CN109142750B - Kit for determining anti-histone antibody IgG and detection method - Google Patents
Kit for determining anti-histone antibody IgG and detection method Download PDFInfo
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Abstract
The invention relates to a kit for determining anti-histone antibody IgG, which is characterized in that: the kit comprises a first reagent and a second reagent, wherein the first reagent comprises a mixed antigen conjugated with biotin, and the mixed antigen comprises a histone antigen, a H1 fragment and a H2A-H2B compound fragment; the second reagent comprises an anti-human IgG antibody conjugated to alkaline phosphatase. The kit and the detection method have the advantages of good stability, high sensitivity, good repeatability and the like, meanwhile, the detection time is short, the total time for completing one test is within 50 minutes, the operation is simple and convenient, the full automation of detection is really realized, and in addition, the kit and the detection method have high positive detection rate of SLE patients.
Description
Technical Field
The invention belongs to the technical field of magnetic particle chemiluminescence immunodiagnosis, and particularly relates to a kit for determining anti-histone antibody IgG and a detection method.
Background
Antinuclear antibodies (ANA), also known as antinuclear antibodies, are a group of autoantibodies that use as target antigens Deoxyriboproteins (DNPs), DNA, Extractable Nuclear Antigens (ENAs), RNA, and the like, which are components of eukaryotic cells. Antinuclear antibodies show different degrees of positive rates in various autoimmune diseases, such as systemic lupus erythematosus (SLE, 95% -100%), rheumatoid arthritis (RA, 10% -20%), mixed connective tissue disease (MCTD, 80% -100%), Sjogren's syndrome (SS, 10% -40%), systemic scleroderma (PSS, 85% -90%), lupus hepatitis (LH, 95% -100%), primary biliary cirrhosis (PBC, 95% -100%), and the like. Among them, Histone (His) is one of the main target antigens of ANA.
Histone is a basic protein which is combined with DNA to form a chromosome, consists of H1, H2A, H2B, H3, H4, H5 and [ H2A-H2B ] -DNA dimer, often exists in a tetramer form to form a nucleosome, has no species specificity and organ specificity, is easy to combine with DNA, antibody and complement components, and is often negative for normal people. Anti-histone antibodies are found in 50-70% SLE and also in 95% of drug-induced lupus patients, which often lack autoantibodies against H2A, H2B. Scholars also reported a positive rate of 35.6% for anti-histone antibodies in SLE patients, but up to 90% for active SLE patients, with 94% of patients having no clear history of taking medication; 5-14% of patients with primary biliary cirrhosis and 24% of rheumatoid arthritis, in particular patients with vasculitis; the histone antibody of anti-H2A-H2B complex has higher positive rate in SLE and procainamide induced SLE, while the histone antibody of anti-H3-H4 complex is mostly seen in hydralazine induced SLE patients.
Because the indirect immunofluorescence method is time-consuming and labor-consuming to detect the anti-histone antibody, an ELISA method is commonly used at present. The histone subtypes are more, and when detecting total histone antibodies, anti-H2A and H2B antibodies are preferably detected at the same time to eliminate drug lupus.
At present, the detection method of the anti-His antibody has been reported, and the detection method of the disease mainly comprises an indirect immunofluorescence method and an enzyme-linked immunosorbent assay, but the methods have defects;
one, indirect immunofluorescence method
The basic principle of the method is that after specific antibodies in a serum sample are specifically combined with antigens in a section, indirect fluorescent antibodies are combined with antigen-antibody complexes to form antigen-antibody fluorescent complexes. Under a fluorescence microscope, the detected result is determined according to the luminescence of the complex. The method comprises the following evaluation: the fluorescence intensity emitted from the antigen-antibody complex is increased due to the increase of the amount of the fluorescein antibody bound to the antigen-antibody complex, and the sensitivity is high. But its deficiencies are also evident:
(1) false positives may occur with this method.
