Disclosure of Invention
The invention aims to provide a detection kit for an anti-mitochondrial antibody M2, which has higher stability and can effectively prolong the storage time of the kit.
In order to solve the technical problems, the invention adopts the following technical scheme:
the detection kit for the anti-mitochondrial antibody M2 comprises a first reagent, a second reagent, a magnetic particle reagent and a chemiluminescent substrate, wherein the first reagent comprises an M2 antigen coupled with biotin and a first buffer solution, the first buffer solution contains 0.005-0.05 g/L of phenylmethylsulfonyl fluoride (PMSF), 0.01-0.1 g/L of dithiothreitol and 1-2 g/L of pyruvic acid and salts or derivatives thereof, and the second reagent comprises an anti-human IgG antibody coupled with alkaline phosphatase and a second buffer solution.
Preferably, the concentration of the phenylmethylsulfonyl fluoride is 0.005 g/L-0.03 g/L, such as 0.006g/L, 0.008g/L, 0.012g/L, 0.016g/L, 0.018g/L, 0.020g/L.
More preferably, the concentration of the phenylmethylsulfonyl fluoride is 0.01g/L to 0.025g/L.
Preferably, the concentration of dithiothreitol is 0.01 g/L-0.05 g/L, such as 0.01g/L, 0.012g/L, 0.014g/L, 0.016g/L, 0.018g/L, 0.020g/L, 0.022g/L, 0.026g/L, 0.030g/L.
Further preferably, the concentration of the dithiothreitol is 0.02-0.025 g/L.
Preferably, the concentration of the pyruvic acid and the salts or derivatives thereof is 1 g/L-1.8 g/L, such as 1.2g/L, 1.4g/L and 1.6g/L.
More preferably, the concentration of the pyruvic acid and the salt or derivative thereof is 1 g/L-1.5 g/L.
Preferably, the first buffer solution further contains 0.1wt% to 3wt% of bovine serum albumin.
Further preferably, the first buffer solution further contains 0.1wt% to 2wt% of bovine serum albumin.
Preferably, the first buffer is a phosphate buffer.
Preferably, the pH of the first buffer is 6.5 to 7.5.
Further preferably, the pH of the first buffer is 6.5 to 7.0.
Preferably, the pyruvic acid and salts or derivatives thereof is pyruvic acid.
Preferably, the pyruvic acid and salts or derivatives thereof are sodium pyruvate.
Preferably, the M2 antigen is a naturally extracted M2 antigen and a recombinant M2 antigen in a mass ratio of 1 (0.025 to 1).
Further preferably, the mass ratio of the naturally extracted M2 antigen to the recombinant M2 antigen is 1: (0.1 to 1), for example, 1:0.2, 1:0.4, 1:0.6, 1:0.8.
preferably, the magnetic particle reagent is a magnetic particle suspension with streptavidin coupled on the surface.
Preferably, the diameter of the magnetic particles in the magnetic particle reagent is 1 to 3 μm.
Preferably, the chemiluminescent substrate is APS-5.
Preferably, the concentration of the biotin-conjugated M2 antigen in the first reagent is 0.5-4 μ g/mL.
Further preferably, the concentration of the biotin-conjugated M2 antigen in the first reagent is 1-3 μ g/mL.
Preferably, the concentration of the anti-human IgG antibody coupled with alkaline phosphatase in the second reagent is 0.1-3 [ mu ] g/mL.
More preferably, the concentration of the anti-human IgG antibody coupled with alkaline phosphatase in the second reagent is 1 to 2.5. Mu.g/mL.
Preferably, the second buffer solution is an MOPS buffer solution containing 0.1wt% to 3wt% of bovine serum albumin and having a pH of 6.5 to 7.5.
More preferably, the second buffer solution is MOPS buffer solution containing 0.1wt% to 2wt% of bovine serum albumin and having pH of 6.5 to 7.0.
