CN111007249A - Herpes simplex virus antibody IgG chemiluminescence immunoassay kit and preparation method thereof - Google Patents
Herpes simplex virus antibody IgG chemiluminescence immunoassay kit and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/035—Herpes simplex virus I or II
Abstract
The invention relates to a herpes simplex virus antibody IgG chemiluminescence immunoassay kit and a preparation method thereof, and belongs to the technical field of immunoassay. The method solves the technical problems of short reagent validity period, complex operation, low sensitivity and high detection cost in the method for detecting the herpes simplex virus IgG antibody in the prior art. The kit comprises an R1 reagent, an R2 reagent and project diluent, wherein the R1 reagent comprises a magnetic bead coated antigen, and the antigen is HSV-1 antigen or HSV-2 antigen; the R2 reagent comprises an anti-human IgG antibody labeled with a chemiluminescent label; the project diluent is a buffer solution containing salt, a surfactant, a preservative and protein, and the pH value of the buffer solution is 6.0-8.0. The kit has the advantages of high sensitivity, strong specificity, good stability, good repeatability, wide detection range, short detection time and relatively low cost, and can be widely used for clinical detection.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a herpes simplex virus antibody IgG chemiluminescence immunodetection kit and a preparation method thereof.
Background
Herpes simplex virus types I and II (HSV-1 and HSV-2) are two members of the herpes virus family. Morphologically similar to other members of the herpesviridae family, having a capsid and a DNA core. The infection rate of HSV-1 in the general population is about 70-80%, and the infection rate of HSV-2 is about 17-25%. HSV-1 and HSV-2 are transmitted by intimate contact between seronegative individuals and individuals secreting the virus. The clinical manifestations of HSV-1 and HSV-2 infection are diverse, as are mucosal and cutaneous lesions and diseases of the eye, gut and Central Nervous System (CNS). Infection of HSV in immunosuppressed patients can present as a serious widespread lesion. Although HSV-1 and HSV-2 are often transmitted by different pathways and infect different parts of the body, there remains much in common the epidemiological characteristics and clinical manifestations between these two viruses. Usually HSV-1 causes mucosal infections, such as those in the eyes, mouth and corners of the mouth, and is also one of the major causes of severe sporadic encephalitis in adults. HSV-2 mainly causes lesions of the skin and mucous membranes of the genital area: genital herpes is now one of the common sexually transmitted diseases. However, the relationship between the type of HSV virus and the site of infection is not absolute. Once HSV infection occurs, the virus can be latent in sensory nerve centers of human bodies, and the virus can recur periodically due to various stimuli, and clinical injury can be caused. Immunosuppressed patients are more susceptible to recurrent infections, suggesting that immune to serum antibodies and virus-specific cellular mediators may contribute to disease recovery.
The probability of abortion or premature delivery of pregnant women infected with genital herpes is 2-3 times of that of normal pregnant women. The active genital tract virus of the pregnant woman can cause the contact of the infant and the infected genital tract during production to cause serious HSV virus infection, if the pregnant woman is infected with HSV during production, 40-60% of the infants can also be infected, and if the pregnant woman is not treated in time, the infant has high morbidity and mortality. 35% of children by age 5 have antibodies to HSV-1 virus, 80% of adults by age 25 have antibodies specific for HSV-1 virus.
Since HSV-1 and HSV-2 share the same antigenic determinants, antibodies to both viruses may cross-react. Both types of viruses frequently relapse despite the presence of anti-viral antibodies in the body. The rapid and accurate diagnosis of HSV infection facilitates early antiviral treatment and reduces infection spread. After infection with HSV, the human immune system will immediately synthesize specific anti-HSV IgM antibody, which can be detected after one week.
The presence of HSV IgM antibodies in general is indicative of recent or recurrent infection. After 2-3 weeks in patients with primary infections, specific IgG antibodies will generally appear in vivo, but after several months, their titers will decline, while those in patients with recurrent infections will not increase. The immune status of the patient can be assessed by detection of IgG and serological evidence of past infection with HSV is provided. The results of seroconversion in conjunction with HSV-1 or HSV-2 antibodies may aid in the diagnosis of recent (primary or secondary) HSV infection.
In the prior art, common methods for detecting herpes simplex virus IgG antibodies include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and immunoblotting (WB), but 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 the defects of low sensitivity, narrow detection range, long detection time, poor repeatability and the like; WB has the disadvantage that reagents for immunoblotting need to be stored at low temperature.