(2) The result of the analysis cannot be distinguished from non-specific reactions according to the molecular weight.
(3) The operation is relatively complex, a fluorescence microscope with higher price is needed, the popularization is difficult in a plurality of primary hospitals, and meanwhile, the method is not suitable for laboratories and diagnosis hospitals with larger sample size.
(4) Background in fluorescence measurement is high, and the use of fluorescence immunoassay technology for quantitative measurement has certain difficulty.
(5) The result judgment requires an experienced professional, and the objectivity of the analysis result is insufficient.
Enzyme linked immunosorbent assay
The ELISA is simple, convenient and easy to implement, has high specificity, is combined with an indirect immunofluorescence method for detection, and can provide more objective experimental basis for clinical diagnosis and treatment of antinuclear antibodies. However, compared with other biological detection or immunoassay, the ELISA detection method, technique, tool or product still has many disadvantages, which make the application thereof limited, and the disadvantages mainly include the following aspects:
(1) the detection reagent is in an open mode in the detection process, is easily influenced by the external environment, and easily causes cross contamination among various reagents to influence the detection result;
(2) the ELISA detection range and sensitivity are low.
(3) The ELISA method has a long detection time, generally needs more than 2 hours to complete a test, and cannot completely meet the clinical requirement of rapid diagnosis.
(4) ELISA method can not random sample injection detection, and the detection result has hysteresis.
CN105954267A discloses a magnetic particle chemiluminescence quantitative determination kit for anti-histone antibody IgG and preparation and detection methods thereof, wherein the kit comprises: an anti-histone antibody IgG calibrator; anti-histone antibody IgG quality control; tris buffer solution containing biotin-labeled histone antigen and bovine serum albumin; tris buffer solution containing sheep anti-human polyclonal antibody marked by alkaline phosphatase and bovine serum albumin; tris buffer solution containing magnetic particles marked by streptavidin and bovine serum albumin; and (5) cleaning the liquid. In this patent, only histone antigens containing biotin labels are used, so that the positive detection rate of SLE patients is low.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit and a detection method for detecting anti-histone antibody IgG, which have high positive detection rate of SLE patients.
In order to solve the technical problems, the invention adopts the following technical scheme:
an object of the present invention is to provide a kit for measuring IgG, which comprises a first reagent and a second reagent, wherein the first reagent comprises a mixed antigen conjugated with biotin, and the mixed antigen comprises a histone antigen, a H1 fragment, and a H2A-H2B complex fragment; the second reagent comprises an anti-human IgG antibody conjugated to alkaline phosphatase.
Preferably, the mass ratio of the histone antigen, the H1 fragment and the H2A-H2B composite fragment is 2-3: 1-2: 1.
preferably, the concentration of the mixed antigen coupled with biotin in the first reagent is 1-5 micrograms/ml.
Preferably, the concentration of the alkaline phosphatase-conjugated anti-human IgG antibody in the second reagent is 0.1-2.5. mu.g/ml.
Preferably, the kit further comprises a magnetic particle separation reagent, a chemiluminescent substrate, a calibrator, a quality control product and a cleaning solution, wherein the magnetic particle separation reagent comprises nano magnetic particles with avidin connected to the surfaces.
Further preferably, said calibrator comprises a first calibrator having a concentration of said anti-histone antibody of 20RU/mL and a second calibrator having a concentration of said anti-histone antibody of 200 RU/mL.
Further preferably, the quality control product comprises a first quality control product with the concentration of the anti-histone antibody of 10RU/mL and a second quality control product with the concentration of the anti-histone antibody of 80 RU/mL.
The chemiluminescent substrate employed in the present invention is an enzymatic chemiluminescent substrate for alkaline phosphatase disclosed in application No. CN 201510359183.0. The chemiluminescence substrate has the advantages of high strength, high sensitivity, long duration, good stability and the like. Since AMPPD can play a role of co-surfactant, the chemiluminescence substrate can be better combined into a chemiluminescence buffer system, so that the chemiluminescence efficiency is greatly improved, and photons are released under the catalysis of alkaline phosphatase.
