CN111007248A - Chemiluminescence immunoassay kit for detecting rubella virus IgM antibody - Google Patents
Chemiluminescence immunoassay kit for detecting rubella virus IgM antibody Download PDFInfo
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Abstract
The invention discloses a chemiluminescence immunoassay kit for detecting rubella virus IgM antibody, belonging to the technical field of immunoassay and comprising a reagent R1, a reagent R2 and a reagent R3; wherein: the reagent R1 is a buffer solution containing a magnetic bead coated anti-human IgM antibody, the concentration of the magnetic bead coated anti-human IgM antibody is 0.5-5%, and the particle size of the magnetic bead is 0.05-3 μm; the reagent R2 is a buffer solution containing a rubella virus antibody marked by a chemiluminescent marker, the concentration of the rubella virus antibody marked by the chemiluminescent marker is 0.1-1.0 mu g/mL, and the marking ratio of the antibody to the marker is 1: 3-1: 20; the reagent R3 is a buffer solution of rubella virus antigen, and the concentration of the rubella virus antigen is 0.1-2.0 mu g/mL. The invention reduces the background blank, improves the sensitivity, does not need enzyme to participate, saves time and cost, can be directly used on a full-automatic biochemical analyzer, does not need large-scale instrument and equipment to be matched, has lower cost and no radioactive pollution, and can be developed and popularized on a large scale.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminescence immunoassay kit for detecting rubella virus IgM antibody, a preparation method and a test method thereof.
Background
Rubella virus (Rubella virus) is the only member of the family togaviridae, the genus Rubella, and humans are the only natural host. The rubella disease itself is not serious, but the greatest threat to public health by rubella virus is its teratogenicity. The infection of the pregnant women with rubella virus can cause congenital damage to the infants, and especially if the pregnant women are infected with rubella virus in the first 3 months of pregnancy, the virus can infect fetuses through blood, so that spontaneous abortion, stillbirth or fetal infection is caused, and serious birth defects of the fetuses are caused: including cataract, deafness, heart disease, and mental retardation, is called Congenital Rubella Syndrome (CRS). Rubella virus can be prevented to a certain extent by vaccination, but once infected, no specific treatment mode exists at present, so that the clinical importance of rubella virus infection, laboratory diagnosis and epidemic characteristics is extremely important.
The detection of the rubella specific antibody can be used for judging the individual immune state, and is helpful for prompting the serological status of the previous infection of the individual and diagnosing and preventing acute rubella infection. After an individual is infected with Rubella virus, the humoral immune reaction firstly generates immune globulin mainly comprising IgM, and then the IgM antibody concentration is reduced, so that the detection of anti-Rubella IgM can be used as an index for early diagnosis of Rubella virus infection; the Rubella IgM antibody is detectable in most Rubella virus acute viral infections and is of great importance to identify pregnant women as being acute, recent or recurrent.
The currently commonly used method for detecting the rubella virus IgM antibody comprises a Radioimmunoassay (RIA), an enzyme-linked immunoassay (ELISA) and an immunoblotting method (WB), but the RIA has the defects of radioactive pollution, short half-life of a marker, radioactive damage to an operator, complex operation, long detection time and the like; ELISA has low sensitivity, narrow detection range, long detection time, poor repeatability and the like; reagents for immunoblotting require low temperature storage, etc.
The detection kit is a chemiluminescence immunoassay, and the method has the advantages of simple operation, no radioactivity risk and short detection time, and can detect target substances such as antigens and antibodies; on the other hand, the rubella virus IgM chemiluminescence immunoassay kit and a chemiluminescence immunoassay instrument form a closed system, so that the error of manual operation is reduced, and the sensitivity and accuracy of the whole system are improved.
Disclosure of Invention
The invention aims to provide a chemiluminescence immunoassay kit for detecting rubella virus IgM antibody.
The invention also aims to provide a preparation method of the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the invention provides a chemiluminescence immunoassay kit for detecting rubella virus IgM antibody, which comprises a reagent R1, a reagent R2 and a reagent R3;
wherein:
the reagent R1 is a buffer solution containing a magnetic bead coated anti-human IgM antibody, the concentration of the magnetic bead coated anti-human IgM antibody is 0.5-5%, and the particle size of the magnetic bead is 0.05-3 μm;
the reagent R2 is a buffer solution containing a rubella virus antibody marked by a chemiluminescent marker, the concentration of the rubella virus antibody marked by the chemiluminescent marker is 0.1-1.0 mu g/mL, and the marking ratio of the antibody to the marker is 1: 3-1: 20;
the reagent R3 is a buffer solution of rubella virus antigen, and the concentration of the rubella virus antigen is 0.1-2.0 mu g/mL.