Disclosure of Invention
In view of the above, in order to solve the technical problems of short reagent validity period, complex operation, low sensitivity and high detection cost of the method for detecting the herpes simplex virus IgG antibody in the prior art, the invention provides a chemiluminescence immunoassay kit for the herpes simplex virus IgG antibody and a preparation method thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows.
The herpes simplex virus antibody IgG chemiluminescence immunoassay kit comprises an R1 reagent, an R2 reagent and a project diluent;
the R1 reagent comprises a magnetic bead coated antigen, and the antigen is HSV-1 antigen or HSV-2 antigen;
the R2 reagent comprises an anti-human IgG antibody labeled with a chemiluminescent label;
the project diluent is a buffer solution containing salt, a surfactant, a preservative and protein, and the pH value of the buffer solution is 6.0-8.0.
Preferably, in the R1 reagent, the concentration of the antigen coated on the magnetic beads is 0.05 wt% to 0.2 wt%.
Preferably, the particle size of the magnetic bead used for coating the antigen is 0.5-3.0 μm.
Preferably, the mass ratio of the magnetic beads to the coating antigen is 10mg (50-500) μ g.
Preferably, the concentration of the anti-human IgG antibody labeled with a chemiluminescent label in the R2 reagent is 0.1 to 1.0. mu.g/ml.
Preferably, the chemiluminescent marker is labeled anti-human IgG antibody, and the labeling molar ratio of the anti-human IgG antibody to the chemiluminescent marker is 1 (3-10); the chemiluminescent marker is acridinium ester or ruthenium terpyridyl; the anti-human IgG is mouse anti-human IgG or sheep anti-human IgG.
Preferably, in the project dilution, the salt is 50-200 mM phosphate, citrate or Bistris propane; the surfactant is 0.02-0.05% of Tween 20 or 0.05-0.1% of choline chloride; the preservative is 0.01-0.03% of sodium azide and 0.01-0.03% of PC300, and the protein is 0.1-3% of bovine serum albumin and 0.01-0.2% of bovine gamma globulin.
Preferably, the kit further comprises one or more of a calibrator, a chemiluminescent excitation liquid and a washing liquid;
the calibrator comprises a positive calibrator and a negative calibrator, wherein the positive calibrator is prepared by inactivating herpes simplex virus IgG positive serum, filtering and diluting with a buffer solution; the negative calibrator is prepared by inactivating herpes simplex virus IgG negative serum, filtering and diluting with a buffer solution;
the chemiluminescence excitation liquid comprises a liquid A and a liquid B, wherein the liquid A is a hydrogen peroxide solution and a nitric acid solution, and the liquid B is a sodium hydroxide solution;
the cleaning solution is PB buffer solution.
The preparation method of the chemiluminescence immunoassay kit for the herpes simplex virus IgG comprises the following steps:
step one, preparing R1 reagent
Uniformly mixing the magnetic bead solution, magnetically separating to remove supernatant, washing the washing solution for multiple times, then carrying out resuspension, adding the coating antigen, uniformly mixing, incubating for 2-4 h at 37 ℃ under continuous rotation and uniform mixing, magnetically separating to remove supernatant, washing the washing solution for multiple times, then sealing by using lysine sealing solution, magnetically separating to remove supernatant, retaining the magnetic bead coating antigen, and carrying out resuspension to the required concentration by using buffer solution to obtain an R1 reagent;
step two, preparing R2 reagent
Firstly, placing an anti-human IgG monoclonal antibody into a centrifugal tube, ensuring that the anti-human IgG monoclonal antibody is positioned at the bottom of the centrifugal tube, adding a carbonic acid buffer solution, fully and uniformly mixing, adding an N, N-dimethylformamide solution of a chemiluminescent marker after uniformly mixing, and centrifuging for 20-40 s at room temperature by using a centrifuge;
then sealing the centrifuge tube with a sealing film, putting the centrifuge tube into a light-proof cassette, putting the cassette into a gas bath constant-temperature oscillator for uniformly mixing, adding lysine sealing liquid, putting the gas bath constant-temperature oscillator for uniformly mixing, and sealing for 0.5-2 h;
purifying the closed anti-human IgG monoclonal antibody on a sephadex G250 column, eluting with a phosphate buffer solution, and respectively collecting;
finally, diluting the collected anti-human IgG monoclonal antibody solution with a buffer solution to obtain an R2 reagent;
step three, preparing project diluent according to the formula of the project diluent;
and step four, subpackaging the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG.