Another object of the present invention is to provide a method for preparing the kit, wherein the method for preparing the first reagent comprises the following steps:
(1) mixing the mixed antigen and biotin activated by N-hydroxysuccinimide according to the mass ratio of 10-50: 1, and standing for 20-40 min at 15-40 ℃;
(2) adding a trihydroxymethyl aminomethane buffer solution with the substance amount concentration of 0.01-0.11 mol/L, standing for 10-20 min at 15-40 ℃, and adding glycerol to obtain a concentrated solution; wherein the addition amount of the tris buffer solution is 15-20 microliters per 1 milligram of the mixed antigen; the addition amount of the glycerol is 400-600 microliters per 1 milligram of the mixed antigen;
(3) diluting the concentrated solution into mixed antigen coupled with biotin, wherein the mixed antigen is 1-5 micrograms/milliliter, and the concentration of the mixed antigen is 1-5 micrograms/milliliter, namely the first reagent, by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of substances of 0.005-0.1 mol/L;
the preparation method of the second reagent comprises the following steps:
(1) adding an anti-human IgG antibody into a 2-iminosulfane hydrochloride coupling agent with the concentration of 4-20 mg/mL, and standing for 18-25 min at 15-40 ℃; wherein the addition amount of the 2-iminothiolane hydrochloride coupling agent is 10-15 ml per 1mg of the anti-human IgG antibody;
(2) adding glycine solution with the concentration of 0.05-0.11 mol/L into the step (1), standing for 4-5 min at 15-40 ℃, desalting by using a G-25 gel column, and collecting the activated anti-human IgG antibody, wherein the addition amount of the glycine solution is 0.5-0.6 ml per 1mg of the anti-human IgG antibody;
(3) adding alkaline phosphatase into a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution with the concentration of 4-5 mg/mL, standing at 15-40 ℃ for 25-35 min, desalting by using a G-25 gel column, and collecting the activated alkaline phosphatase, wherein the addition amount of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution is 0.25-0.9 mL per 1mg of the alkaline phosphatase;
(4) mixing the anti-human IgG antibody activated in the step (2) with the alkaline phosphatase activated in the step (3), standing for 12-24 h at 2-8 ℃, and purifying by using a Superdex 200 gel purification column to obtain a concentrated solution of a conjugate, wherein the feeding mass ratio of the anti-human IgG antibody to the alkaline phosphatase is 1: 0.5-1.5;
(5) and (3) diluting the concentrated solution of the connecting object by using a trihydroxymethyl aminomethane buffer solution containing 0.1-3% of bovine serum albumin by mass, the pH value of the buffer solution being 7.8-8.0 and the mass concentration of the substance being 0.01-0.11 mol/L to the concentration of the anti-human IgG antibody coupled with the alkaline phosphatase being 0.1-2.5 micrograms/ml, thus obtaining the second reagent.
Preferably, the preparation method of the calibrator comprises the following steps: diluting an anti-histone antibody with a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of 0.005-0.1 mol/L into a first calibrator with the concentration of 20RU/mL and a second calibrator with the concentration of 200RU/mL respectively.
Preferably, the preparation method of the quality control product comprises the following steps: diluting the anti-histone antibody into a first quality control product with the concentration of 10RU/mL and a second quality control product with the concentration of 80RU/mL by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of a substance of 0.005-0.1 mol/L.
