CN112903997B - EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit and detection method thereof - Google Patents
EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit and detection method thereof Download PDFInfo
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Abstract
The application provides an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit and a detection method thereof, and the kit comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum solution, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate, wherein the coating plate is coated with EB virus capsid antigen recombinant antigens, the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA, a microporous plate coated with purified gene recombinant EB virus capsid antigens and enzyme-labeled anti-human IgA and other reagents are used for preparing the kit, the IgA antibody of the EB virus capsid antigens in human serum is qualitatively detected by utilizing an ELISA indirect method principle, and the kit has the advantages of high sensitivity, strong specificity and strong anti-interference capability, and meanwhile, the kit is simple in structure, low in detection cost and good in accuracy.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit and a detection method thereof, and particularly relates to an IgA antibody for qualitatively detecting EB Virus Capsid Antigen (VCA) in human serum by utilizing an enzyme-linked immunoassay method of an indirect method principle.
Background
EB virus is gamma herpes virus of human B lymphocyte, which mainly invades B lymphocyte and has affinity to human B lymphocyte, epithelial cell (including parotid gland, pharynx, cervix etc.) and gland cell. The current research finds that EB virus infection is related to various human tumors such as nasopharyngeal carcinoma, lymph cancer and the like. Capsid Antigen (VCA) is a structural protein synthesized during the late phase of epstein-barr virus proliferation, and is present in the cytoplasm and nucleus. VCA and viral DNA form a nucleocapsid that eventually assembles into complete virions. The abundant expression of VCA marks the EB virus to enter the replication state.
The existing IgA antibody detection reagent for EB Virus Capsid Antigen (VCA) has the defects of low sensitivity, poor specificity and weak anti-interference capability, so that the troubles of false negative and false positive diagnosis of the reagent often occur, and accurate diagnosis is not available, so that accurate treatment is difficult, and the problem to be solved is urgent.
Disclosure of Invention
The invention aims to provide an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit which has high sensitivity, strong specificity and strong anti-interference capability.
Another purpose of the invention is to provide an EB virus capsid antigen IgA antibody enzyme-linked immunoassay method.
The technical scheme provided by the invention is as follows: an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate;
the coating plate is coated with EB virus capsid antigen recombinant antigen;
the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA.
The application provides an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit, which comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate, wherein the EB virus capsid antigen recombinant antigen is coated on the coating plate, the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA, the kit is prepared by using a microporous plate coated by a purified gene recombinant EB Virus Capsid Antigen (VCA), enzyme-labeled anti-human IgA and other reagents, the IgA antibody of the EB virus nuclear antigen (VCA) in human serum is qualitatively detected by using an ELISA indirect method principle, and the kit has the advantages of high sensitivity, strong specificity and strong anti-interference capability, has good specificity on EB virus NA1 IgA positive, EB virus VCA positive, EB virus ZTa positive sample, and giant cell, herpes simplex virus type 1, The reagent has the advantages that the reagent has no obvious cross reaction in diseases such as II type, liver cancer, intestinal cancer, head and neck cancer, lung cancer, lymphoma and the like, and interference substances (such as hyperlipidemia, hyperbilirubicin, hemolysis, anticoagulation of sodium citrate and anticoagulation of EDTA-2Na samples) do not interfere the detection of the reagent.
As a further improvement of the invention, the EB virus capsid antigen recombinant antigen is prepared by adopting a genetic engineering technology.
As a further improvement of the invention, the EB virus capsid antigen recombinant antigen is prepared by adopting the following method: selecting the rear 176 amino acid parts of EB virus capsid antigen (open reading frame BFRF3) protein as target antigens, taking mRNA sequences corresponding to the EB virus capsid antigen as templates, obtaining target gene fragments through RT-PCR, then transferring the target gene fragments into a pGEX-2T gene fusion expression system, carrying out enzyme digestion sequencing identification, carrying out IPTG induced expression on recombinant colonies, purifying an expression product through GST affinity chromatography, and cutting off GST parts by thrombin to finally obtain the recombinant EB virus capsid antigen. Preferably, the EB VCA DNA sequence has 430 base pairs in total and codes 176 amino acids, and the coating antigen has the advantages of high sensitivity and good specificity.
As a further improvement, the molecular weight of the EB virus capsid antigen recombinant antigen is 25-27 kD, and the purity is more than or equal to 90%. The purity and molecular weight of EB virus capsid antigen recombinant envelope antigen are detected by SDS-polyacrylamide gel electrophoresis, and the result is shown in figure 1, wherein "VCA" is the EB virus capsid antigen recombinant antigen identified by SDS-PAGE electrophoresis: 5 mu g of the product shows a band, the molecular weight is 26kD, and the purity is more than 90 percent.