Preferably, in the first step, the resuspension buffer is 25M Tris buffer with pH 7.2; in the second step, the buffer solution for dilution is the same as the item diluent.
Compared with the prior art, the invention has the beneficial effects that:
1. the herpes simplex virus IgG antibody chemiluminescence immunoassay kit adopts a chemiluminescence immunoassay method for detection, and the method has the advantages of simple operation, no radioactivity risk, short detection time, and capability of detecting target substances such as antigens and antibodies.
2. The chemiluminescence immunoassay kit for herpes simplex virus IgG selects chemiluminescent markers such as acridinium ester and the like as the marker materials of a chemiluminescence immunoassay method, the energy transition generated when the excited state returns to the ground state of the marker materials is direct chemiluminescence, enzyme participation is not needed, and time and cost are saved.
3. The chemiluminescence immunoassay kit for the herpes simplex virus IgG antibody can complete the detection of the herpes simplex virus IgG by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool; the kit is matched with an instrument to form a closed system, and the system error is small; the numerical value can be directly given, and the time required by clinical detection is shortened; the artificial operation error is reduced, and the detection precision and sensitivity are higher; and the unattended operation is realized, and the cost is low.
In conclusion, the chemiluminescence immunoassay kit for herpes simplex virus IgG has the advantages of high sensitivity, strong specificity, good stability, good repeatability, wide detection range, short detection time and relatively low cost, and can be widely used for clinical detection.
Detailed Description
For a further understanding of the invention, reference will now be made in detail to the specific embodiments of the invention, but it is to be understood that the description is intended to illustrate further features and advantages of the invention, and not to limit the scope of the invention as claimed.
The herpes simplex virus antibody IgG chemiluminescence immunoassay kit comprises an R1 reagent, an R2 reagent and a diluent;
the R1 reagent comprises a magnetic bead coated antigen, and the antigen is HSV-1 antigen or HSV-2 antigen; in order to stably store the magnetic bead coated antigen, the R1 reagent also comprises a buffer solution;
the R2 reagent comprises a chemiluminescent marker labeled anti-human IgG antibody, and in order to stably store the chemiluminescent marker labeled anti-human IgG antibody, the R2 reagent also comprises a buffer solution;
the project diluent is a buffer solution containing salt, a surfactant, a preservative and protein, and the pH value of the buffer solution is 6.0-8.0.
In the above technical solution, the buffer solution in the R1 reagent and the buffer solution in the R2 reagent are not particularly limited, and may be used to stably store the anti-human IgG antibody labeled with the magnetic bead coated antigen and the chemiluminescent label, for example, the buffer solution in the R1 reagent is a 25M Tris solution with pH of 7.2, and the buffer solution in the R2 reagent is the same as the item diluent.
In the technical scheme, the concentration of the antigen coated by the magnetic beads in the R1 reagent is preferably 0.05 wt% -0.2 wt%. The particle size of the magnetic beads is 0.5-3.0 μm, and the more preferable particle size is 3 μm; the mass ratio of the magnetic beads to the coating antigen is 10mg (50-500) mu g.
In the technical scheme, the concentration of the anti-human IgG antibody marked by the chemiluminescence marker in the R2 reagent is 0.1-1.0 mu g/ml. An anti-human IgG antibody marked by a chemiluminescent marker, wherein the molar ratio of the anti-human IgG antibody to the marker of the chemiluminescent marker is 1 (3-10); the chemiluminescent marker is acridinium ester or ruthenium terpyridyl, preferably acridinium ester; the anti-human IgG is mouse anti-human IgG or sheep anti-human IgG.
According to the technical scheme, in the project diluent, the salt is 50-200 mM of phosphate, citrate or Bistris propane; the surfactant is 0.02-0.05% of Tween 20 or 0.05-0.1% of choline chloride; the preservative is 0.01-0.03% of sodium azide and 0.01-0.03% of PC 300; the protein is 0.1-3% bovine serum albumin and 0.01-0.2% bovine gamma globulin.
