Human bloodstain detection kit based on hHb / hMPO double detection method and detection method

Abstract

The invention discloses a human bloodstain detection kit based on an hHb / hMPO double detection method and a detection method. The kit comprises a fluorescently-labeled anti-human hemoglobin specific monoclonal antibody, a fluorescently-labeled anti-human MPO specific monoclonal antibody, a fluorescently-labeled DNP-BSA, an anti-human hemoglobin specific monoclonal antibody for coating, an anti-human MPO specific monoclonal antibody for coating, a rabbit anti-DNP polyclonal antibody, a chromatographic test strip and other related materials. The kit disclosed by the invention can be applied to accurate and rapid identification of human bloodstains in criminal reconnaissance, and interference of other animal bloodstains is eliminated by specifically detecting human hemoglobin and human MPO in the bloodstains; whether nucleated leukocytes exist or not is judged by measuring human MPO in bloodstains through a fluorescent quantitative method, a basis is provided for DNA detection, and the sample sampling amount of DNA detection can be adjusted according to the content of MPO.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a human blood stain detection kit and a detection method based on the dual detection of hHb / hMPO. Background Art

[0002] Blood (stains) are the most common forensic physical evidence samples, and blood stain inspection is the most frequently encountered and important detection item in forensic physical evidence inspection, accounting for about 80% of forensic physical evidence inspection. All suspicious blood or blood stains found at the scene, on the injury-causing objects, and clothing (victims and suspects) are related to blood (stain) inspection.

[0003] Once blood leaves the human body, it is subject to the effects and influences of external physical, chemical, and biological factors. The degradation, contamination, and corruption processes of blood are very rapid. The longer the time since leaving the body, the greater the change in blood components. In particular, the destruction of macromolecular antigen proteins and DNA increases the difficulty of individual identification of blood stains. Therefore, blood stain detection methods are more complex than clinical tests and more difficult than fresh blood detection. The older the blood stain, the lower the positive rate of the test. Therefore, it is important to promptly discover and extract blood stains and conduct tests immediately.

[0004] Blood stain preliminary test is a screening test, the purpose of which is to screen out non-blood stain samples from a large number of suspicious blood stains. Many stains are similar in appearance to blood stains, such as paint, pigment, sauce, soil, rust, vegetable, and fruit juice stains. Through the preliminary test, such stain samples can be excluded. There are many methods for the preliminary test. Currently, most of them are to determine hemoglobin in blood stains to achieve the purpose of screening blood stains, which have the characteristics of high sensitivity, simple operation, and rapidity. However, hemoglobin exists in red blood cells, and mature human red blood cells have no nucleus and cannot be used for DNA detection; therefore, it is very necessary to develop a kit with a detection index based on specific markers of blood nucleated cells to provide a basis for blood stain DNA detection. Summary of the Invention

[0005] Aiming at the above technical problems existing in the prior art, the purpose of the present invention is to provide a human blood stain detection kit and a detection method based on the dual detection of hHb / hMPO.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The preparation method of the anti-human MPO specific monoclonal antibody is as follows: Immunize mice with human-derived MPO antigen protein, and use the mouse monoclonal antibody technology to prepare anti-human-derived MPO monoclonal antibody. In antibody screening, human blood stains and blood stain samples of different animals such as pigs, cows, sheep, dogs, cats, rabbits, and mice are respectively used for testing to obtain a pair of anti-human blood stain MPO specific monoclonal antibodies that do not react with blood stains of different animals such as pigs, cows, sheep, dogs, cats, rabbits, and mice.

[0008] Further, one of the pair of anti-human blood stain MPO specific monoclonal antibodies is labeled with quantum dot fluorescent microspheres, designated as Q-hMPO-01, and the other is used as a coating antibody, designated as C-hMPO-02.