As a further improvement of the invention, the preparation method of the EB virus capsid antigen recombinant antigen coated on the coating plate comprises the following steps:
(1) coating: diluting an EB virus capsid antigen recombinant antigen with a coating buffer solution, coating the diluted EB virus capsid antigen recombinant antigen into the holes of a coating plate according to 95-105 mu L/hole, and incubating and adsorbing for 18-24 hours at 18-28 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate, and then carrying out incubation and sealing for 18-22 hours at 2-8 ℃ by using sealing solution according to 145-155 mu L/hole;
(3) drying and storing: removing the confining liquid, drying at 30-37 ℃ for more than 4 hours, and sealing for storage.
As a further improvement of the invention, the confining liquid 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;
in the step (2), the plate washing is carried out by using 10mmol/L washing solution at a rate of 300. mu.L/hole.
Determination of the coating time:
preparing coating liquid according to determined concentration, adding the coating liquid into a microporous plate, respectively placing the plate at 18-28 ℃ for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 hours, carrying out experiments by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and determining the optimal coating time by analyzing the A value of the in-plant quality control products. The results are shown in table 1:
TABLE 1. quality control A values in the factory for different coating times
As can be seen from Table 1, when the coating time is more than or equal to 16 hours, the A values of 3 positive quality control products tend to be flat, which indicates that the maximum adsorption capacity of the coated antigen is achieved; the A values of 2 negative quality control products are less than 0.15, and the display specificity is good. According to the above analysis, and in order to ensure the product quality, the coating time was set to 18 to 24 hours.
Determination of coating liquid addition:
coating plates are prepared by respectively using 25 muL/hole, 50 muL/hole, 75 muL/hole, 100 muL/hole, 150 muL/hole and 200 muL/hole of coating liquid, the coating plates are placed for 20 hours at the temperature of 18-28 ℃, and the in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 are used as research materials to carry out experiments, and the optimal coating liquid adding amount is determined by analyzing the A value of the in-plant quality control products. The results are shown in table 2:
TABLE 2 quality control A values of different coating solutions in-plant
As can be seen from Table 2, when the liquid adding amount of the coating solution is more than or equal to 75 μ L/well, the A values of the 3 positive quality control products tend to be gentle, which indicates that the maximum adsorption amount of the coating antigen is achieved; the A values of 2 negative quality control products are less than 0.15, and the display specificity is good. According to the analysis, the liquid adding amount of the coating liquid is determined to be 95-105 mu L/hole in order to ensure the product quality.
Determination of coating temperature:
the microporous plate added with the coating liquid is respectively placed in a refrigerator (2-8 ℃), a room temperature (18-28 ℃) and a warm box (35-37 ℃) for coating, experiments are carried out by taking quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 in the factory as research materials, and the optimal coating temperature is determined by analyzing the A value of the quality control products in the factory. The results are shown in table 3:
TABLE 3. quality control A values of in-plant quality control articles for different coating temperatures
As can be seen from table 3, the sensitivity was highest with the coated plate coated at room temperature; under the three coating conditions, the A values of 2 negative quality control products are less than 0.15, and the display specificity is good. According to the above analysis, the coating temperature was set to room temperature (18-28 ℃).
As a further improvement of the invention, the enzyme conjugate is an enzyme-labeled anti-human IgA obtained by adopting a sodium periodate method and labeling by peroxidase, experiments are carried out by taking in-factory quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and when the concentration of the enzyme-labeled anti-human IgA is more than or equal to 1:25000, the A value of the positive sample tends to be flat, the sensitivity reaches the upper limit, and when the concentration of enzyme-labeled anti-human IgA is less than or equal to 1: at 25000, the A values of the negative samples are all less than 0.15, the difference is not large, and the display specificity is good, so in conclusion, the use concentration of the enzyme-labeled anti-human IgA is set to be 1:25000, and the invitrogen enzyme-labeled anti-human IgA provided by Guangzhou Yingwei Chujin biotechnology limited is preferably used, so that the advantages of high sensitivity and good specificity are achieved. The results are shown in Table 4.