The herpes simplex virus antibody IgG chemiluminescence immunoassay kit can also comprise one or more of a calibrator, chemiluminescence excitation liquid and cleaning liquid according to actual conditions:
the calibrator comprises a positive calibrator and a negative calibrator. The positive calibrator is prepared by inactivating herpes simplex virus IgG positive serum, filtering and diluting with a buffer solution; the negative calibrator is prepared by inactivating herpes simplex virus IgG negative serum, filtering and diluting with buffer solution, wherein the negative calibrator is a plurality of solutions with different concentrations of the negative herpes simplex virus IgG; the S/CO value of the positive calibrator is generally 2.5, and the S/CO value of the negative calibrator is 0. Preferably, the inactivation is carried out at 0 ℃ for 1h, and the filtration is carried out by using a 0.22 mu m filter membrane. The buffer was 100mM PBS buffer containing 20% calf serum.
The chemiluminescence excitation liquid comprises a liquid A and a liquid B, wherein the liquid A is a hydrogen peroxide solution and a nitric acid solution, and the liquid B is a sodium hydroxide solution.
The cleaning solution is PB buffer solution.
The preparation method of the chemiluminescence immunoassay kit for the herpes simplex virus IgG comprises the following steps:
step one, preparing R1 reagent
Uniformly mixing the magnetic bead solution, magnetically separating to remove supernatant, washing the washing solution for multiple times, then carrying out resuspension, adding the coating antigen, uniformly mixing, incubating for 2-4 h at 37 ℃ under continuous rotation and uniform mixing, magnetically separating to remove supernatant, washing the washing solution for multiple times, then sealing by using lysine sealing solution, magnetically separating to remove supernatant, retaining the magnetic bead coating antigen, and carrying out resuspension to the required concentration by using buffer solution to obtain an R1 reagent;
step two, preparing R2 reagent
Firstly, placing an anti-human IgG monoclonal antibody into a centrifugal tube, ensuring that the anti-human IgG monoclonal antibody is positioned at the bottom of the centrifugal tube, adding a carbonic acid buffer solution, fully and uniformly mixing, adding an N, N-Dimethylformamide (DMF) solution of a chemiluminescent marker after uniformly mixing, and centrifuging for 20-40 s at room temperature by using a centrifuge;
then sealing the centrifuge tube with a sealing film, putting the centrifuge tube into a light-proof cassette, putting the cassette into a gas bath constant-temperature oscillator for uniformly mixing, adding lysine sealing liquid, putting the gas bath constant-temperature oscillator for uniformly mixing, and sealing for 0.5-2 h;
purifying the closed anti-human IgG monoclonal antibody on a sephadex G250 column, eluting with a phosphate buffer solution, and respectively collecting;
finally, diluting the collected anti-human IgG monoclonal antibody solution with a buffer solution to obtain an R2 reagent;
step three, preparing project diluent according to the formula of the project diluent;
step four, subpackaging the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG;
if the kit also comprises one or more of a calibrator, a chemiluminescent excitation liquid and a cleaning liquid, then one or more of the calibrator, the chemiluminescent excitation liquid and the cleaning liquid are correspondingly prepared respectively, and then subpackaging is carried out.
According to the technical scheme, in the first step, the devices adopted for mixing are vortex mixing instruments, the devices adopted for magnetic separation are magnetic separation frames, the magnetic separation time is generally 1-3 min, and the magnetic separation time can be longer, so that liquid layering and supernatant liquid generation are achieved. The magnetic bead solution is a Tosyl magnetic bead solution. The concentration of the magnetic bead solution is not particularly limited, but is preferably 10 mg/mL. The washing solution is PB buffer solution, the concentration is preferably 0.02M, and the number of washing times is generally three. The concentration of the lysine solution was 20 wt%. The mass ratio of the magnetic beads to the coating antigen is 10mg (50-500) mu g. The resuspension buffer used was 25M Tris buffer, pH 7.2. The R1 reagent needs to be stored at 2-8 ℃.
In the technical scheme, in the second step, the technical means for ensuring that the anti-human IgG monoclonal antibody is positioned at the bottom of the centrifugal tube is to centrifuge the centrifugal machine for 20-40 s at room temperature generally; the preferred labeling molar ratio of the anti-human IgG monoclonal antibody to the chemiluminescent label is 1 (3-10); the concentration of the DMF solution of acridinium ester is generally 2 mg/mL; the temperature of the gas bath constant temperature oscillator is preferably 25 ℃, the mixing in the gas bath constant temperature oscillator generally needs 2-4 h, and the speed generally adopts medium speed; the concentration of the lysine confining liquid is 20 wt%, and the proportion of the added lysine confining liquid to the antihuman IgG monoclonal antibody is as follows: 500 mu g of 1 mL; the R2 reagent needs to be stored at 2-8 ℃.
