CN112213483A - Chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and preparation method thereof - Google Patents
Chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and a preparation method thereof, belonging to the technical field of immunoassay. The chemiluminescence immunoassay kit comprises an R1 reagent containing a buffer solution of magnetic beads coated by herpes simplex virus type 1 antigen and magnetic beads coated by human herpes simplex virus type 2 antigen; the R2 reagent contains buffer solution of anti-human IgM antibody labeled with a chemiluminescent label; the R3 reagent contained a blocking agent and a buffer of human IgG adsorbent. The kit is a chemiluminescence immunoassay, and the method has the advantages of simple operation, wide detection range, short detection time, no enzyme participation, accurate quantification and capability of detecting target substances such as antigens and antibodies; on the other hand, the herpes simplex virus 1+2 type 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 the accuracy of the whole system are improved.
Description
Technical Field
The invention belongs to the technical field of immunoassay, and particularly relates to a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody 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, 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.
The currently common methods for detecting the herpes simplex virus 1+2 IgM antibody include an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence enzyme immunoassay and a colloidal gold method, but the ELISA has the advantages of long operation time, narrow detection range, long detection time, poor repeatability and the like; chemiluminescent enzyme immunoassays are susceptible to enzymatic activity; the gold labeling method has low sensitivity, and can only be qualitative and not quantitative.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody and a preparation method thereof.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody, which comprises: r1 reagent, R2 reagent and R3 reagent;
the R1 reagent is a buffer solution containing magnetic beads coated by herpes simplex virus type 1 antigens and magnetic beads coated by human herpes simplex virus type 2 antigens;
the R2 reagent is a buffer solution containing an anti-human IgM antibody marked by a chemiluminescent marker;
the R3 reagent is a buffer solution containing a blocking agent and a human IgG adsorbent.
In the technical scheme, in the magnetic beads coated with the herpes simplex virus type 1 antigen or the magnetic beads coated with the human herpes simplex virus type 2 antigen in the R1 reagent, the concentration mass ratio of the herpes simplex virus type 1 antigen or the human herpes simplex virus type 2 antigen to the coated magnetic beads is 0.5-5%; the magnetic beads have a particle size of 1.0 to 3.0 μm.
In the technical scheme, the magnetic beads for coating in the R1 reagent are magnetic particles with Tosyl functional groups, the antigens used for coating the magnetic beads are herpes simplex virus 1 antigen and human herpes simplex virus 2 antigen respectively, and the mass ratio of the reagent is 0.01-1%.
In the technical scheme, the labeling ratio of the anti-human IgM antibody to the chemiluminescent label in the R2 reagent is 1: 3-1: 10.
In the above technical scheme, the anti-human IgM antibody in the R2 reagent is a murine anti-human IgM antibody or a ovine anti-human IgM antibody.
In the technical scheme, the chemiluminescent marker in the R2 reagent is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
In the technical scheme, the structural formula of the acridine ester is as follows:
in the technical scheme, the blocker in the R3 reagent is a non-specific mouse IgG antibody or a sheep IgG antibody; the human IgG adsorbent is a goat anti-human IgG antibody or a rabbit anti-human IgG antibody.
In the technical scheme, the buffer solution of the R1 reagent is 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide, the buffer solution with the pH value of 7.2, and the total concentration of magnetic beads is 0.01-1%; the buffer solution of the R2 reagent is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 6.0-7.0, and the final concentration of the chemiluminescent marker anti-human IgM antibody is 0.1-1.0 mu g/mL; the buffer solution of the R3 reagent is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocking agent is 0.1-1.0 mu g/mL, and the final concentration of the human IgG adsorbent is 50-200 mu g/mL.