Preferably, the preparation method of the first reagent comprises the following steps:
(1) Mixing a naturally extracted M2 antigen with biotin activated by N-hydroxysuccinimide, standing for 20min to 40min at 15-40 ℃, adding a phosphate buffer solution with the substance amount concentration of 0.01-0.11 mol/L, standing for 10min to 20min at 15-40 ℃, adding glycerol to obtain a biotinylated naturally extracted M2 antigen, and storing at-30-0 ℃ for later use;
(2) Mixing a recombinant M2 antigen with biotin activated by N-hydroxysuccinimide, standing for 20min to 40min at 15-40 ℃, adding a phosphate buffer solution with the substance amount concentration of 0.01-0.11 mol/L, standing for 10min to 20min at 15-40 ℃, adding glycerol to obtain a biotinylated recombinant M2 antigen, and storing at-30-0 ℃ for later use;
(3) Mixing the biotinylated naturally-extracted M2 antigen prepared in the step (1) with the biotinylated recombinant M2 antigen prepared in the step (2), and diluting with a first buffer solution to obtain the first reagent.
Preferably, the use method of the detection kit comprises the following steps:
(1) Sequentially adding the magnetic particle reagent, the first reagent and a sample to be detected into a detection tube, uniformly mixing, and incubating for 5min to 10min at 36-38 ℃, wherein the volume ratio of the magnetic particle reagent to the first reagent to the sample to be detected is 1: (0.8 to 1.2): (0.4 to 0.8);
(2) Adding a magnetic field to enable the system in the step (1) to settle in the magnetic field, removing supernatant, and then washing for multiple times by using a washing liquid;
(3) Removing the magnetic field, adding the second reagent into the system in the step (2), uniformly mixing, and incubating for 5min to 10min at the temperature of 36-38 ℃, wherein the volume ratio of the sample to be detected to the second reagent is 0.4: (1~2);
(4) Adding a magnetic field to enable the system in the step (3) to settle in the magnetic field, removing supernatant, and then washing for multiple times by using a washing liquid;
(5) And (4) removing the magnetic field, adding 200 mu L of the chemiluminescence substrate into the system in the step (4), uniformly mixing, incubating at 36-38 ℃ for 5-10 min, and detecting the relative luminescence intensity value.
Further preferably, the volume ratio of the magnetic particle reagent, the first reagent and the sample to be detected is 1: (0.8 to 1): (0.5 to 0.6).
Preferably, the detection kit further comprises a calibrator, a quality control material and a washing solution.
Further preferably, the concentration of the calibrator is 10 AU/mL-160 AU/mL, for example, 20AU/mL, 50AU/mL, 100AU/mL, 150AU/mL.
Further preferably, the concentration of the quality control substance is 20 AU/mL-150 AU/mL, for example, 30AU/mL, 50AU/mL, 80AU/mL, 100AU/mL, 140AU/mL.
The detection method adopts a full-automatic chemiluminescence determinator to carry out testing, and the detection steps are fully automatic.
Compared with the prior art, the invention has the following advantages:
according to the invention, the phenylmethylsulfonyl fluoride, the dithiothreitol, the pyruvic acid and the salt or derivative thereof are added in the first reagent, so that the stability of the first reagent is greatly improved, the storage time of the kit is prolonged, and the sensitivity, the precision and the accuracy of the kit can be equivalent to or even higher than those of the conventional chemiluminescence kit.
Detailed Description
The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
In order to provide a detection kit for the anti-mitochondrial antibody M2, the inventors have surprisingly found through extensive research and experimental verification that the stability of the kit can be improved by adjusting and optimizing the first reagent, particularly by adding a composite stabilizer (phenylmethylsulfonyl fluoride, dithiothreitol and pyruvic acid and salts or derivatives thereof) to the first reagent, thereby prolonging the storage time of the kit. In addition, for the detection of the anti-mitochondrial antibody in the detection sample, the buffer solution with a specific formula is used and is combined with other reagents for application, so that the stability of the kit is improved, and the detection accuracy, sensitivity, linear result, precision and the like of the kit can be improved. Therefore, the detection kit for the anti-mitochondrial antibody M2, which has good stability, high sensitivity and good specificity, is developed. The detection kit for the anti-mitochondrial antibody M2 can take a full-automatic chemiluminescence determinator as a detection instrument, the time for completing all processes of quantitative detection of the anti-mitochondrial antibody M2 is 30min, and the reaction time is greatly shortened compared with an enzyme-linked immunosorbent assay. The chemiluminescence immunoassay kit is matched with an instrument, is simple and convenient to operate, shortens the time required by clinical detection, avoids result deviation caused by manual operation, and effectively improves the detection efficiency and saves the labor cost.