The chemiluminescence immunoassay kit for the herpes simplex virus IgG antibody can be used for detecting the herpes simplex virus IgG antibody. During detection, a full-automatic chemiluminescence immunoassay analyzer (CM180) is used for detecting a negative calibrator and a positive calibrator, and the analyzer automatically calculates cutoff according to a measured chemiluminescence signal value; and then testing a sample to be tested according to requirements, comparing a chemiluminescence signal value obtained by the sample to be tested with a cutoff value obtained by calibration by an instrument, automatically calculating a detection result, expressing the result of the sample to be tested by reactivity or non-reactivity and a cutoff index (sample signal value/cutoff value), and finally evaluating the performance of the herpes simplex virus IgG antibody chemiluminescence immunoassay kit. The detection process of each analyte is as follows: incubating the object to be tested (10 mu L), the R1 reagent (40 mu L) and the project diluent (50 mu L) for 18min, cleaning for 5min, reacting with the R2 reagent (50 mu L) to form a magnetic microsphere-HSV antigen-HSV antibody-anti-human IgG antibody-chemiluminescent marker compound, separating the immune compound by a magnetic separator, washing off free components, exciting the chemiluminescent marker to emit light by a chemiluminescent exciting liquid, and recording the relative luminous intensity (RLU).
The present invention is further illustrated by the following examples.
Example 1
Step one, preparation of calibration product
Positive calibrator:
inactivating IgG positive serum of the herpes simplex virus at 60 ℃ for 1h, filtering, and diluting with a buffer solution until the S/CO value is 2.5; the buffer was 100mM PBS buffer containing 20% calf serum.
Negative calibrator:
inactivating simplex herpes virus IgG negative serum for 1h at 60 ℃, filtering, and diluting with a buffer solution until the S/CO value is 0; the buffer was 100mM PBS buffer containing 20% calf serum.
Step two, preparation of R1 reagent
Using a vortex mixer to mix 1mL of Tosyl magnetic bead (10mg/mL) solution, placing the mixture on a magnetic separation rack for 1min, removing supernatant, washing the mixture with 0.02M PB buffer solution for three times, then suspending the mixture, adding 0.05mg of coated antigen, mixing the mixture evenly, incubating the mixture at 37 ℃ for 2h under continuous rotation and mixing evenly, washing the separated supernatant with 0.02M PB buffer solution for three times, then sealing the separated supernatant with 20 wt% lysine sealing solution, magnetically separating the separated supernatant, reserving the coated antigen of the magnetic bead, using 25M Tris buffer solution with pH7.2 to resuspend the mixture until the concentration of the coated antigen of the magnetic bead is 0.05 wt%, obtaining R1 reagent, and storing the reagent at 2-8 ℃.
Step three, preparation of R2 reagent
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 carbonic acid buffer solution, fully and uniformly mixing, adding 0.75 mu L 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, collecting, diluting by the buffer solution until the final concentration is 0.1 mu G/mL, and storing at 2-8 ℃;
the buffer solution is a buffer solution with pH6.0 containing 50mM citric acid, 0.02% Tween 20, 0.01% sodium azide, 0.01% PC300, 0.1% bovine serum albumin and 0.01% bovine gamma globulin.
Step four, preparation of project diluent
The item diluent was a buffer solution of ph6.0 containing 50mM citric acid, 0.02% tween 20, 0.01% sodium azide, 0.01% PC300, 0.1% bovine serum albumin, and 0.01% bovine gamma globulin, and was prepared according to the formulation of the item diluent.
And fifthly, subpackaging the calibration product, the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG.
Example 2
Step one, preparation of calibration product
The same as in example 1.
Step two, preparation of R1 reagent
Using a vortex mixer to mix 1mL of Tosyl magnetic bead (10mg/mL) solution, placing the mixture on a magnetic separation rack for 2min, removing supernatant, washing the mixture with 0.02M PB buffer solution for three times, then suspending the mixture, adding 0.25mg of coated antigen, mixing the mixture evenly, incubating the mixture at 37 ℃ for 3h under continuous rotation and mixing evenly, washing the separated supernatant with 0.02M PB buffer solution for three times, then sealing the separated supernatant with 20 wt% lysine sealing solution, magnetically separating the separated supernatant, reserving the coated antigen of the magnetic bead, using 25M Tris buffer solution with pH7.2 to resuspend the mixture until the concentration of the coated antigen of the magnetic bead is 0.1 wt%, obtaining R1 reagent, and storing the reagent at 2-8 ℃.