The invention provides a preparation method of a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody, which comprises the following steps:
step 1, preparation of R1 reagent
Taking magnetic particles, adding a self-contained cleaning solution, cleaning, adding a herpes simplex virus 1 antigen and a herpes simplex virus 2 antigen respectively, suspending for 4-12 hours at room temperature, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution, adding BSA for sealing, suspending for 4-6 hours, carrying out magnetic separation, and cleaning to obtain magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen respectively; mixing magnetic beads coated with a herpes simplex virus type 1 antigen and magnetic beads coated with a human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide and a buffer solution with the pH of 7.2 to prepare a solid phase reagent with the total concentration of the magnetic beads being 0.01-1%, namely an R1 reagent;
step 2, preparation of R2 reagent
Replacing the anti-human IgM antibody buffer solution with PBS by using an ultrafiltration centrifugal tube, adding a chemiluminescence marker DMF solution, fully and uniformly mixing, standing at room temperature for 2-4 hours, and purifying and marking the anti-human IgM antibody by using an AKTA protein purifier; placing the collected chemiluminescence marker labeled anti-human IgM antibody at 2-8 ℃ for storage, and diluting the purified chemiluminescence marker labeled anti-human IgM antibody concentrated solution with 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% PC300, 0.01-0.03% sodium azide, or 0.05-0.1% Tween-20, 1-3% BSA, 0.5-2% BGG, pH 6.0-7.0 buffer solution to obtain the chemiluminescence marker labeled anti-human IgM antibody with the final concentration of 0.1-1.0 mu g/mL, namely R2 reagent;
step 3, preparation of R3 reagent
Preparing a buffer solution containing a blocking agent and a human IgG adsorbent by using the buffer solution, namely an R3 reagent;
the buffer solution is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocker is 0.1-1.0 mu g/mL, and the final concentration of the human IgG adsorbent is 50-200 ug/mL.
The detection principle of the kit is indirect method. Incubating the sample with R3 solution (50 μ L) and R1 solution (40 μ L) of magnetic particles coated with herpes simplex virus type 1 antigen and herpes simplex virus type 2 antigen for 18min, washing for 5min, reacting with chemiluminescence marker labeled anti-human IgM antibody solution (50 μ L) to form herpes simplex virus coated magnetic particles-human HSV-1+2 IgM antibody-acridinium ester labeled anti-human IgM antibody complex, separating immune complex by magnetic separator, and washing off free components. The relative luminescence intensity (RLU) is recorded by exciting the chemiluminescent label to luminesce with an acid-base excitation solution, and the measurement results are automatically calculated by the instrument (comparing the chemiluminescent signal value obtained from the sample reaction product with the cutoff value obtained from a previous calibration).
When the chemiluminescence immunoassay kit for detecting the herpes simplex virus 1+2 IgM antibody is used for detecting the HSV-1+2 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 formula 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 by reactivity or non-reactivity and a cutoff index (sample signal value/cutoff value, S/CO), and finally, the performance of the herpes simplex virus type 1+2 IgM antibody detection kit is evaluated.
The invention 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 magnetic particles are used as solid phase carriers, the specific surface area is large, and the detection sensitivity is further improved.
2. The chemiluminescence immunoassay kit for the herpes simplex virus 1+2 IgM antibody is a full-automatic measurement sample, 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 calibrator and the quality control material used in the herpes simplex virus 1+2 type IgM antibody detection kit are assigned by other commercialized herpes simplex virus 1+2 type IgM antibody detection kits (herpes simplex virus 1+2 type IgM antibody detection kits produced by Soxhlet's diagnosis (Italy) Co., Ltd.) to ensure the accuracy of the test result.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a flow chart of the preparation of the chemiluminescence immunoassay kit for detecting the herpes simplex virus type 1+2 IgM antibody of the present invention.
Detailed Description
The invention provides a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody, which comprises: r1 reagent, R2 reagent and R3 reagent; the R1 reagent is a buffer solution containing magnetic beads coated by herpes simplex virus type 1 antigens and magnetic beads coated by human herpes simplex virus type 2 antigens; the R2 reagent is a buffer solution containing an anti-human IgM antibody marked by a chemiluminescent marker; the R3 reagent is a buffer solution containing a blocking agent and a human IgG adsorbent.
In the magnetic beads coated with the herpes simplex virus type 1 antigen or the magnetic beads coated with the human herpes simplex virus type 2 antigen in the R1 reagent, the concentration mass ratio of the herpes simplex virus type 1 antigen or the human herpes simplex virus type 2 antigen to the coated magnetic beads is 0.5-5%; the particle size of the magnetic beads is 1.0-3.0 μm; the magnetic beads for coating are magnetic particles with Tosyl functional groups, the antigens used by the magnetic beads for coating are herpes simplex virus 1 antigen and human herpes simplex virus 2 antigen respectively, and the mass ratio of the reagents is 0.01-1%; the buffer solution is 25Mm Tris, 0.05 percent of Tween-20, 0.05 percent of Proclin300 and 0.02 percent of sodium azide, the pH value is 7.2, and the total concentration of the magnetic beads is 0.01 to 1 percent.