Preferably, the anti-human IgM antibody in reagent R1 is an anti-human IgM murine monoclonal antibody.
Preferably, the rubella virus antibody in the reagent R2 is a rubella virus E1 protein monoclonal antibody.
Preferably, the chemiluminescent label in R2 is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
Preferably, the rubella virus antigen in R3 is inactivated rubella virus.
Preferably, the buffer solution and the storage solution used by the reagent are buffer solution which contains a surfactant, a preservative and protein and has the pH value of 6.0-8.0; wherein the buffer salt is citrate, Bistris propane or phosphate.
Preferably, the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody further comprises 2.0S/CO and 0.0S/CO calibrators.
Preferably, the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody further comprises quality control substances of 2.0S/CO and 0.5S/CO.
Preferably, the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody further comprises chemiluminescence substrate solution A and chemiluminescence substrate solution B, wherein the solution A is hydrogen peroxide and nitric acid solution, and the solution B is sodium hydroxide solution.
Preferably, the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody further comprises a cleaning solution, and the cleaning solution is a PB buffer solution.
The detection principle of the kit is a capture method. Incubating the sample with an anti-human IgM antibody R1 solution (40 mu L) and an R3 antigen solution (50 mu L) coated by magnetic beads for 18min, cleaning for 5min, reacting with an acridinium ester labeled rubella virus antibody solution (50 mu L) to form a magnetic microsphere-anti-human IgM antibody-rubella virus-acridinium ester compound, separating the immune compound by a magnetic separator, and washing off free components. The acridinium ester is excited to emit light by an acid-base excitation liquid, the relative luminous intensity (RLU) is recorded, and the instrument (comparing the chemiluminescence signal value obtained from the reaction product of the sample with the cutoff value obtained by previous calibration) automatically calculates the detection result.
When the chemiluminescence immunoassay kit for detecting the Rubella virus IgM antibody is used for detecting the Rubella IgM antibody, a large number of samples are detected by using a full-automatic chemiluminescence immunoassay analyzer, a formula is calculated by a statistical method, and the chemiluminescence immunoassay kit is arranged in a radio frequency card; then, testing two-point calibration, and calibrating the main calibration curve; and then testing the quality control product, and judging that the calibration is qualified when the test result of the quality control product falls within the target value range. Testing an actual clinical sample, and calculating the concentration of the sample according to the luminous value of the sample; the results of the samples are expressed as reactivity or non-reactivity and a cutoff index (sample signal value/cutoff value, S/CO), and finally the performance of the rubella virus IgM antibody detection kit is evaluated.
Compared with the prior art, the chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody has the beneficial effects that:
1. acridinium ester and the like are selected as the labeling materials of a chemiluminescence immunoassay system, and the materials have the energy transition generated when an excited state returns to a ground state as direct chemiluminescence, so that the background blank is reduced, and the sensitivity is improved; does not need enzyme participation, and saves time and cost. The paramagnetic particles are used as solid phase carriers, the specific surface area is large, and the detection sensitivity is further improved.
2. The rubella virus IgM antibody chemiluminescence immunoassay kit is a full-automatic measurement sample, and directly gives numerical values, reduces artificial operation errors, and realizes unattended operation. If the device can be directly used on a full-automatic biochemical analyzer, large-scale instrument and equipment cooperation is not needed, the cost is lower, radioactive pollution is avoided, and large-scale development and popularization can be realized.
3. The reagent and the instrument form a closed system, and the system error is small. The chemiluminescence immunoassay kit can replace kits such as ELISA methods on the market in the aspect of accuracy, and provides more rapid and accurate measurement data for customers.
4. The calibration material and the quality control material used in the rubella virus IgM antibody detection kit are assigned by other commercial rubella virus IgM antibody detection kits (rubella virus IgM detection kits produced by Yapei trade (Shanghai) Co., Ltd.) to ensure the accuracy of the test result.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a preparation process of the chemiluminescence immunoassay kit for detecting rubella virus IgM antibody.