Specifically, the detection kit for the anti-mitochondrial antibody M2 comprises a first reagent, a second reagent, a magnetic particle reagent and a chemiluminescent substrate.
The first reagent includes a biotin-conjugated M2 antigen, and a first buffer. The first buffer solution is a phosphate buffer solution (pH is 6.5-7.5), and the first buffer solution contains 0.005-0.05 g/L of phenylmethylsulfonyl fluoride (PMSF), 0.01-0.1 g/L of dithiothreitol, 1-2 g/L of pyruvic acid, salts or derivatives thereof and 0.1-3 wt% of bovine serum albumin. The second reagent comprises an anti-human IgG antibody coupled to alkaline phosphatase, and a second buffer. The second buffer solution is MOPS buffer solution (pH is 6.5-7.5), and the MOPS buffer solution comprises 0.1-3 wt% of bovine serum albumin. The mass ratio of the M2 antigen is 1:1, and a recombinant M2 antigen. The magnetic particle reagent is a magnetic particle suspension with streptavidin coupled on the surface, wherein the diameter of the magnetic particle is 1-3 mu m. The chemiluminescent substrate is APS-5.
The present invention will be described in further detail with reference to specific examples.
In the following examples and comparative examples, unless otherwise specified, the starting materials, reagents and the like used were conventional commercial products, in which tetramethylethylenediamine was available from Amresco (Amresco) in America under the trade designation 0761-100mL.
In the following examples and comparative examples, "%" is defined as mass% unless otherwise specified.
Example 1
This example provides a detection kit for anti-mitochondrial antibody M2, comprising
A first reagent (at a concentration of 1. Mu.g/mL); a second reagent (at a concentration of 2. Mu.g/mL); a magnetic microparticle reagent; a chemiluminescent substrate; a calibrator; and (5) quality control.
The preparation method of the first reagent comprises the following steps:
the first reagent includes a biotin-conjugated M2 antigen, and a first buffer.
Step 1: 1mg of naturally extracted M2 antigen (America A Gu Taike (AROTEC) Inc., model number: ATM02-10] and 0.025mg of N-hydroxysuccinimide activated biotin were mixed and left to stand at 25 ℃ for 30min; the volume of a reaction system is 1mL by phosphate buffer solution with the quantity concentration of the supplementary substances of 0.05mol/L, the reaction system is kept stand for 15min at 25 ℃, the mixture is desalted by zeba centrifugal desalting columns, purified water and the phosphate buffer solution are firstly used for treatment in the desalting process, finally the obtained biotinylation naturally extracted M2 antigen is added, liquid in a centrifuge tube is collected to a preservation tube to obtain the biotinylation naturally extracted M2 antigen, 1mL of glycerol is added, and 0.1mL of each bottle is subpackaged and preserved at-20 ℃ for later use. Obtaining biotinylated natural extracted M2 antigen, and storing at-20 ℃ for later use;
step 2: mixing 1mg of biotin activated by recombinant M2 antigen (Shanghai Ferma Biotech Co., ltd., model No. ATG 001) and 0.025mg of N-hydroxysuccinimide, and standing at 25 deg.C for 30min; the volume of a reaction system is 1mL by phosphate buffer solution with the quantity concentration of the supplementary substances of 0.05mol/L, the reaction system is kept stand for 15min at 25 ℃, the mixture is desalted by zeba centrifugal desalting columns, purified water and the phosphate buffer solution are firstly used for treatment in the desalting process, the obtained biotinylation recombination M2 antigen is added at last, liquid in a centrifuge tube is collected to a preservation tube to obtain the biotinylation recombination M2 antigen, 1mL of glycerol is added, and 0.1mL of each bottle is subpackaged and preserved at-20 ℃ for later use. Obtaining biotinylated recombinant M2 antigen, and storing at-20 ℃ for later use;
and step 3: preparing phosphate buffer solution which contains 0.025g/L of phenylmethylsulfonyl fluoride, 0.025g/L of dithiothreitol, 1.0g/L of sodium pyruvate and 1% of bovine serum albumin by mass percent, has pH of 7.0 and has the mass concentration of 0.01 mol/L;
and 4, step 4: diluting the biotinylated natural extracted M2 antigen prepared in the step 1 and the biotinylated recombinant M2 antigen prepared in the step 2 into a mixed solution with the total concentration of the biotinylated M2 antigen being 1 mu g/mL by using the buffer solution prepared in the step 3, and obtaining a first reagent, wherein the mass ratio of the biotinylated natural extracted M2 antigen to the biotinylated recombinant M2 antigen is 1:1.