The labeling ratio of the anti-human IgM antibody to the chemiluminescent label in the R2 reagent is 1: 3-1: 10; the anti-human IgM antibody is a mouse anti-human IgM antibody or a sheep anti-human IgM antibody; the chemiluminescent marker is acridinium ester, luminol, isoluminol or ruthenium terpyridyl, preferably acridinium ester, and further preferably acridinium ester with the structural formula shown below; the buffer solution of the R2 reagent is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 6.0-7.0, and the final concentration of the chemiluminescent marker anti-human IgM antibody is 0.1-1.0 mu g/mL;
the blocker in the R3 reagent is a non-specific mouse IgG antibody or a sheep IgG antibody; the human IgG adsorbent is a goat anti-human IgG antibody or a rabbit anti-human IgG antibody, the buffer solution is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocker is 0.1-1.0 mu g/mL, and the final concentration of the human IgG adsorbent is 50-200 ug/mL.
Preferably, the chemiluminescence immunoassay kit for detecting the herpes simplex virus type 1+2 IgM antibody further comprises 2.0S/CO and 0.0S/CO calibrators.
Preferably, the chemiluminescence immunoassay kit for detecting the herpes simplex virus type 1+2 IgM antibody further comprises quality control substances of 2.0S/CO and 0.5S/CO.
Preferably, the chemiluminescence immunoassay kit for detecting the herpes simplex virus type 1+2 IgM antibody further comprises a chemiluminescence substrate solution A and a chemiluminescence substrate solution B, wherein the solution A is a hydrogen peroxide solution and a nitric acid solution, and the solution B is a sodium hydroxide solution.
Preferably, the chemiluminescence immunoassay kit for detecting the herpes simplex virus 1+2 type IgM antibody further comprises a cleaning solution, wherein the cleaning solution is a PB buffer solution.
The detection principle of the kit is indirect method. Incubating the sample with R3 solution (50 μ L) and R1 solution (40 μ L) of magnetic particles coated with herpes simplex virus type 1 antigen and herpes simplex virus type 2 antigen for 18min, washing for 5min, reacting with chemiluminescence marker labeled anti-human IgM antibody solution (50 μ L) to form herpes simplex virus coated magnetic particles-human HSV-1+2 IgM antibody-acridinium ester labeled anti-human IgM antibody complex, separating immune complex by magnetic separator, and washing off free components. The relative luminescence intensity (RLU) is recorded by exciting the chemiluminescent label to luminesce with an acid-base excitation solution, and the measurement results are automatically calculated by the instrument (comparing the chemiluminescent signal value obtained from the sample reaction product with the cutoff value obtained from a previous calibration).
When the chemiluminescence immunoassay kit for detecting the herpes simplex virus 1+2 IgM antibody is used for detecting the HSV-1+2 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 formula 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 by reactivity or non-reactivity and a cutoff index (sample signal value/cutoff value, S/CO), and finally, the performance of the herpes simplex virus type 1+2 IgM antibody detection kit is evaluated.