TABLE 4 enzyme-labeled anti-human IgA quality control product A values in different dilution ratios
As a further improvement of the invention, the sample diluent is a solution containing a protein stabilizer and a preservative; preferably, the sample diluent is 10mmol/L PBS (pH 7.4) containing 20 wt% goat serum, 0.2 wt% sodium caseinate, 5wt% PEG6000, 4 wt% sucrose, 0.1wt% ProClin 300. The test sample type is serum or plasma.
The concentrated washing solution is PBS buffer solution containing Tween-20, preferably the concentrated washing solution comprises the components of 0.25mol/LpH6.0-6.5PB containing 20 wt% NaCl, 0.5 wt% KCl and 3 wt% Tween-20;
the substrate A solution is a solution containing carbamide peroxide;
the substrate B solution is a solution containing TMB;
the stop solution is 1mol/L sulfuric acid solution;
the negative control solution is EB virus capsid antigen IgA antibody negative human serum with the A value less than 0.12, a healthy human blood sample from tumor research institute in Zhongshan city, the kit is used for detecting the human serum with the A value less than 0.12, the human serum is treated at 60 ℃ for 1 hour, and then preservative is added and stored at-20 ℃;
the quality control serum is inactivated EB virus capsid antigen lgA antibody positive human serum with the A value range of 0.8-2.4 and is from a patient blood sample diagnosed by tumor research institute in Zhongshan city, the kit is used for detecting the human serum with the A value range of 0.8-2.4, the human serum is treated at 60 ℃ for 1 hour, and then preservative is added and stored at-20 ℃.
An EB virus capsid antigen IgA antibody enzyme-linked immunoassay method comprises the EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit, and the detection method comprises the following steps:
s1, sample adding: taking a coating plate coated with EB virus capsid antigen recombinant antigen, arranging a blank control hole, adding 100 mu L of sample diluent, additionally arranging a negative control hole and a quality control serum hole, respectively adding 100 mu L of negative control liquid and 100 mu L of quality control serum, arranging a sample hole to be detected, adding 100 mu L of sample diluent and 5 mu L of sample to be detected, uniformly mixing, covering with a sealing plate adhesive, and placing at 37 ℃ for incubation for 30 minutes;
s2, adding enzyme conjugate: washing the plate with concentrated washing solution, adding 100 μ L/well of enzyme conjugate, covering with sealing plate glue, and incubating at 37 deg.C for 30 min;
s3, color development and termination: washing the plate with concentrated washing solution, adding 50 μ L of each of the substrate A solution and the substrate B solution, developing in a dark place at 37 deg.C for 15 min, and adding 50 μ L of stop solution;
s4, absorbance measurement: selecting the wavelength of 450nm by using a microplate reader, calibrating zero by using blank holes, and measuring the A value of each hole;
s5, analyzing and judging results: and (5) testing the result through step S4 to verify the effectiveness of the test, if the following conditions are met: the blank control A value is less than or equal to 0.08, the quality control serum A value is less than 2.4 and the negative control A value is less than 0.12, so the test is effective; and judging the detection result through a critical value, wherein the critical value is the average A value of the quality control serum multiplied by 20 percent, and is judged to be negative when the detection A value of the sample to be detected is less than the critical value, and is judged to be positive when the detection A value of the sample is more than or equal to the critical value.
As a further improvement of the invention, the coating buffer solution is 0.05mol/L carbonate buffer solution with pH9.6 and containing 0.02 wt% of sodium azide, and EB virus capsid antigen is diluted according to the volume proportion of 1: 500-1: 1000.
Determination of sample dilution ratio:
5 parts of the in-plant quality control product is diluted by the sample diluent from the original time, and then diluted by time, and the sample diluent and the sample adding amount of the sample are determined by analyzing the A value of the in-plant quality control product. The results are shown in Table 5:
TABLE 5. dilution of different samples and A value of quality control product in the sample adding amount factory
As can be seen from Table 5, when the sample dilution ratio is 1:20, the value of the positive quality control A is the largest in3 parts, and the value of the negative quality control A in 2 parts is less than 0.15, which shows good specificity. The sample dilution ratio was set to 1:20 according to the above analysis.
Determination of the amount of enzyme conjugate loaded:
the enzyme conjugate with the use concentration is added into a micropore plate according to 25 muL, 50 muL, 75 muL, 100 muL, 150 muL and 200 muL per hole, experiments are carried out by taking the quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 in factories as research materials, and the adding amount of the enzyme conjugate is determined by analyzing the A value of the quality control products in the factories. The results are shown in Table 6:
TABLE 6 quality control A values in the factory for different enzyme conjugate addition
As can be seen from Table 6, when the amount of the enzyme conjugate added was 100. mu.L/well, 3 parts of the positive control A showed a high value, while 2 parts of the negative control A showed a good specificity, all of which were less than 0.15. Therefore, the amount of the enzyme conjugate added was determined to be 100. mu.L/well.