The third purpose of the invention is to provide a detection method of the kit, which comprises the following steps:
(1) sequentially adding a magnetic particle separation reagent and a first reagent into a detection tube, then adding a sample to be detected, uniformly mixing, incubating for 10-25 min at 36-38 ℃, and then cleaning with a cleaning solution under the action of a magnetic field, wherein the feeding volume ratio of the magnetic particle separation reagent to the first reagent to the sample to be detected is 1: 0.5-2: 0.2 to 0.5;
(2) removing the magnetic field, adding a second reagent, uniformly mixing, incubating for 10-25 min at 36-38 ℃, and then cleaning with a cleaning solution under the action of the magnetic field, wherein the feeding volume ratio of the sample to be detected to the second reagent is 1: 4-8;
(3) removing the magnetic field, adding a chemiluminescence substrate, fully suspending, incubating at 36-38 ℃ for 5-10 min, and detecting the relative luminescence intensity value.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the kit and the detection method have the advantages of good stability, high sensitivity, good repeatability and the like, meanwhile, the detection time is short, the total time for completing one test is within 50 minutes, the operation is simple and convenient, the full automation of detection is really realized, and in addition, the kit and the detection method have high positive detection rate of SLE patients.
Drawings
FIG. 1 is a schematic diagram of the detection;
FIG. 2 is a linear regression curve.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, the implementation conditions which are not indicated are conventional conditions in the industry, and the reagents in the invention can be commercially obtained.
Example 1: preparation of the first reagent
1. Materials and instruments:
materials: histone antigen, H1 fragment, H2A-H2B complex fragment, N-hydroxysuccinimide activated biotin, tris buffer, glycerol, phosphate buffer;
the instrument comprises the following steps: a refrigerator.
2. The preparation method comprises the following steps:
step 1: mixing 0.5mg mixed antigen (histone antigen, H1 fragment, H2A-H2B composite fragment mass ratio is 2: 1: 1) with 0.01mg N-hydroxysuccinimide activated biotin, standing at 25 deg.C for 30 min;
step 2: adding 10uL of trihydroxymethyl aminomethane buffer solution with the substance concentration of 0.01mol/L, standing at 25 deg.C for 15min, adding 300uL of glycerol to obtain biotinylated His antigen, and storing at-20 deg.C;
and step 3: and diluting the biotinylated His antigen into a solution with the concentration of 1ug/mL by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of the substance of 0.01mol/L to obtain the first reagent.
Example 2: preparation of the second reagent
1. Materials and instruments:
materials: anti-human IgG antibody, 2-iminothiolane hydrochloride coupling agent, glycine, alkaline phosphatase, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution, bovine serum albumin, tris buffer solution;
the instrument comprises the following steps: refrigerator, G-25 gel column, Superdex 200 gel purification column;
2. the preparation method comprises the following steps:
step 1: adding 5mg of anti-human IgG antibody into 50mL of 2-iminothiolane hydrochloride coupling agent with the concentration of 10mg/mL, and standing for 20min at 25 ℃;
step 2: adding 3mL of 0.1mol/L glycine solution, standing at 25 deg.C for 5min, desalting with G-25 gel column, collecting activated anti-human IgG antibody, and storing at 2-8 deg.C;
and step 3: adding 5mg alkaline phosphatase into 2.5mL of 5mg/mL 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester solution, standing at 25 deg.C for 30min, desalting with G-25 gel column, collecting activated alkaline phosphatase, and storing at 2-8 deg.C;
and 4, step 4: mixing the activated anti-human IgG antibody and the activated alkaline phosphatase, standing at 5 ℃ for 20h, purifying by a Superdex 200 gel purification column to obtain a concentrated solution of a conjugate, and storing at 2-8 ℃ for later use;
and 5: and (3) diluting the concentrated solution of the connecting substance by using a tris buffer solution containing 1% by mass of bovine serum albumin, pH 7.9 and the mass concentration of substances of 0.1mol/L until the concentration of the alkaline phosphatase-labeled anti-human IgG antibody is 1 mu g/ml, thus obtaining the second reagent.