As shown in FIG. 1, the preparation process of the R1, R2 and R3 reagents of the invention comprises the steps of firstly screening antigen and antibody, then respectively coating magnetic beads, labeling the antibody with chemiluminescent marker (such as acridinium ester), preparing R3 reagent, then preparing according to the concentration of the antigen coated magnetic beads and the labeled antibody with chemiluminescent marker (such as acridinium ester), and finally subpackaging the reagent. The invention provides a preparation method of a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody, which comprises the following steps:
step 1, preparation of R1 reagent
Taking magnetic particles (the product is commercially available) with 10mg/mL and Tosyl functional groups, adding 5mL of self-contained cleaning solution, cleaning, respectively adding herpes simplex virus 1 antigen and herpes simplex virus 2 antigen, suspending at room temperature for 4-12 hours, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution of 0.1-10mM PB and pH6.0-8.0, adding 5% BSA (bovine serum albumin) for sealing, suspending for 4-6 hours, carrying out magnetic separation, cleaning, and respectively obtaining magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen; mixing magnetic beads coated with a herpes simplex virus type 1 antigen and magnetic beads coated with a human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide and a buffer solution with the pH of 7.2 to prepare a solid phase reagent with the total concentration of the magnetic beads being 0.01-1%, namely an R1 reagent;
the magnetic beads coated with the herpes simplex virus 1-type antigen and the magnetic beads coated with the human herpes simplex virus 2-type antigen are respectively prepared according to the step 1, and then the obtained magnetic beads coated with the herpes simplex virus 1-type antigen and the obtained magnetic beads coated with the human herpes simplex virus 2-type antigen are mixed according to the mixing ratio of 1:1, and a buffer solution is used for preparing a solid phase reagent with the total concentration of the magnetic beads being 0.01-1%, namely an R1 reagent. Wherein the total concentration of the magnetic beads is the sum of the concentration of the magnetic beads coated with the herpes simplex virus type 1 antigen and the concentration of the magnetic beads coated with the human herpes simplex virus type 2 antigen.
Step 2, preparation of R2 reagent
Replacing the buffer solution of the anti-human IgM antibody with PBS (the antibody amount is 500 mu g) by using an ultrafiltration centrifugal tube, adding a chemiluminescent marker DMF solution, fully and uniformly mixing, standing at room temperature for 2-4 hours, and purifying and marking the anti-human IgM antibody by using an AKTA protein purifier; placing the collected chemiluminescence marker labeled anti-human IgM antibody at 2-8 ℃ for storage, and diluting the purified chemiluminescence marker labeled anti-human IgM antibody concentrated solution with 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% PC300, 0.01-0.03% sodium azide, or 0.05-0.1% Tween-20, 1-3% BSA, 0.5-2% BGG, pH 6.0-7.0 buffer solution to obtain the chemiluminescence marker labeled anti-human IgM antibody with the final concentration of 0.1-1.0 mu g/mL, namely R2 reagent;
step 3, preparation of R3 reagent
Preparing a buffer solution containing a blocking agent and a human IgG adsorbent by using the buffer solution, namely an R3 reagent;
the buffer solution is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocker is 0.1-1.0 mu g/mL, and the final concentration of the human IgG adsorbent is 50-200 ug/mL.
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments. 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.
The structural formula of the acridinium esters used in the following examples is as follows:
example 1 chemiluminescence immunoassay kit for detecting herpes simplex virus type 1+2 IgM antibody of the present invention
(1) Preparation of herpes simplex virus 1+2 IgM antibody calibrator and quality control product
A commercial herpes simplex virus 1+2 type IgM antibody detection kit (a herpes simplex virus 1+2 type IgM antibody detection kit produced by Song diagnosis (Italy) Co., Ltd.) is used for assignment, and a herpes simplex virus 1+2 type IgM antibody protein solution with an assignment result of 5.0S/CO is 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 herpes simplex virus 1+2 type IgM antibody protein solution with the assignment result of 5.0S/CO, and diluting the 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 herpes simplex virus 1+2 IgM antibody protein solution with the assignment result of 5.0S/CO, and diluting the solution into 0.5S/CO by using a quality control substance matrix to serve as a low-concentration quality control substance.
(2) Preparation of reagent R1
Taking magnetic particles (the product is commercially available) with 10mg/mL and Tosyl functional groups, adding 5mL of self-contained cleaning solution, respectively adding a herpes simplex virus 1 antigen and a herpes simplex virus 2 antigen, suspending at room temperature for 4 hours, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution of 0.1mM PB and pH6.0, adding 5% BSA (bovine serum albumin) for sealing, suspending for 4 hours, carrying out magnetic separation, and cleaning to respectively obtain magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen; mixing magnetic beads coated with the herpes simplex virus type 1 antigen and magnetic beads coated with the human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide and a buffer solution with the pH value of 7.2 to prepare a solid phase reagent with the total concentration of the magnetic beads of 0.05%, and storing at the temperature of 2-8 ℃.