Determination of the sample addition amounts of the substrate A solution and the substrate B solution:
adding the substrate A liquid and the substrate B liquid into a microporous plate according to 25 muL +25 muL, 50 muL +50 muL, 75 muL +75 muL and 100 muL +100 muL per hole, carrying out experiments by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and determining the adding amount of the color developing liquid by analyzing the A value of the in-plant quality control products. The results are shown in Table 7:
TABLE 7 quality control A values in the factory for different substrate A and B liquid sample amounts
As can be seen from Table 7, when the sample amounts of the substrate A solution and the substrate B solution were 50. mu.L + 50. mu.L, 3 positive quality controls had high A values, while 2 negative quality controls had A values of less than 0.15, showing good specificity. Therefore, the amounts of the substrate A solution and the substrate B solution added were set to 50. mu.L + 50. mu.L.
Determination of the reaction temperature:
the method is characterized in that the method is respectively operated at 4 ℃, 25 ℃, 37 ℃ and 42 ℃, experiments are carried out by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and the optimal reaction temperature is determined by analyzing the A value of the in-plant quality control products. The results are shown in Table 8:
TABLE 8. quality control A values in the plant for different reaction temperatures
As is clear from Table 8, when the reaction temperature is 37 ℃, the reaction temperature is set to 37 ℃ because the in-plant quality control products have high sensitivity and good specificity.
Determination of the reaction time of the sample:
after sample adding, the reaction is carried out for 5, 10, 20, 30, 40 and 50 minutes respectively, experiments are carried out by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and the optimal sample reaction time is determined by analyzing the A value of the in-plant quality control products. The results are shown in Table 9:
TABLE 9 in-plant quality control A values for different sample reaction times
As can be seen from Table 9, when the reaction time of the sample amount is more than or equal to 30 minutes, the A values of 3 positive quality control substances tend to be flat, and the A values of 2 negative quality control substances are less than 0.15, so that the display specificity is good. According to the above analysis, and also in order to ensure the product quality, the sample reaction time was set to 30 minutes.
Determination of enzyme conjugate reaction time:
after the enzyme conjugates are added, the reaction is carried out for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes respectively, experiments are carried out by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 minutes and ZSN2 as research materials, and the optimal reaction time of the enzyme conjugates is determined by analyzing the A value of the in-plant quality control products. The results are shown in Table 10:
TABLE 10 quality control A values in the plant for different enzyme conjugate reaction times
As can be seen from Table 10, when the reaction time of the enzyme conjugate is longer than or equal to 20 minutes, the A values of 3 positive quality control substances tend to be flat, and the A values of 2 negative quality control substances are less than 0.15, showing that the specificity is very good. According to the above analysis, and also in order to ensure the product quality, the enzyme conjugate reaction time was set to 30 minutes.
Determination of reaction time of substrate solution A and substrate solution B:
after adding the substrate A liquid and the substrate B liquid, respectively reacting for 5, 10, 15, 20, 25 and 30 minutes, carrying out experiments by taking in-plant quality control products ZS1#, ZS2#, ZS3#, ZSN1 and ZSN2 as research materials, and determining the optimal reaction time of the substrate A liquid and the substrate B liquid by analyzing the A value of the in-plant quality control products. The results are shown in Table 11:
TABLE 11 quality control A values in the plant for the reaction times of the different substrates A and B
As can be seen from Table 11, when the reaction time of the substrate A solution and the substrate B solution is longer than or equal to 10 minutes, the values of 3 positive quality control substances A tend to be flat, and the values of 2 negative quality control substances A are less than 0.15, showing that the specificity is very good. According to the above analysis, the reaction time of the substrate A liquid and the substrate B liquid was set to 15 minutes in order to ensure the product quality.
As a further improvement of the invention, the blank control hole, the negative control hole and the quality control serum hole are respectively provided with 2 holes;
the concentrated washing solution needs to be prepared by using distilled water or deionized water as a solvent in a ratio of 1:25, diluting;
the plate washing specifically comprises the following operations: the plate was washed 5 times with concentrated wash solution, each time for 1 minute, patted dry. When reading the result with a microplate reader, the blank wells should be zeroed. In order to ensure the accurate result, the result is judged within 5-10 minutes after the reaction is ended.