Example 3: preparation of calibrator
1. Materials and instruments:
materials: anti-histone antibody, phosphate buffer solution and standard substance;
2. the preparation method comprises the following steps:
selecting anti-histone antibody, diluting with phosphate buffer solution with pH of 7-7.5 and substance amount concentration of 0.01mol/L in a certain proportion, and making into calibrator with concentration of 20RU/mL and 200RU/mL respectively by reference to standard.
Example 4: preparation of quality control product
1. Materials and instruments:
materials: anti-histone antibody, phosphate buffer solution and standard substance;
2. the preparation method comprises the following steps:
diluting the anti-histone antibody by phosphate buffer solution with pH of 7-7.5 and substance amount concentration of 0.01mol/L according to a certain proportion, and preparing quality control substances with concentrations of 10RU/mL and 80RU/mL respectively according to standard substances.
Example 5:
magnetic particle separating reagent and cleaning liquid.
The chemiluminescent substrate is shown in the formula II in Table 1.2APSH-1 of an enzymatic chemiluminescent substrate of alkaline phosphatase disclosed in application No. CN201510359183.0, namely
Example 6: testing steps of full-automatic chemiluminescence tester
The full-automatic chemiluminescence determinator test sequentially comprises the following steps:
step 1: the detection kit needs to be matched with a full-automatic chemiluminescence determinator of the company for use, the kit is placed in a corresponding position of a reagent bin of the full-automatic chemiluminescence determinator, and information of the kit is input into an instrument system through a bar code scanner or is set through instrument matching software.
Step 2: the calibrator is placed in the instrument sample compartment. The calibrator information is identified by a bar code scanner and the calibrator position is assigned in the instrument system.
And step 3: and (3) placing the quality control material/sample to be detected in an instrument sample bin, and editing corresponding detection information through instrument matching software.
And 4, step 4: the running program is started, and all the steps of the calibrator/quality control material/sample to be tested are automatically executed.
Taking the example of detecting a sample to be detected by the equipment, the automatic execution of the equipment comprises the following steps:
step 1: sequentially adding 50 mu L of magnetic particle separation reagent and 50 mu L of first reagent into a detection tube, then adding 20 mu L of sample to be detected, uniformly mixing, and incubating for 20min at 37 ℃;
step 2: adding a magnetic field, allowing the system incubated in the step 1 to settle in the magnetic field, removing supernatant, and adding 500 mu L of cleaning solution for 3-5 times of cleaning;
and step 3: removing the magnetic field, adding 100 μ L of the second reagent into the system cleaned in step 2, mixing, and incubating at 37 deg.C for 15 min;
and 4, step 4: adding a magnetic field, allowing the system incubated in the step 3 to settle in the magnetic field, removing the supernatant, adding 500 mu L of cleaning solution, cleaning for 3-5 times, removing the magnetic field, and oscillating to fully suspend the magnetic particles;
and 5: and (4) adding a magnetic field, repeating the step 4, cleaning again, settling the suspended magnetic particles in the magnetic field, removing the supernatant, removing the magnetic field, adding 150 mu L of chemiluminescence substrate, removing the magnetic field, fully suspending, and incubating at 37 ℃ for 5min to detect the relative luminescence intensity value.
When the detection kit is matched with a full-automatic chemiluminescence determinator for use, full automation is realized from the steps of dilution, sample addition, incubation, cleaning and detection, and unattended running water operation can be realized. The full-automatic closed operation system is simple and convenient to operate, high in reliability, good in stability and good in repeatability of detection results, avoids result deviation caused by manual operation, and effectively improves detection efficiency and saves labor cost.
Performance evaluation of the kit:
1. the kit of the invention
(1) Sample comparison
The negative and positive coincidence rate:
the kit detects the content of the anti-His antibody in clinical serum, and clinical comparison is carried out between the kit and similar products ELISA of foreign well-known companies, and the result shows that the anti-His antibody detection kit has a negative coincidence rate of 95.3 percent (242/254) and a positive coincidence rate of: 88.0% (88/100), see Table 1 for specific data; individual histone antigen negative compliance: 89.4% (227/254), positive match rate: 70% (70/100), see table 2 for specific data; h1 antigen negative match rate alone: 87% (221/254), positive match rate: 77% (77/100), see table 3 for specific data; the antigen negative coincidence rate of the H2A-H2B complex fragment alone is 90.6% (230/254), and the positive coincidence rate is: 82% (82/100), see Table 4 for specific data.