(3) Preparation of reagent R2
Replacing the buffer solution of the anti-human IgM antibody with PBS (the antibody amount is 500. mu.g) by using an ultrafiltration centrifugal tube, adding an acridinium ester DMF solution, and enabling the labeling ratio of the antibody to the acridinium ester to be 1: and 3, fully and uniformly mixing, standing at room temperature for 2 hours, and purifying and marking the anti-human IgM antibody by an AKTA protein purifier. And (3) storing the collected acridinium ester labeled anti-human IgM antibody at 2-8 ℃, and diluting the purified anti-human IgM antibody concentrated solution with a buffer solution of 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG and pH6.5 to obtain an antibody final concentration of 0.1 mu g/mL, namely the R2 reagent.
(4) Preparation of reagent R3
The final concentrations were 0.1. mu.g/mL of nonspecific mouse IgG antibody and 50. mu.g/mL of goat anti-human IgG, 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG, 100mM PB solution pH 8.0.
Example 2 chemiluminescent immunoassay kit for detecting herpes simplex virus type 1+2 IgM antibody according to the present invention
(1) Preparation of calibrator and quality control material
The same as in example 1.
Preparation of reagent R1
Taking magnetic particles (the product is commercially available) with 10mg/mL and Tosyl functional groups, adding 5mL of self-contained cleaning solution, respectively adding a herpes simplex virus 1 antigen and a herpes simplex virus 2 antigen, suspending at room temperature for 8 hours, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution of 5mM PB and pH7.0, adding 5% BSA (bovine serum albumin) for sealing, suspending for 5 hours, carrying out magnetic separation, and cleaning to respectively obtain magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen; mixing magnetic beads coated with a herpes simplex virus type 1 antigen and magnetic beads coated with a human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide and a buffer solution with the pH value of 7.2 to prepare a solid phase reagent with the magnetic bead concentration of 0.007%, and storing at the temperature of 2-8 ℃.
(3) Preparation of reagent R2
Replacing the buffer solution of the anti-human IgM antibody with PBS (the antibody amount is 500. mu.g) by using an ultrafiltration centrifugal tube, adding an acridinium ester DMF solution, and enabling the labeling ratio of the antibody to the acridinium ester to be 1: and 5, fully and uniformly mixing, standing at room temperature for 3 hours, and purifying and marking the anti-human IgM antibody by an AKTA protein purifier. And (3) storing the collected acridinium ester labeled anti-human IgM antibody at 2-8 ℃, and diluting the purified anti-human IgM antibody concentrated solution with a buffer solution of 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG and pH6.5 to obtain an antibody final concentration of 0.1 mu g/mL, namely the R2 reagent.
(4) Preparation of reagent R3
The final concentration was 0.5. mu.g/mL of nonspecific mouse IgG antibody and 100ug/mL of goat anti-human IgG, 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG, 100mM PB solution pH 8.0.
Example 3 chemiluminescent immunoassay kit for detecting herpes simplex virus type 1+2 IgM antibody according to the present invention
(1) Preparation of calibrator and quality control material
The same as in example 1.
(2) Preparation of reagent R1
Taking magnetic particles (the product is commercially available) with 10mg/mL and Tosyl functional groups, adding 5mL of self-contained cleaning solution, respectively adding a herpes simplex virus 1 antigen and a herpes simplex virus 2 antigen, suspending at room temperature for 12 hours, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution of 10mM PB and pH8.0, adding 5% BSA (bovine serum albumin) for sealing, suspending for 6 hours, carrying out magnetic separation, and cleaning to respectively obtain magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen; mixing magnetic beads coated with the herpes simplex virus type 1 antigen and magnetic beads coated with the human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300, 0.02% sodium azide and a buffer solution with the pH value of 7.2 to prepare a solid phase reagent with the total concentration of the magnetic beads of 1%, and storing at the temperature of 2-8 ℃.
(3) Preparation of reagent R2
Replacing the buffer solution of the anti-human IgM antibody with PBS (the antibody amount is 500. mu.g) by using an ultrafiltration centrifugal tube, adding an acridinium ester DMF solution, and enabling the labeling ratio of the antibody to the acridinium ester to be 1: and 10, fully and uniformly mixing, standing at room temperature for 4 hours, and purifying and marking the anti-human IgM antibody by an AKTA protein purifier. And (3) storing the collected acridinium ester labeled anti-human IgM antibody at 2-8 ℃, and diluting the purified anti-human IgM antibody concentrated solution with a buffer solution of 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG and pH6.5 to obtain an antibody final concentration of 0.1 mu g/mL, namely the R2 reagent.