Step three, preparation of R2 reagent
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 30s) and then adding carbonic acid buffer solution, fully and uniformly mixing, adding 2.5 mu L of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging the mixture at room temperature for 30s 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 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, collecting, diluting by the buffer solution until the final concentration is 0.5 mu G/mL, and storing at 2-8 ℃;
the buffer solution is a buffer solution with pH7.0 containing 100mM Bistris propane, 0.05% choline chloride, 0.02% sodium azide, 0.02% PC300, 1% bovine serum albumin and 0.1% bovine gamma globulin.
Step four, preparation of project diluent
The project diluent was prepared according to the formulation of the project diluent, which was a buffer solution of ph7.0 containing 100mM Bistris propane, 0.05% choline chloride, 0.02% sodium azide, 0.02% PC300, 1% bovine serum albumin, and 0.1% bovine gamma globulin.
And fifthly, subpackaging the calibration product, the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG.
Example 3
Step one, preparation of calibration product
The same as in example 1.
Step two, preparation of R1 reagent
Using a vortex mixer to mix 1mL of Tosyl magnetic bead (10mg/mL) solution, placing the mixture on a magnetic separation rack for 3min, removing supernatant, washing the mixture with 0.02M PB buffer solution for three times, then suspending the mixture, adding 0.5mg of coating antigen, mixing the mixture evenly, incubating the mixture at 37 ℃ for 4h under continuous rotation and mixing evenly, washing the separated supernatant with 0.02M PB buffer solution for three times, then sealing the separated supernatant with 20 wt% lysine sealing solution, magnetically separating the separated supernatant, reserving the coating antigen of the magnetic bead, using 25M Tris buffer solution with pH7.2 to resuspend the mixture until the concentration of the coating antigen of the magnetic bead is 0.2 wt%, obtaining R1 reagent, and storing the reagent at 2-8 ℃.
Step three, preparation of R2 reagent
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 40s), adding a carbonic acid buffer solution, fully and uniformly mixing, adding 5 mu L of 2mg/mL DMF solution of acridinium ester after uniformly mixing, and centrifuging the mixture at room temperature for 40s 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 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, collecting, diluting by the buffer solution until the final concentration is 1.0 mu G/mL, and storing at 2-8 ℃;
the buffer solution is a buffer solution with pH8.0 containing 200mM phosphate, 0.05% Tween 20, 0.03% sodium azide, 0.03% PC300, 3% bovine serum albumin and 0.2% bovine gamma globulin.
Step four, preparation of project diluent
The item diluent was prepared from a formulation of the item diluent, which was a buffer solution of ph8.0 containing 200mM phosphate, 0.05% tween 20, 0.03% sodium azide, 0.03% PC300, 3% bovine serum albumin, and 0.2% bovine gamma globulin.
And fifthly, subpackaging the calibration product, the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG.
The herpes simplex virus IgG antibody chemiluminescence detection kit of example 1 was tested.
The rubella virus herpes simplex virus IgG antibody detection kit is used for respectively detecting the national reference (purchase inspection institute, lot number: 370015-201801) of the HSV-1IgG antibody detection kit and the national reference (purchase inspection institute, lot number: 370016-201801) of the HSV-2IgG antibody detection kit, and the results are as follows:
performance evaluation of HSV-1IgG antibody detection kit
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are given in table 1: the test results are negative and completely accord with national negative reference products.
TABLE 1 negative reference assay results
(2) Positive reference compliance rate
The kit was used to assay national positive references with the results shown in table 2: the test results are positive and completely accord with national positive reference products.
TABLE 2 Positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 3: the test result meets the requirements of national detection limit reference products.
TABLE 3 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, Coefficient of Variation (CV) was not more than 15% calculated according to formula (1), and the test results are shown in table 4: the coefficient of variation was 1.31%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 4 results of reproducibility measurement
HSV-2IgG antibody detection kit performance evaluation
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are given in table 5: the test results are negative and completely accord with national negative reference products.
TABLE 5 negative reference assay results
(2) Positive reference compliance rate
The national positive reference was determined using the kit and the results are given in table 6: the test results are positive and completely accord with national positive reference products.
TABLE 6 positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 7: the test result meets the requirements of national detection limit reference products.
TABLE 7 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, Coefficient of Variation (CV) was not more than 15% calculated according to formula (1), and the test results are shown in table 8: the coefficient of variation was 2.21%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 8 results of reproducibility measurement
The herpes simplex virus IgG antibody chemiluminescence detection kit of example 2 was tested.