the preparation method of the second reagent comprises the following steps:
the second reagent comprises an anti-human IgG antibody coupled to alkaline phosphatase, and a second buffer.
And (2) diluting the anti-human IgG antibody coupled with alkaline phosphatase [ Sigma (Sigma) company in USA ] by using MOPS buffer solution containing 1.0% of bovine serum albumin by mass and having pH of 6.8 to obtain the second reagent, wherein the concentration of the anti-human IgG antibody marked by alkaline phosphatase in the second reagent is 2.0 mu g/mL.
The preparation method of the calibrator comprises the following steps:
selecting an anti-mitochondrial antibody (Shanghai Fermat Biotechnology Co., ltd.), diluting with a tetramethylethylenediamine buffer solution with pH of 7-7.5 and substance concentration of 0.1mol/L according to a certain proportion, and preparing into calibrators with concentrations of 10AU/mL and 160AU/mL respectively.
The preparation method of the quality control product comprises the following steps:
selecting an anti-mitochondrial antibody (Shanghai Fermat Biotechnology Co., ltd.), diluting with a tetramethylethylenediamine buffer solution with pH of 7-7.5 and substance concentration of 0.1mol/L according to a certain proportion, and preparing into quality control products with concentrations of 20AU/mL and 150AU/mL respectively.
The detection method comprises the following steps:
the kit adopts a full-automatic chemiluminescence determinator for testing, and specifically comprises the following steps:
step 1: the detection kit of the embodiment 1 is matched with a matched full-automatic chemiluminescence determinator 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: placing the calibrator in an instrument sample bin, identifying calibrator information through a bar code scanner, and allocating calibrator positions in an instrument system;
and step 3: placing a quality control product/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 calibrator/quality controller/sample to be tested processing steps will be automatically executed.
The full-automatic chemiluminescence determinator is matched with a detection reagent for use, and the detection steps are fully automated. 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.
Example 2
This example is substantially the same as example 1 except that in this example, phenylmethylsulfonyl fluoride was present at a concentration of 0.01g/L, dithiothreitol was present at a concentration of 0.02g/L and sodium pyruvate was present at a concentration of 1.5g/L.
Comparative example 1
This comparative example is essentially the same as example 1 except that the first reagent does not contain phenylmethylsulfonyl fluoride, dithiothreitol, and sodium pyruvate.
Comparative example 2
This comparative example is essentially the same as example 1 except that the first reagent does not contain sodium pyruvate.
Comparative example 3
This comparative example is essentially the same as example 1 except that the first reagent does not contain phenylmethylsulfonyl fluoride and dithiothreitol.
And (3) detection results:
the performance test of the detection kit for the anti-mitochondrial antibody M2 prepared in example 1, example 2 and comparative example 1~3 was performed by a conventional method in the art unless otherwise specified.
The detection kit for the anti-mitochondrial antibody M2 can be matched with a full-automatic chemiluminescence immunoassay analyzer (Suzhou Huizhong Biotechnology Co., ltd., type: IMS 1200) for detection, and the detection principle is a double-antibody sandwich method.
After the detection reagent for the anti-mitochondrial antibody M2 in example 1, example 2 and comparative example 1~3 was prepared, the sample was stored in a refrigerator at a temperature of 0 ℃ to 2 ℃ for 365 days. Then, various performances of the kit are tested.