(4) Preparation of reagent R3
The final concentration was 1.0. mu.g/mL of nonspecific mouse IgG antibody and 200ug/mL of goat anti-human IgG, 100mM PB, 0.05% Tween-20, 1% BSA, 0.5% BGG, 100mM PB solution at pH 8.0.
Example 4 evaluation of the Performance of the chemiluminescence immunoassay kit for detecting the IgM antibody of herpes simplex virus type 1+2 of the present invention
The chemiluminescence immunoassay kit for detecting the herpes simplex virus type 1+2 IgM antibody of the embodiment 1 is adopted to detect a clinical sample of the herpes simplex virus type 1+2 IgM antibody, and a commercial herpes simplex virus type 1+2 IgM antibody detection kit (a herpes simplex virus type 1+2 IgM antibody detection kit produced by Soxhlet diagnostics (Italy) Limited) is used as a contrast reagent.
The detection method comprises the following steps:
the method uses a dire full-automatic chemiluminescence immunoassay analyzer (CM-180) as a detection instrument, and an indirect method, wherein the sample volume is 10 mu L, the magnetic particle reagent (R1) coated by the herpes simplex virus 1 antigen and the herpes simplex virus 2 antigen and the intermediate reagent (R3) are 40 mu L, and the anti-human IgM antibody reagent (R2) marked by a chemiluminescent 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). Instrument for measuring the position of a moving objectThe reactant is sent into a dark room, and the luminous excitation liquid A (H) is added 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 clinical sample compliance rate
The prepared kit is used for measuring negative clinical samples, the results are shown in table 1, the test results are negative, and the test results completely accord with the control reagent.
TABLE 1 measurement results of sexual reference
(2) Positive clinical sample compliance rate
The prepared kit is used for determining positive clinical samples, the results are shown in table 2, the test results are all positive, and the test results completely accord with the control reagent.
TABLE 2 Positive reference assay results
(4) Repetitive reference (R)
The kit is used for detecting the configured repetitive samples, the measurement is repeated 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 percent according to the formula (1), and the test results are shown in the table 4: the coefficient of variation was 4.44%.
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
TABLE 4 results of reproducibility measurement
The kit is a chemiluminescence immunoassay, and the method has the advantages of simple operation, wide detection range, short detection time, no enzyme participation, accurate quantification and capability of detecting target substances such as antigens and antibodies; on the other hand, the herpes simplex virus 1+2 type 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 the accuracy of the whole system are improved.
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 herpes simplex virus 1+2 IgM antibody is characterized by comprising: r1 reagent, R2 reagent and R3 reagent;
the R1 reagent is a buffer solution containing magnetic beads coated by herpes simplex virus type 1 antigens and magnetic beads coated by human herpes simplex virus type 2 antigens;
the R2 reagent is a buffer solution containing an anti-human IgM antibody marked by a chemiluminescent marker;
the R3 reagent is a buffer solution containing a blocking agent and a human IgG adsorbent.
2. The chemiluminescent immunoassay kit for detecting the IgM antibody of herpes simplex virus type 1+2 according to claim 1, wherein the magnetic beads coated with the herpes simplex virus type 1 antigen or the human herpes simplex virus type 2 antigen in the R1 reagent have a concentration of the herpes simplex virus type 1 antigen or the human herpes simplex virus type 2 antigen to the coated magnetic beads by mass ratio of 0.5% to 5%, and the particle size of the magnetic beads is 1.0 to 3.0 μm.
3. The chemiluminescent immunoassay kit for detecting the IgM antibody of herpes simplex virus type 1+2 according to claim 1, wherein the magnetic beads coated in the R1 reagent are magnetic particles with Tosyl functional groups.
4. The chemiluminescence immunoassay kit for detecting the herpes simplex virus 1+2 IgM antibody according to claim 1, wherein the labeling ratio of the anti-human IgM antibody to the chemiluminescent label in the R2 reagent is 1:3 to 1: 10.