Detailed Description
The chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody comprises a reagent R1, a reagent R2 and a reagent R3;
wherein:
the reagent R1 is a buffer solution containing a magnetic bead coated anti-human IgM antibody, the concentration of the magnetic bead coated anti-human IgM antibody is 0.5-5%, and the particle size of the magnetic bead is 0.05-3 μm;
the reagent R2 is a buffer solution containing a rubella virus antibody marked by a chemiluminescent marker, the concentration of the rubella virus antibody marked by the chemiluminescent marker is 0.1-1.0 mu g/mL, and the marking ratio of the antibody to the marker is 1: 3-1: 20;
the reagent R3 is a buffer solution of rubella virus antigen, and the concentration of the rubella virus antigen is 0.1-2.0 mu g/mL.
The preparation process of the reagents R1, R2 and R3 is shown in fig. 1, and includes screening antigen and antibody, preparing antigen solution by respectively coating antibody on magnetic beads and labeling antibody with chemiluminescent marker (such as acridinium ester), mixing according to the concentration of the antibody coated on magnetic beads and the concentration of the antibody labeled with chemiluminescent marker (such as acridinium ester), and packaging the reagents.
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
Example 1 chemiluminescence immunoassay kit for detecting rubella virus IgM antibody of the invention
(1) Preparation of rubella virus IgM antibody calibrator and quality control product
A protein solution of a rubella virus IgM antibody is taken, a commercial rubella virus IgM antibody detection kit (a rubella virus IgM detection kit produced by Yapei trade (Shanghai) Co., Ltd.) is used for assignment, and a rubella virus IgM antibody protein solution with the assignment result of 5.0S/CO is taken and diluted into 2.0S/CO by a calibrator matrix to be used as a high-concentration calibrator.
The base material of the calibrator was assigned a value of 0.0S/CO and used as a low concentration calibrator.
And taking the rubella virus IgM antibody protein solution with the assignment result of 5.0S/CO, and diluting the rubella virus IgM antibody protein solution into 2.0S/CO by using a quality control substance matrix to be used as a high-concentration quality control substance.
And taking the rubella virus IgM antibody protein solution with the assignment result of 5.0S/CO, and diluting the rubella virus IgM antibody protein solution into 0.5S/CO by using a quality control substance matrix to be used as a low-concentration quality control substance.
(2) Preparation of reagent R1
Uniformly mixing a solution of 10mg of Tosyl magnetic beads (with the particle size of 0.05-3 um), carrying out magnetic separation to remove supernatant, washing the mixture with 0.02M PB buffer solution for three times, then carrying out resuspension, adding 0.05mg of coated antibody, uniformly mixing, incubating at 37 ℃ for 2-4 h under continuous rotation and uniform mixing, washing the supernatant after magnetic separation with 0.02M PB buffer solution for three times, then sealing with 20% lysine, carrying out magnetic separation to remove the supernatant, using storage buffer solution to resuspend the magnetic beads, wherein the concentration of the coated antibody of the magnetic beads is 0.5%, and collecting and storing at 2-8 ℃ for subsequent experiments.
(3) Preparation of reagent R2
Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging the antibody at room temperature for 20s), adding a carbonic acid buffer solution, fully and uniformly mixing, adding 0.75mL of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging the mixture at room temperature for 20s by using a centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 2 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 deg.C), mixing at medium speed, and blocking for 0.5 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And storing the collected antibody solution at the temperature of 2-8 ℃. When in use, the purified rubella virus antibody concentrated solution is diluted by a buffer solution of 100mM PB, 0.05% Tween, 0.05% Proclin300 and pH8.0 to a final concentration of 0.1 mu g/mL, and is stored at 2-8 ℃.
(4) Preparation of reagent R3
The inactivated rubella virus was dissolved at a concentration of 0.1ug/mL in a pH8.0 phosphate buffer containing surfactant, preservative and protein, and the rubella virus antigen was present at a concentration of 0.1 ug/mL.
Example 2 chemiluminescent immunoassay kit for detecting rubella virus IgM antibody of the present invention
(1) Preparation of calibrator and quality control material
The same as in example 1.
(2) Preparation of reagent R1
Uniformly mixing a solution of 10mg of Tosyl magnetic beads (with the particle size of 0.05-3 um), carrying out magnetic separation to remove supernatant, washing the mixture with 0.02M PB buffer solution for three times, then carrying out resuspension, adding 0.25mg of coated antibody, uniformly mixing, incubating for 2-4 h at 37 ℃ under continuous rotation and uniform mixing, washing the supernatant after magnetic separation with 0.02M PB buffer solution for three times, then sealing with 20% lysine, carrying out magnetic separation to remove the supernatant, using storage buffer solution to resuspend the magnetic beads, wherein the concentration of the coated antibody of the magnetic beads is 2.5%, and collecting and storing at 2-8 ℃ for subsequent experiments.