The detection principle of the application is as follows: the EB virus capsid antigen recombinant antigen is coated on a coating plate, when in detection, the serum to be detected is incubated in the plate hole, if specific EB Virus Capsid Antigen (VCA) antibody exists, the antibody is combined with solid phase EB virus capsid antigen recombinant antigen, unbound components are removed by washing, then enzyme-labeled anti-human IgA is added, incubating in a plate hole, combining IgA component in a solid-phase specific antigen-antibody compound with an enzyme-labeled antibody, removing the unbound components by washing, adding a substrate (TMB) for incubating in the plate hole, reacting an enzyme (HRP) in the solid-phase compound with the substrate to generate a color (blue), adding sulfuric acid to terminate the reaction (the color is changed from blue to yellow), measuring the absorbance (A) value at a wavelength of 450nm by using an enzyme-labeling instrument, thereby qualitatively determining whether the EB virus capsid antigen IgA antibody exists in the sample so as to judge whether the EB virus is infected.
Meanwhile, the application is provided with a control group, the blank control group, the negative control liquid, the quality control blood clear liquid and the blood sample to be detected are subjected to absorbance measurement together to verify the effectiveness of the test, if the blank control A value is less than or equal to 0.08, the quality control serum A value is less than 2.4 and the negative control A value is less than 0.12, the test is established, and the detection accuracy is further improved.
Compared with the prior art, the invention has the following advantages:
the application provides an EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit, which comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate, wherein the EB virus capsid antigen recombinant antigen is coated on the coating plate, the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA, the kit is prepared by using a microporous plate coated by a purified gene recombinant EB virus nuclear antigen and enzyme-labeled anti-human IgA and other reagents, the IgA antibody for qualitatively detecting the EB virus capsid antigen in human serum by using the principle of an ELISA indirect method has the advantages of high sensitivity, strong specificity and strong anti-interference capacity, has good specificity on EB virus NA1 positive IgA, EB virus VCA IgM positive samples and EB virus ZTa positive samples, and has good specificity on giant cells, herpes simplex virus types 1, II, liver cancer, and the like, Intestinal cancer, head and neck cancer, lung cancer, lymphoma disease and other diseases have no obvious cross reaction, and do not interfere the detection of the product on hyperlipemia, hyperbilirubinemia, hemolysis, anticoagulation of sodium citrate and EDTA-2Na anticoagulation samples.
The invention relates to an EB virus capsid antigen IgA antibody enzyme-linked immunosorbent assay method, which detects through the immune specific reaction of an antigen antibody, realizes qualitative detection of the IgA antibody of EB Virus Capsid Antigen (VCA) in human serum by an enzyme-linked immunosorbent assay method based on the principle of indirect method, has the characteristics of high specificity and strong accuracy, has simple and convenient steps and reasonable design, and is suitable for popularization.
Drawings
FIG. 1 shows the purity and molecular weight identification results of EB virus capsid antigen recombinant antigen of the present invention.
Detailed Description
The following describes specific embodiments of the present invention with reference to the specific embodiments.
The kit comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coating plate, wherein the coating plate is coated with an EB virus capsid antigen recombinant antigen, and the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA prepared according to the working concentration of 1: 25000.
The preparation method of the EB virus capsid antigen recombinant antigen coated on the coating plate comprises the following steps:
(1) coating: diluting the EB virus capsid antigen recombinant antigen with a coating buffer solution, coating the EB virus capsid antigen recombinant antigen into the hole of a coating plate according to 95 mu L/hole, and incubating and adsorbing for 18 hours at the temperature of 28 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate, and then incubating and sealing at 2 ℃ for 22 hours by using a sealing solution according to 155 mu L/hole;
(3) and (3) drying and storing: removing the sealing liquid, drying at 30 deg.C for more than 4 hr, sealing, and storing.
The sample diluent is a solution containing a protein stabilizer and a preservative;
the concentrated washing solution is PBS buffer solution containing Tween-20;
the substrate A solution is a solution containing carbamide peroxide;
the substrate B solution is a solution containing TMB;
the stop solution is 1mol/L sulfuric acid solution;
the negative control solution is EB virus capsid antigen IgA antibody negative human serum with the A value less than 0.12;
the quality control serum is inactivated EB virus capsid antigen lgA antibody positive human serum with the A value range of 0.8-2.4.