5. The chemiluminescent immunoassay kit for detecting an IgM antibody of herpes simplex virus type 1+2 according to claim 1, wherein the anti-human IgM antibody in the R2 reagent is a murine anti-human IgM antibody or a goat anti-human IgM antibody.
6. The chemiluminescent immunoassay kit for detecting an IgM antibody of herpes simplex virus type 1+2 according to claim 1, wherein the chemiluminescent label in the R2 reagent is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
7. The chemiluminescent immunoassay kit for detecting an IgM antibody of herpes simplex virus type 1+2 according to claim 6, wherein the acridinium ester has the following structural formula:
8. the chemiluminescent immunoassay kit for detecting the IgM antibody of herpes simplex virus type 1+2 according to claim 1, wherein the blocking agent in the R3 reagent is a non-specific mouse IgG antibody or a sheep IgG antibody; the human IgG adsorbent is a goat anti-human IgG antibody or a rabbit anti-human IgG antibody.
9. The chemiluminescence immunoassay kit for detecting the IgM antibody of herpes simplex virus type 1+2 of claim 1, wherein the buffer solution of the R1 reagent is 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300 and 0.02% sodium azide, the buffer solution with pH7.2, and the total concentration of magnetic beads is 0.01% -1%; the buffer solution of the R2 reagent is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 6.0-7.0, and the final concentration of the chemiluminescent marker anti-human IgM antibody is 0.1-1.0 mu g/mL; the buffer solution of the R3 reagent is 100-400 mM PB, Bistris propane or HE PES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocking agent is 0.1-1.0 mu G/mL, and the final concentration of the human Ig G adsorbent is 50-200 mu G/mL.
10. A preparation method of a chemiluminescence immunoassay kit for detecting herpes simplex virus 1+2 IgM antibody is characterized by comprising the following steps:
step 1, preparation of R1 reagent
Taking magnetic particles, adding a self-contained cleaning solution, cleaning, adding a herpes simplex virus 1 antigen and a herpes simplex virus 2 antigen respectively, suspending for 4-12 hours at room temperature, carrying out magnetic separation, removing supernatant, cleaning with a buffer solution, adding BSA for sealing, suspending for 4-6 hours, carrying out magnetic separation, and cleaning to obtain magnetic beads coated with the herpes simplex virus 1 antigen or magnetic beads coated with the human herpes simplex virus 2 antigen respectively; mixing magnetic beads coated with a herpes simplex virus type 1 antigen and magnetic beads coated with a human herpes simplex virus type 2 antigen according to the mixing ratio of 1:1 by using 25Mm Tris, 0.05% Tween-20, 0.05% Proclin300 and 0.02% sodium azide and a buffer solution with the pH value of 7.2 to prepare a solid phase reagent with the total concentration of the magnetic beads being 0.01-1%, namely an R1 reagent;
step 2, preparation of R2 reagent
Replacing the anti-human IgM antibody buffer solution with PBS by using an ultrafiltration centrifugal tube, adding a chemiluminescent marker DMF solution, standing at room temperature for 2-4 hours, and purifying and marking the anti-human IgM antibody by using an AKTA protein purifier; placing the collected chemiluminescence marker labeled anti-human IgM antibody at 2-8 ℃ for storage, and diluting the purified chemiluminescence marker labeled anti-human IgM antibody concentrated solution with 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% PC300, 0.01-0.03% sodium azide, or 0.05-0.1% Tween-20, 1-3% BSA, 0.5-2% BGG, pH 6.0-7.0 buffer solution to obtain the chemiluminescence marker labeled anti-human IgM antibody with the final concentration of 0.1-1.0 mu g/mL, namely R2 reagent;
step 3, preparation of R3 reagent
Preparing a buffer solution containing a blocking agent and a human IgG adsorbent by using the buffer solution, namely an R3 reagent;
the buffer solution is 100-400 mM PB, Bistris propane or HEPES, 0.05-0.1% of PC300, 0.01-0.03% of sodium azide, or 0.05-0.1% of Tween-20, 1-3% of BSA, 0.5-2% of BGG, the pH value is 7.0-8.0, the final concentration of the blocker is 0.1-1.0 mu g/mL, and the final concentration of the human IgG adsorbent is 50-200 ug/mL.
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