1. Stability of
The reagents for detecting anti-mitochondrial antibody M2 in examples 1, 2 and 5363 in 1~3 were stored under the above-mentioned storage conditions for 365 days, and then left at 37 ℃ for 7 days to measure the signal retention rates of 7 kinds of quality control concentrations from low to high, as shown in Table 1.
As can be seen from Table 1, the relative deviation results of the test of the kits of the embodiment 1 and the embodiment 2 on the 7 th day are within 10%, and are greatly improved compared with the results of the comparative examples 1, 2 and 3, which shows that the kit has good stability, meets the clinical requirements, and can be stored for 2 years.
2. Sample comparison
The kit of example 1 was used to detect the anti-mitochondrial antibody content in 200 sera (100 negatives and 100 positives), and was compared clinically with anti-mitochondrial antibody ELISA test kits from foreign known companies and national known brand luminescence kits, and the experimental results are shown in Table 2. Wherein, the domestic famous brand luminescence kit is a kit which is three months away from the production date, and the kit is stored in a refrigerator at 0-2 ℃.
As can be seen from Table 2, the negative coincidence rate of the anti-mitochondrial antibody M2 reagent of the example 1 is 96% (96/100), and the positive coincidence rate is 96% (96/100), which indicates that the anti-mitochondrial antibody kit of the invention has high clinical coincidence rate, and the difference between the clinical coincidence rate of the kit of the invention after being placed for 1 year and the detection result coincidence rate of similar products of domestic well-known companies after being placed for 3 months is small, and even better.
3. Sensitivity of the probe
The LOD of the detection reagent of example 1 was 0.5AU/mL, that of the detection reagent of the domestic known brand was 0.7AU/mL, and that of the enzyme-linked immunosorbent assay was 2AU/mL.
The above results indicate that the sensitivity of the detection kit of example 1 is superior to that of the domestic famous brand.
4. Linear range
Diluting a part of high-value serum according to the proportion of 1, 1/2, 1/4, 1/8, 1/16 and 1/40, 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 was found, and the result is shown in fig. 1.
As can be seen from fig. 1, the linear correlation coefficient of the anti-mitochondrial antibody kit was 0.9997 and greater than 0.9900.
5. Precision degree
The reagents in example 1 were used to test three quality control substances with different concentrations twice a day, and the test was repeated 4 times in the afternoon, for 10 days, and each concentration was measured 80 times, and the results of calculating the coefficient of variation are shown in table 3.
As is clear from Table 3, the results show that the Coefficient of Variation (CV) is within 10%, indicating that the accuracy of the kit is high.
The above results show that:
1. stability: after the anti-mitochondrial antibody M2 detection kit prepared by the invention is placed at 0-2 ℃ for 365 days, is placed at 37 ℃ for 7 days, and then is used for measuring the signal retention rates of 7 quality control concentrations, the relative deviation in the examples 1 and 2 is within 10%, and the performances of the comparative examples 1, 2 and 3 are reduced along with the prolonging of time.
2. Sample comparison: the negative coincidence rate of the anti-mitochondrial antibody M2 reagent of the example 1 is 96% (96/100), and the positive coincidence rate is 96% (96/100), which shows that the anti-mitochondrial antibody kit of the invention has high clinical coincidence rate.
3. Sensitivity: in example 1, the LOD of the test agent is 0.5AU/mL, which are less than the values in the kits of the national known brand and the instruction of the enzyme linked immunosorbent assay.
4. Linear range: the linear correlation coefficient of the anti-mitochondrial antibody kit is 0.9997 and is greater than 0.9900, which is superior to the prior art.
5. Precision: the Coefficient of Variation (CV) of the quality control products with three different concentrations is within 10 percent, which indicates that the precision of the kit is high.
In conclusion, after the anti-mitochondrial antibody M2 detection kit provided by the invention is placed at 0-2 ℃ for 365 days, the stability of each component of the kit is high, and the detection sample compliance of the kit is improved, so that the sensitivity, the linear result and the precision are improved.
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.