The rubella virus herpes simplex virus IgG antibody detection kit is used for respectively detecting the national reference (purchase inspection institute, lot number: 370015-201801) of the HSV-1IgG antibody detection kit and the national reference (purchase inspection institute, lot number: 370016-201801) of the HSV-2IgG antibody detection kit, and the results are as follows:
performance evaluation of HSV-1IgG antibody detection kit
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are given in table 9: the test results are negative and completely accord with national negative reference products.
TABLE 9 negative reference assay results
(2) Positive reference compliance rate
The national positive reference was determined using the kit and the results are given in table 10: the test results are positive and completely accord with national positive reference products.
TABLE 10 Positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 11: the test result meets the requirements of national detection limit reference products.
TABLE 11 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, Coefficient of Variation (CV) was not more than 15% calculated according to formula (1), and the test results are shown in table 12: the coefficient of variation was 2.05%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 12 results of reproducibility measurement
HSV-2IgG antibody detection kit performance evaluation
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are shown in table 13: the test results are negative and completely accord with national negative reference products.
TABLE 13 negative reference assay results
(2) Positive reference compliance rate
The national positive reference was determined using the kit and the results are given in table 14: the test results are positive and completely accord with national positive reference products.
TABLE 14 positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 15: the test result meets the requirements of national detection limit reference products.
TABLE 15 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, Coefficient of Variation (CV) was not more than 15% calculated according to formula (1), and the test results are shown in table 16: the coefficient of variation was 1.87%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 16 results of reproducibility measurement
The herpes simplex virus IgG antibody chemiluminescence detection kit of example 3 was tested.
The rubella virus herpes simplex virus IgG antibody detection kit is used for respectively detecting the national reference (purchase inspection institute, lot number: 370015-201801) of the HSV-1IgG antibody detection kit and the national reference (purchase inspection institute, lot number: 370016-201801) of the HSV-2IgG antibody detection kit, and the results are as follows:
performance evaluation of HSV-1IgG antibody detection kit
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are given in table 17: the test results are negative and completely accord with national negative reference products.
TABLE 17 negative reference assay results
(2) Positive reference compliance rate
The national positive reference was determined using the kit and the results are given in table 18: the test results are positive and completely accord with national positive reference products.
TABLE 18 Positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 19: the test result meets the requirements of national detection limit reference products.
TABLE 19 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, calculating the coefficient of variation according to formula (1)(CV) not greater than 15%, test results as in Table 20: the coefficient of variation was 5.1%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 20 results of reproducibility measurement
HSV-2IgG antibody detection kit performance evaluation
(1) Negative reference product compliance rate
The national negative reference was determined using the kit and the results are shown in table 21: the test results are negative and completely accord with national negative reference products.
TABLE 21 negative reference assay results
(2) Positive reference compliance rate
The kit was used to assay national positive references with the results shown in table 22: the test results are positive and completely accord with national positive reference products.
TABLE 22 Positive reference assay results
(3) Minimum detection limit
The national minimum detection limit reference was determined using the kit and the results are shown in table 23: the test result meets the requirements of national detection limit reference products.
TABLE 23 measurement results of minimum examination limit test article
(4) Repeatability of
Detecting the national repetitive reference substance by using the kit, repeatedly determining for 10 times, and calculating the average value of 10 measurement results
And standard deviation SD, Coefficient of Variation (CV) was not more than 15% as calculated according to formula (1), and the test results are shown in table 24: the coefficient of variation was 1.92%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 24 repeatability measurements
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The herpes simplex virus antibody IgG chemiluminescence immunoassay kit is characterized by comprising an R1 reagent, an R2 reagent and a project diluent;
the R1 reagent comprises a magnetic bead coated antigen, and the antigen is HSV-1 antigen or HSV-2 antigen;
the R2 reagent comprises an anti-human IgG antibody labeled with a chemiluminescent label;
the project diluent is a buffer solution containing salt, a surfactant, a preservative and protein, and the pH value of the buffer solution is 6.0-8.0.
2. The IgG chemiluminescent immunoassay kit for herpes simplex virus antibodies according to claim 1, wherein the concentration of the antigen coated by the magnetic beads in the R1 reagent is 0.05 wt% to 0.2 wt%.