(3) Preparation of reagent R2
Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging the antibody for 30s at room temperature by a centrifuge), adding carbonic acid buffer solution, fully and uniformly mixing, adding 2.5mL of 2mg/mL luminol DMF solution after uniformly mixing, and centrifuging the mixture for 30s at room temperature by the centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 3 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 ℃), and mixing at medium speed for 1 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And storing the collected antibody solution at the temperature of 2-8 ℃. When in use, the purified rubella virus antibody concentrated solution is diluted by a buffer solution of 100mM citric acid, 0.05% Tween, 0.05% Proclin300 and pH6.5 until the final concentration is 0.5 mu g/mL, and the solution is stored at the temperature of 2-8 ℃.
(4) Preparation of reagent R3
Inactivated rubella virus at a concentration of 0.5ug/mL was dissolved in a citrate buffer ph6.5, containing surfactant, preservative and protein, with a rubella virus antigen concentration of 1.0 μ g/mL.
Example 3 chemiluminescent immunoassay kit for detecting rubella virus IgM antibody of the present invention
(1) Preparation of calibrator and quality control material
The same as in example 1.
(2) Preparation of reagent R1
Uniformly mixing a solution of 10mg of Tosyl magnetic beads (with the particle size of 0.05-3 um), carrying out magnetic separation to remove supernatant, washing the mixture with 0.02M PB buffer solution for three times, then carrying out resuspension, adding 0.5mg of coated antibody, uniformly mixing, incubating at 37 ℃ for 2-4 h under continuous rotation and uniform mixing, washing the supernatant after magnetic separation with 0.02M PB buffer solution for three times, then sealing with 20% lysine, carrying out magnetic separation to remove the supernatant, using storage buffer solution to resuspend the magnetic beads, wherein the concentration of the coated antibody of the magnetic beads is 5%, and collecting and storing at 2-8 ℃ for subsequent experiments.
(3) Preparation of reagent R2
Putting 500 mu g of the antibody into a centrifuge tube, ensuring that the antibody is positioned at the bottom of the centrifuge tube (centrifuging for 40s at room temperature by a centrifuge), adding carbonic acid buffer solution, fully and uniformly mixing, adding 5mL of 2mg/mL DMF solution of terpyridyl ruthenium after uniformly mixing, and centrifuging for 40s at room temperature by the centrifuge. Sealing the centrifuge tube with sealing film, placing in dark cassette, placing the cassette in gas bath constant temperature oscillator (25 deg.C), and mixing for 4 hr. Adding 1mL of 20% lysine blocking solution, placing into a gas bath constant temperature oscillator (25 ℃), and mixing at medium speed for 2 h. Purifying the blocked antibody by a sephadex G250 column, eluting by a PB buffer solution, and collecting step by step. And storing the collected antibody solution at the temperature of 2-8 ℃. When in use, the purified rubella virus antibody concentrated solution is diluted by a buffer solution of 100mM BisTris propane, 0.05% Tween, 0.05% Proclin300 and pH6.0 until the final concentration is 1.0 mu g/ml, and the solution is stored at the temperature of 2-8 ℃.
(4) Preparation of reagent R3
The inactivated rubella virus at a concentration of 2.0ug/mL was dissolved in a pH6.0 Bistris propane buffer containing surfactant, preservative and protein, and the rubella virus antigen concentration was 2.0 μ g/mL.
Example 4 evaluation of the Performance of the chemiluminescence immunoassay kit for detecting the IgM antibody of rubella virus of the present invention
The rubella virus IgM antibody chemiluminescence immunoassay kit of the invention example 1 is adopted to detect rubella virus IgM antibody national reference (procurement inspection institute, batch number: 360006-201702).
