid to remove unbound components, adding 50 mu L of substrate A liquid and substrate B liquid into each hole of the coated plate respectively, and developing for 15 minutes at 37 ℃ in a dark place;
s5, terminating the reaction: adding 50 mu L of stop solution into each hole of the coated plate to stop the reaction;
s6, absorbance measurement: sequentially measuring the absorbance value of each hole of the coated plate at the wavelength of 450nm/630nm by using an enzyme-labeling instrument;
s7, analyzing and judging results: the test effectiveness is verified through an S6 absorbance determination result, and if the absorbance average value of the positive control solution/the absorbance average value of the negative control solution is more than or equal to 2.1, the test is established; judging the detection result by a critical value, wherein the critical value =0.15+ the absorbance average value of the negative control solution, and when the absorbance average value of the negative control solution is less than 0.05, calculating according to 0.05; if the absorbance value of the blood sample to be detected is larger than the critical value, the blood sample to be detected is a positive result, and if the absorbance value of the blood sample to be detected is smaller than the critical value, the blood sample to be detected is a negative result.
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