Compared with a fluorescence method, the kit has the negative coincidence rate of 97.5 percent (195/200) and the positive coincidence rate of 90.0 percent (135/150), and the specific data are shown in a table 5; individual histone antigen negative compliance: 92.5% (185/200), positive match rate: 83.3% (125/150), see Table 6 for specific data; h1 antigen negative match rate alone: 95% (190/200), positive match rate: 86.7% (130/150), see table 7 for specific data; the antigen negative coincidence rate of the H2A-H2B complex fragment alone is 95% (190/200), and the positive coincidence rate is: 90% (135/150), see Table 8 for specific data.
It can be seen that the kit has high consistency with the existing anti-His antibody detection reagent on the market, and the clinical comparison coincidence rate is improved by a lot compared with the reagent of a single antigen.
Meanwhile, the clinical compliance rate of the kit is detected, 150 SLE patients, 50 rheumatoid patients, 50 ankylosing spondylitis and 100 health physical examination persons are selected, and the results show that the SLE sample positive detection rate is 90% (135/150), the rheumatism patient sample positive detection rate is 30% (15/50), the ankylosing spondylitis sample positive detection rate is 0% (0/50) and the health physical examination sample positive detection rate is 3% (3/100), the sensitivity of the kit on the SLE patients is 90%, the specificity of the kit is 96.5% (193/200), and specific data are shown in Table 9.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
TABLE 5
TABLE 6
TABLE 7
TABLE 8
TABLE 9
(2) Sensitivity: the LOD of the kit is 0.084RU/mL, and the sensitivity of the enzyme-linked immunosorbent assay is 2 RU/mL.
(3) Linearity: diluting a part of high-value serum according to the proportion of 1/2, 1/4, 1/8, 1/16, 1/40, 1/80, 1/200 and 1/400, detecting the diluted sample by using a kit, and making a regression curve according to the dilution proportion and the detection concentration. The square value of the correlation coefficient R is obtained. The results are shown in fig. 2, which shows that the linear correlation coefficient of the anti-histone antibody kit is 0.9987, greater than 0.99, and the slope is 1.01.
(4) Accuracy: adding high value serum and median serum to a base serum according to a ratio of 9:1 to form serum addition samples with 2 concentration levels, detecting the sample concentration, and calculating the recovery rate according to the following formula. The recovery rate of the serum sample application of the method is between 85 and 115 percent, and the data are shown in a table 10.
Sample recovery after addition/(0.9 × sample a +0.1 × sample B) × 100%
In the formula: sample a was basal serum and sample B was spiked high value, median serum.
Watch 10
(5) Precision: the quality control products with three different concentrations are detected twice a day, the detection is carried out in the afternoon, 4 times of repetition are carried out each time, the detection lasts for 10 days, the detection is carried out for 80 times of each concentration, the coefficient of variation is calculated, the result shows that the coefficient of variation is within 10%, and the result is shown in a table 11.
TABLE 11
| Concentration (RU/mL) | Number of measurements | Inter-assay CV (%) |
| 10 | 80 | 3.6 |
| 20 | 80 | 4.5 |
| 100 | 80 | 3.1 |
(6) Stability: after the anti-His antibody detection kit is placed at 37 ℃ for 7 days, the quality control of high, medium and low concentrations is determined, and the result shows that the detection concentrations of the 3 quality controls are all in the quality control concentration range. The anti-His antibody detection kit is proved to have good stability and meet the clinical requirements.