Embodiment 2, an EB virus capsid antigen IgA antibody enzyme-linked immunosorbent assay kit, comprising an enzyme conjugate, a sample diluent, a negative control solution, a quality control blood clear solution, a substrate a solution, a substrate B solution, a stop solution, a concentrated washing solution, and a coating plate, wherein an EB virus capsid antigen recombinant antigen is coated on the coating plate, and the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA prepared at a working concentration of 1: 25000.
The preparation method of the EB virus capsid antigen recombinant antigen coated on the coating plate comprises the following steps:
(1) coating: diluting the EB virus capsid antigen recombinant antigen with a coating buffer solution, coating the EB virus capsid antigen recombinant antigen into the hole of a coating plate according to 105 mu L/hole, and carrying out incubation and adsorption for 24 hours at 23 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate, and then incubating and blocking for 20 hours at 4 ℃ by using a blocking solution according to 145 mu L/hole;
(3) drying and storing: removing the sealing liquid, drying at 35 deg.C for more than 4 hr, sealing, and storing.
The sample diluent is a solution containing a protein stabilizing agent and a preservative;
the concentrated washing solution is PBS buffer solution containing Tween-20;
the substrate A solution is a solution containing carbamide peroxide;
the substrate B solution is a solution containing TMB;
the stop solution is 1mol/L sulfuric acid solution;
the negative control solution is EB virus capsid antigen IgA antibody negative human serum with the A value less than 0.12;
the quality control serum is inactivated EB virus capsid antigen lgA antibody positive human serum with the A value range of 0.8-2.4.
Embodiment 3, an EB virus capsid antigen IgA antibody enzyme-linked immunosorbent assay kit, comprising an enzyme conjugate, a sample diluent, a negative control solution, a quality control blood clear solution, a substrate a solution, a substrate B solution, a stop solution, a concentrated washing solution, and a coating plate, wherein an EB virus capsid antigen recombinant antigen is coated on the coating plate, and the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA prepared at a working concentration of 1: 25000.
The preparation method of the EB virus capsid antigen recombinant antigen coated on the coating plate comprises the following steps:
(1) coating: diluting EB virus capsid antigen recombinant antigen with coating buffer solution, coating the EB virus capsid antigen recombinant antigen into the hole of a coating plate according to 100 mu L/hole, and carrying out incubation and adsorption for 20 hours at 25 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate, and then incubating and blocking the plate for 18 hours at 8 ℃ by using a blocking solution according to 150 mu L/hole;
(3) drying and storing: removing the blocking liquid, drying at 37 deg.C for more than 4 hr, sealing, and storing.
The sample diluent is a solution containing a protein stabilizing agent and a preservative;
the concentrated washing solution is PBS buffer solution containing Tween-20;
the substrate A solution is a solution containing carbamide peroxide;
the substrate B solution is a solution containing TMB;
the stop solution is 1mol/L sulfuric acid solution;
the negative control solution is EB virus capsid antigen IgA antibody negative human serum with the A value less than 0.12;
the quality control serum is inactivated EB virus capsid antigen lgA antibody positive human serum with the A value range of 0.8-2.4.
Clinical test experiments were conducted on the kits prepared in examples 1 to 3 above
The detection steps are as follows:
s1, sample adding: taking a coating plate coated with an EB virus capsid antigen recombinant antigen, setting 2 holes of blank control, adding 100 mu L of sample diluent, additionally setting 2 holes of a negative control hole and a quality control serum hole, respectively adding 100 mu L of negative control liquid and 100 mu L of quality control serum, setting a sample hole to be detected, adding 100 mu L of sample diluent and 5 mu L of sample to be detected, uniformly mixing, covering with a sealing plate adhesive, and then placing at 37 ℃ for incubation for 30 minutes;
s2, adding enzyme conjugate: washing the plate with concentrated washing solution for 5 times, standing for 1 min each time, drying, adding enzyme conjugate 100 μ L/hole, covering with sealing plate glue, and incubating at 37 deg.C for 30 min;
s3, color development and termination: washing the plate with concentrated washing solution for 5 times, standing for 1 min each time, drying, adding 50 μ L/well of substrate A solution and substrate B solution, developing at 37 deg.C in dark for 15 min, and adding 50 μ L of stop solution;
s4, absorbance measurement: selecting the wavelength of 450nm by using a microplate reader, calibrating zero by using blank holes, and measuring the A value of each hole;
s5, analyzing and judging results: and (5) testing the result through step S4 to verify the effectiveness of the test, if the following conditions are met: the blank control A value is less than or equal to 0.08, the quality control serum A value is less than 2.4 and the negative control A value is less than 0.12, so the test is effective; and judging the detection result through a critical value, wherein the critical value is the average A value of the quality control serum multiplied by 20 percent, and is judged to be negative when the detection A value of the sample to be detected is less than the critical value, and is judged to be positive when the detection A value of the sample is more than or equal to the critical value.