3. The IgG chemiluminescent immunoassay kit for herpes simplex virus antibodies according to claim 1, wherein the magnetic beads used for the antigen coated by the magnetic beads have a particle size of 0.5-3.0 μm.
4. The IgG chemiluminescent immunoassay kit for herpes simplex virus antibodies according to claim 1, wherein the mass ratio of the magnetic beads to the coating antigen is 10mg (50-500) μ g.
5. The IgG chemiluminescent immunoassay kit for a herpes simplex virus antibody according to claim 1, wherein the concentration of the anti-human IgG antibody labeled with a chemiluminescent label in the R2 reagent is 0.1 to 1.0. mu.g/ml.
6. The IgG chemiluminescent immunoassay kit for a herpes simplex virus antibody according to claim 1, wherein the labeling molar ratio of the anti-human IgG antibody to the chemiluminescent label of the anti-human IgG antibody labeled with the chemiluminescent label is 1 (3-10); the chemiluminescent marker is acridinium ester or ruthenium terpyridyl; the anti-human IgG is mouse anti-human IgG or sheep anti-human IgG.
7. The IgG chemiluminescent immunoassay kit for a herpes simplex virus antibody according to claim 1, wherein in the dilution of the item, the salt is 50-200 mM of phosphate, citrate or Bistris propane; the surfactant is 0.02-0.05% of Tween 20 or 0.05-0.1% of choline chloride; the preservative is 0.01-0.03% of sodium azide and 0.01-0.03% of PC300, and the protein is 0.1-3% of bovine serum albumin and 0.01-0.2% of bovine gamma globulin.
8. The IgG chemiluminescent immunoassay kit for a herpes simplex virus antibody of claim 1, further comprising one or more of a calibrator, a chemiluminescent excitation fluid, and a washing fluid;
the calibrator comprises a positive calibrator and a negative calibrator, wherein the positive calibrator is prepared by inactivating herpes simplex virus IgG positive serum, filtering and diluting with a buffer solution; the negative calibrator is prepared by inactivating herpes simplex virus IgG negative serum, filtering and diluting with a buffer solution;
the chemiluminescence excitation liquid comprises a liquid A and a liquid B, wherein the liquid A is a hydrogen peroxide solution and a nitric acid solution, and the liquid B is a sodium hydroxide solution;
the cleaning solution is PB buffer solution.
9. The method for preparing a chemiluminescent immunoassay kit for herpes simplex virus IgG of any of claims 1 to 8, comprising the steps of:
step one, preparing R1 reagent
Uniformly mixing the magnetic bead solution, magnetically separating to remove supernatant, washing the washing solution for multiple times, then carrying out resuspension, adding the coating antigen, uniformly mixing, incubating for 2-4 h at 37 ℃ under continuous rotation and uniform mixing, magnetically separating to remove supernatant, washing the washing solution for multiple times, then sealing by using lysine sealing solution, magnetically separating to remove supernatant, retaining the magnetic bead coating antigen, and carrying out resuspension to the required concentration by using buffer solution to obtain an R1 reagent;
step two, preparing R2 reagent
Firstly, placing an anti-human IgG monoclonal antibody into a centrifugal tube, ensuring that the anti-human IgG monoclonal antibody is positioned at the bottom of the centrifugal tube, adding a carbonic acid buffer solution, fully and uniformly mixing, adding an N, N-dimethylformamide solution of a chemiluminescent marker after uniformly mixing, and centrifuging for 20-40 s at room temperature by using a centrifuge;
then sealing the centrifuge tube with a sealing film, putting the centrifuge tube into a light-proof cassette, putting the cassette into a gas bath constant-temperature oscillator for uniformly mixing, adding lysine sealing liquid, putting the gas bath constant-temperature oscillator for uniformly mixing, and sealing for 0.5-2 h;
purifying the closed anti-human IgG monoclonal antibody on a sephadex G250 column, eluting with a phosphate buffer solution, and respectively collecting;
finally, diluting the collected anti-human IgG monoclonal antibody solution with a buffer solution to obtain an R2 reagent;
step three, preparing project diluent according to the formula of the project diluent;
and step four, subpackaging the R1 reagent, the R2 reagent and the project diluent to complete the preparation of the chemiluminescence immunoassay kit for the herpes simplex virus IgG.
10. The method for preparing a chemiluminescent immunoassay kit for herpes simplex virus IgG according to claim 9, wherein in the first step, the resuspension buffer is 25M Tris buffer with pH 7.2; in the second step, the buffer solution for dilution is the same as the item diluent.
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