(7) Specificity: the results of the detection results of bilirubin, hemoglobin, rheumatoid factors, triglycerides and human anti-mouse antibodies with different concentrations added to samples with different concentration values of high, medium and low are shown in table 12, and the results show that the additives have no influence on the detection results of the anti-His antibody detection kit.
TABLE 12
| Interfering substance | Concentration of addition | Cross reaction Rate (%) |
| Bilirubin | 20mg/dL | 0.13 |
| Hemoglobin | 1000mg/dL | 0.47 |
| Triglycerides | 2000mg/dL | 0.50 |
| Human anti-mouse antibodies | 2000ng/mL | 0.76 |
| Rheumatoid factor | 1000IU/mL | 1.08 |
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (6)
1. A kit for measuring IgG, which is an anti-histone antibody, characterized in that: the kit comprises a first reagent and a second reagent, wherein the first reagent comprises a mixed antigen conjugated with biotin, and the mixed antigen comprises a histone antigen, a H1 fragment and a H2A-H2B compound fragment; the second reagent comprises an anti-human IgG antibody coupled with alkaline phosphatase,
the mass ratio of the histone antigen, the H1 fragment and the H2A-H2B compound fragment is 2: 1: 1,
the preparation method of the first reagent comprises the following steps:
(1) mixing the mixed antigen and biotin activated by N-hydroxysuccinimide according to the mass ratio of 10-50: 1, and standing for 20-40 min at 15-40 ℃;
(2) adding a trihydroxymethyl aminomethane buffer solution with the substance amount concentration of 0.01-0.11 mol/L, standing for 10-20 min at 15-40 ℃, and adding glycerol to obtain a concentrated solution; wherein the addition amount of the tris buffer solution is 15-20 microliters per 1 milligram of the mixed antigen; the addition amount of the glycerol is 400-600 microliters per 1 milligram of the mixed antigen;
(3) diluting the concentrated solution into mixed antigen coupled with biotin, wherein the mixed antigen is 1-5 micrograms/milliliter, and the concentration of the mixed antigen is 1-5 micrograms/milliliter, namely the first reagent, by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of substances of 0.005-0.1 mol/L;
the kit also comprises a magnetic particle separation reagent, a chemiluminescent substrate, a calibrator, a quality control material and a cleaning solution,
the magnetic particle separating reagent comprises nano magnetic particles with avidin connected to the surface,
the calibrator comprises a first calibrator with the concentration of the anti-histone antibody being 20RU/mL and a second calibrator with the concentration of the anti-histone antibody being 200RU/mL,
the quality control product comprises a first quality control product with the concentration of the anti-histone antibody of 10RU/mL and a second quality control product with the concentration of the anti-histone antibody of 80 RU/mL.
2. The kit for the determination of anti-histone antibody IgG according to claim 1, characterized in that: the concentration of the mixed antigen coupled with biotin in the first reagent is 1-5 micrograms/ml.
3. The kit for the determination of anti-histone antibody IgG according to claim 1, characterized in that: the concentration of the anti-human IgG antibody coupled with alkaline phosphatase in the second reagent is 0.1-2.5 micrograms/ml.