The test instrument is as follows:
a ThermoLabsystemsMultskanMk3 microplate reader; a ThermoWELLWASH4MK2 plate washer; PYX-DHS-40X 50-B water-resisting electric heating constant temperature incubator.
The test trials included the following items:
(1) the accuracy is as follows:
testing a sample: 15 parts of positive factory quality control material and 15 parts of negative factory quality control material.
The test method comprises the following steps: the EB virus capsid antigen IgA antibody diagnostic kits prepared in the embodiments 1-3 are used for respectively carrying out accuracy test on test samples, and judging that the test samples are negative or positive, and the test results are shown in a table 12:
TABLE 12 results of the Performance test of examples 1 to 3
As shown in the test results in Table 12, the positive reference sample coincidence rate is 15/15, and the negative reference sample coincidence rate is 15/15. The kit of the application has good accuracy.
(2) And (3) specific detection:
cross reaction of other detection indexes of EBV
Testing a sample: EB virus NA1 IgA positive, EB virus VCAIgM positive, EB virus ZTaIgA positive sample.
The test method comprises the following steps: and adding excessive EB virus capsid antigen recombinant antigen into EB virus NA1 IgA positive, EB virus VCAIgM positive and EB virus ZTaIgA positive samples to neutralize the possibly existing EB virus capsid antigen IgA antibody. The following tests were then performed and the results are shown in table 13 below:
TABLE 13 test results for each sample
The sample added with the excessive EBV VCA antigen is detected by the kit prepared in the embodiment 1-3 of the application, the specificity of the kit is checked by statistically analyzing the experimental results, and the results are shown in Table 14:
TABLE 14 Cross-reaction test results
The above results show that: the specificity of the EB virus capsid antigen IgA kit to EB virus NA1 IgA positive samples, EB virus VCAIgM positive samples and EB virus ZTaIgA positive samples is 100%, so the EB virus NA1 IgA positive samples, EB virus VCAIgM positive samples and EB virus ZTaIgA positive samples do not influence the detection result of the kit.
b. Cross-reaction of other related diseases
The test method comprises the following steps: fluorescent quantitative PCR detection of cytomegalovirus and herpes simplex virus type 1, II using EB virus
Samples of type, liver cancer, intestinal cancer, head and neck cancer, lung cancer and lymphoma were detected according to the detection method, the specificity of the kits of examples 1-3 was examined, and the test results are shown in table 15:
TABLE 15 results of the specificity test of examples 1 to 3
The above results show that: the specificity of the EB virus capsid antigen IgA kit to giant cells, herpes simplex virus type 1, type II, liver cancer, intestinal cancer, head and neck cancer, lung cancer and lymphoma diseases is similar to that of physical examination of healthy people, namely the kit has no obvious cross reaction to the diseases.
(3) Anti-interference experiment:
testing an interference sample:
taking 5 parts of quality control products in the factory, and respectively adding 5 mu L of triglyceride into the quality control products, wherein the volume percentage (V/V) is 5 percent, which indicates that the content of the triglyceride in the serum is 21.54 mmol/L;
taking 0.1mL of each of 5 parts of in-plant quality control products, respectively adding 6 mu L of bilirubin solution (5mg/mL), wherein the volume percentage (V/V) is 6 percent, which indicates that the content of bilirubin solution in serum is 818 mu mol/L;
taking 5 parts of factory quality control products, 0.5mL each, and adding 9mg of hemoglobin respectively to make the content of hemoglobin in serum 18 mg/mL;
taking 5 parts of factory quality control products, 0.5mL each, and adding 3% sodium citrate solution according to a ratio of 1:9 respectively;
0.5mL of each of 5 parts of in-plant quality control materials was added with 1.5% EDTA-2Na solution at a ratio of 0.4: 5.
The test method comprises the following steps:
the samples added with the interference substances with different concentrations are detected by using the kit of the embodiments 1-3 according to the detection method, the accuracy of the samples is detected by using the detection kit under the condition that the interference substances (hyperlipidemia, hyperbilirubicin, hemolysis, anticoagulation of sodium citrate and anticoagulation of EDTA-2 Na) exist, and the test results are shown in tables 16, 17 and 18.