The detection method comprises the following steps:
a dire full-automatic chemiluminescence immunoassay analyzer (CM-180) is used as a detection instrument, the methodology is a capture method, the sample volume is 10 mu L, the reagent volume of the anti-human IgM monoclonal antibody coated by the magnetic beads is 40 mu L, the reagent volume of the rubella virus antigen solution is 50 mu L, and the reagent volume of the rubella virus antibody marked by the chemiluminescence substance is 50 mu L. Firstly, 10 μ L of sample is added, 90 μ L of diluent is added, the sample is diluted by 10 times, then R3 reagent and R1 reagent are respectively added, the incubation is carried out for 18min, the washing is carried out for 5min (the washing solution is PB buffer solution), the R2 reagent is added, the incubation is carried out for 5min, and the washing is carried out for 5min (the washing solution is PB buffer solution). The instrument sends the reactant into a dark room, and adds the luminous excitation liquid A (H) once2O2And HNO3Solution) and solution B (NaOH solution) were reacted and finally the relative luminescence intensity (RLU) was recorded. The instrument (compare chemiluminescence signal values obtained from sample reaction products with cutoff values obtained from previous calibrations) automatically calculates the detection results.
The detection results are as follows:
(1) negative reference product compliance rate
The prepared kit is used for measuring the national negative reference products, the results are shown in table 1, the test results are all negative, and the test results completely accord with the national negative reference products.
TABLE 1 measurement results of sexual reference
(2) Positive reference compliance rate
The prepared kit is used for determining the national positive reference substance, the results are shown in table 2, and the test results are all positive and completely accord with the national positive reference substance.
TABLE 2 Positive reference assay results
(3) Reference product for detection limit
The prepared kit is used for measuring the national detection limit reference substance, the result is shown in table 3, and the test result meets the requirement of the national detection limit reference substance.
TABLE 3 measurement results of detection limit reference
(4) Repetitive reference (R)
The kit is used for carrying out repeated detection on national reference substances, the repeated detection is carried out for 10 times, the average value and the standard deviation SD of 10 measurement results are calculated, the Coefficient of Variation (CV) is not more than 15% according to the formula (1), and the test results are shown in the table 4: the coefficient of variation was 3.80%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 4 results of reproducibility measurement
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.
Claims (10)
1. A chemiluminescence immunoassay kit for detecting rubella virus IgM antibody is characterized by comprising a reagent R1, a reagent R2 and a reagent R3;
wherein:
the reagent R1 is a buffer solution containing a magnetic bead coated anti-human IgM antibody, the concentration of the magnetic bead coated anti-human IgM antibody is 0.5-5%, and the particle size of the magnetic bead is 0.05-3 μm;
the reagent R2 is a buffer solution containing a chemiluminescent marker labeled rubella virus antibody, the concentration of the chemiluminescent marker labeled rubella virus antibody is 0.1-1.0 mu g/mL, and the labeling ratio of the antibody to the marker is 1: 3-1: 20;
the reagent R3 is a buffer solution of rubella virus antigen, and the concentration of the rubella virus antigen is 0.1-2.0 mu g/mL.
2. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibody according to claim 1, wherein the anti-human IgM antibody in the reagent R1 is an anti-human IgM murine monoclonal antibody.
3. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibodies of claim 1, wherein the rubella virus antibody in the reagent R2 is rubella virus E1 protein monoclonal antibody.
4. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibody according to claim 1, wherein the chemiluminescent label in R2 is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
5. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibodies of claim 1 wherein the rubella virus antigen in R3 is inactivated rubella virus.
6. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibodies according to claim 1, wherein the buffer and storage solution used in the reagent are buffer solution with pH 6.0-8.0 containing surfactant, preservative and protein; wherein the buffer salt is citrate, Bistris propane or phosphate.
7. The chemiluminescent immunoassay kit for the detection of rubella virus IgM antibodies of claim 1 further comprising calibrators of 2.0S/CO and 0.0S/CO.
8. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibodies of claim 1 further comprising quality controls for 2.0S/CO and 0.5S/CO.
9. The chemiluminescence immunoassay kit for detecting the rubella virus IgM antibody according to claim 1, further comprising chemiluminescence substrate solution A and solution B, wherein the solution A is hydrogen peroxide and nitric acid solution, and the solution B is sodium hydroxide solution.
10. The chemiluminescent immunoassay kit for detecting rubella virus IgM antibody according to claim 1, further comprising a washing solution, wherein the washing solution is PB buffer solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112710840A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | S100 beta protein detection kit and S100 beta protein detection method |
| CN112763721A (en) * | 2020-12-23 | 2021-05-07 | 迪瑞医疗科技股份有限公司 | Rubella virus IgG antibody chemiluminescence immunoassay kit and preparation method thereof |
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