4. A method of preparing a kit according to any one of claims 1 to 3, wherein:
the preparation method of the first reagent comprises the following steps:
(1) mixing the mixed antigen and biotin activated by N-hydroxysuccinimide according to the mass ratio of 10-50: 1, and standing for 20-40 min at 15-40 ℃;
(2) adding a trihydroxymethyl aminomethane buffer solution with the substance amount concentration of 0.01-0.11 mol/L, standing for 10-20 min at 15-40 ℃, and adding glycerol to obtain a concentrated solution; wherein the addition amount of the tris buffer solution is 15-20 microliters per 1 milligram of the mixed antigen; the addition amount of the glycerol is 400-600 microliters per 1 milligram of the mixed antigen;
(3) diluting the concentrated solution into mixed antigen coupled with biotin, wherein the mixed antigen is 1-5 micrograms/milliliter, and the concentration of the mixed antigen is 1-5 micrograms/milliliter, namely the first reagent, by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of substances of 0.005-0.1 mol/L;
the preparation method of the second reagent comprises the following steps:
(1) adding an anti-human IgG antibody into a 2-iminosulfane hydrochloride coupling agent with the concentration of 4-20 mg/mL, and standing for 18-25 min at 15-40 ℃; wherein the addition amount of the 2-iminothiolane hydrochloride coupling agent is 10-15 ml per 1mg of the anti-human IgG antibody;
(2) adding glycine solution with the concentration of 0.05-0.11 mol/L into the step (1), standing for 4-5 min at 15-40 ℃, desalting by using a G-25 gel column, and collecting the activated anti-human IgG antibody, wherein the addition amount of the glycine solution is 0.5-0.6 ml per 1mg of the anti-human IgG antibody;
(3) adding alkaline phosphatase into a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution with the concentration of 4-5 mg/mL, standing at 15-40 ℃ for 25-35 min, desalting by using a G-25 gel column, and collecting the activated alkaline phosphatase, wherein the addition amount of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution is 0.25-0.9 mL per 1mg of the alkaline phosphatase;
(4) mixing the anti-human IgG antibody activated in the step (2) with the alkaline phosphatase activated in the step (3), standing for 12-24 h at 2-8 ℃, and purifying by using a Superdex 200 gel purification column to obtain a concentrated solution of a conjugate, wherein the feeding mass ratio of the anti-human IgG antibody to the alkaline phosphatase is 1: 0.5-1.5;
(5) and (3) diluting the concentrated solution of the connecting object by using a trihydroxymethyl aminomethane buffer solution containing 0.1-3% of bovine serum albumin by mass, the pH value of the buffer solution being 7.8-8.0 and the mass concentration of the substance being 0.01-0.11 mol/L to the concentration of the anti-human IgG antibody coupled with the alkaline phosphatase being 0.1-2.5 micrograms/ml, thus obtaining the second reagent.
5. The method for preparing a kit according to claim 4, wherein:
the preparation method of the calibrator comprises the following steps: diluting an anti-histone antibody into a first calibrator with the concentration of 20RU/mL and a second calibrator with the concentration of 200RU/mL respectively by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of a substance of 0.005-0.1 mol/L;
the preparation method of the quality control product comprises the following steps: diluting the anti-histone antibody into a first quality control product with the concentration of 10RU/mL and a second quality control product with the concentration of 80RU/mL by using a phosphate buffer solution with the pH of 7-7.5 and the mass concentration of a substance of 0.005-0.1 mol/L.
6. A test method using the kit according to any one of claims 1 to 3 for diagnosis and treatment of non-diseases, characterized in that: the method comprises the following steps:
(1) sequentially adding a magnetic particle separation reagent and a first reagent into a detection tube, then adding a sample to be detected, uniformly mixing, incubating for 10-25 min at 36-38 ℃, and then cleaning with a cleaning solution under the action of a magnetic field, wherein the feeding volume ratio of the magnetic particle separation reagent to the first reagent to the sample to be detected is 1: 0.5-2: 0.2 to 0.5;
(2) removing the magnetic field, adding a second reagent, uniformly mixing, incubating for 10-25 min at 36-38 ℃, and then cleaning with a cleaning solution under the action of the magnetic field, wherein the feeding volume ratio of the sample to be detected to the second reagent is 1: 4-8;
(3) removing the magnetic field, adding a chemiluminescence substrate, fully suspending, incubating at 36-38 ℃ for 5-10 min, and detecting the relative luminescence intensity value.
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| CN114316016B (en) * | 2021-12-14 | 2023-11-10 | 江苏浩欧博生物医药股份有限公司 | Method for biotinylation of Jo-1 antigen and anti-Jo-1 antibody detection kit |
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