TABLE 16 anti-interference test results of the kit of example 1
TABLE 17 anti-interference test results of the kit of example 2
TABLE 18. results of the anti-interference test of the kit of example 3
The above results show that: the samples of hyperlipemia, hyperbilirubicin, hemolysis, sodium citrate anticoagulation and EDTA-2Na anticoagulation have no obvious influence on the detection result of the EB virus capsid antigen IgA kit prepared in the embodiment 1-3.
In summary, the present application performs analytical performance detection on the EB virus capsid antigen IgA antibody diagnostic kit prepared in examples 1 to 3, and it can be seen from the research test results that the minimum detection limit of the kit meets the requirement, the accuracy and precision are good, both the positive compliance rate and the negative compliance rate meet the requirement, common interfering substances and cross diseases do not affect the detection result, and all indexes can reach the product quality standard, and the kit has the characteristics of high specificity and strong accuracy.
Claims (4)
1. An EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit is characterized in that: comprises an enzyme conjugate, a sample diluent, a negative control solution, a quality control serum, a substrate A solution, a substrate B solution, a stop solution, a concentrated washing solution and a coated plate;
the coating plate is coated with EB virus capsid antigen recombinant antigen;
the enzyme conjugate is horse radish peroxidase-labeled anti-human IgA;
the enzyme conjugate is enzyme-labeled anti-human IgA obtained by labeling with peroxidase by adopting a sodium periodate method, and the use concentration of the enzyme-labeled anti-human IgA is 1: 25000;
the preparation method of the EB virus capsid antigen recombinant antigen coated on the coating plate comprises the following steps:
(1) coating: diluting the EB virus capsid antigen recombinant antigen with a coating buffer solution, coating the EB virus capsid antigen recombinant antigen into the holes of a coating plate according to 95-105 mu L/hole, and carrying out incubation and adsorption for 18-24 hours at 18-28 ℃;
(2) and (3) sealing: discarding the coating solution, washing the plate, and then incubating and sealing for 18-22 hours at 2-8 ℃ by using sealing solution according to 145-155 mu L/hole;
(3) drying and storing: removing the sealing liquid, drying at 30-37 ℃ for more than 4 hours, and sealing for storage;
the EB virus capsid antigen recombinant antigen is prepared by adopting a gene engineering technology, and is prepared by adopting the following method: selecting the rear 176 amino acid parts of the EB virus capsid antigen protein as target antigens, taking mRNA sequences corresponding to the EB virus capsid antigen as templates, obtaining target gene fragments through RT-PCR, then transferring the target gene fragments into a pGEX-2T gene fusion expression system, carrying out enzyme digestion sequencing identification, carrying out IPTG induced expression on recombinant colonies, purifying an expression product through GST affinity chromatography, cutting off GST parts by thrombin, and finally obtaining the recombinant EB virus capsid antigen.
2. The EB virus capsid antigen IgA antibody enzyme-linked immunoassay kit according to claim 1, wherein: the molecular weight of the EB virus capsid antigen recombinant antigen is 33-35 kD, and the purity is more than or equal to 90%.
3. The EB virus capsid antigen IgA 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;
in the step (2), the plate is washed by using 10mmol/L concentrated washing solution according to 300 mu L/hole.
4. The kit for ELISA detection of EB virus capsid antigen IgA antibody according to claim 1, wherein: the sample diluent is a solution containing a protein stabilizing agent and a preservative;
the concentrated washing solution is PBS buffer solution containing Tween-20;
the substrate A solution is a solution containing carbamide peroxide;
the substrate B solution is a solution containing TMB;
the stop solution is 1mol L sulfuric acid solution;
the negative control solution is EB virus capsid antigen IgA antibody negative human serum with the A value less than 0.12;
the quality control serum is inactivated EB virus capsid antigen lgA antibody positive human serum with the A value range of 0.8-2.4.
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| CN101144817A (en) * | 2003-08-20 | 2008-03-19 | 香港神农有限公司 | EB virus protein ELISA diagnosis reagent kit and its preparation method |
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101144817A (en) * | 2003-08-20 | 2008-03-19 | 香港神农有限公司 | EB virus protein ELISA diagnosis reagent kit and its preparation method |
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
Non-Patent Citations (1)
| Title |
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| Evaluation of seven recombinant VCA-IgA ELISA kits for the diagnosis of nasopharyngeal carcinoma in China: a case-control trial;Rui Gao et al.;《BMJ OPEN》;20171231;第7卷;1-8 * |
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