[0009] The preparation method of the anti-human hemoglobin specific monoclonal antibody is as follows: Immunize mice with human hemoglobin, and use mouse monoclonal antibody technology to prepare anti-human hemoglobin monoclonal antibody. In antibody screening, blood stain samples of different animals such as human blood stain, and pig, cow, sheep, dog, cat, rabbit, mouse, etc. are used for testing respectively to obtain a pair of anti-human hemoglobin specific monoclonal antibodies that do not react with blood stains of different animals such as pig, cow, sheep, dog, cat, rabbit, mouse, etc.

[0010] Further, one of the pair of anti-human hemoglobin specific monoclonal antibodies is labeled with quantum dot fluorescent microspheres, designated as Q-hHb-01, and the other is used as a coating antibody, designated as C-hHb-02.

[0011] A human blood stain detection kit based on hHb / hMPO double detection method, the kit further includes an immunochromatographic test strip, and the immunochromatographic test strip includes a backing, an NC membrane, a sample pad, a release pad and a blotting paper; the sample pad, the release pad, the NC membrane and the blotting paper are overlapped and assembled on the backing, the release pad and the blotting paper are respectively laminated at both ends of the NC membrane, and a detection area is formed on the surface of the NC membrane, and the sample pad is laminated on the release pad; on the NC membrane of the detection area, a test line area T1 line, a T2 line close to the release pad and a control line area C line close to the blotting paper are provided; DNP-BSA is conjugated to carboxyl quantum dot microspheres, designated as Q-DNP; Q-hHb-01, Q-hMPO-01, and Q-DNP are mixed and sprayed on the release pad, and C-hHb-02 is coated in the T1 line area on the NC membrane, C-hMPO-02 is coated in the T2 line area, and rabbit anti-DNP polyclonal antibody is coated in the C line area; the kit uses a dry fluorescence chromatography analyzer for quantitative analysis.

[0012] Further, a human blood stain detection kit based on hHb / hMPO double detection method, the detection limit of human hemoglobin in the kit is set at 1 ng / mL, the detection limit of human MPO protein is set at 0.1 ng / mL, and a dry fluorescence chromatography analyzer is used for quantitative analysis, and a quantitative standard curve of human hemoglobin and human MPO protein is preset in the detection instrument, and then the corresponding protein content in the actual sample detection is calculated according to the corresponding standard curve; the kit can be applied to the accurate and rapid identification of human blood stains in criminal investigation.

[0013] Further, the production of the standard curve includes the following process:

[0014] Step 1. Prepare Standard Curve 1: Using human MPO antigen protein as the standard, a series of standard solutions with different concentrations in the range of 1 ng / mL - 1024 ng / mL are prepared with PBS buffer at pH 7.4. Using PBS buffer at pH 7.4 as the blank control, the above-mentioned kit is used for detection respectively. After detection chromatography, a dry fluorescence chromatography analyzer is used to measure the fluorescence value of the T1 line and the fluorescence value of the C line. A standard curve 1 is plotted with the ratio of the fluorescence value of the T1 line to the fluorescence value of the C line, i.e., the T1 / C fluorescence value ratio, as the ordinate and the concentration of human MPO antigen protein as the abscissa;

[0015] Step 2. Prepare Standard Curve 2: Using human Hb protein as the standard, a series of standard solutions with different concentrations in the range of 1 ng / mL - 1024 ng / mL are prepared with PBS buffer at pH 7.4. Using PBS buffer at pH 7.4 as the blank control, the above-mentioned kit is used for detection respectively. After detection chromatography, a dry fluorescence chromatography analyzer is used to measure the fluorescence value of the T2 line and the fluorescence value of the C line. A standard curve 2 is plotted with the ratio of the fluorescence value of the T2 line to the fluorescence value of the C line, i.e., the T2 / C fluorescence value ratio, as the ordinate and the concentration of human Hb protein as the abscissa.

[0016] Furthermore, the usage method of the kit is as follows: Immerse the suspected bloodstain sample in PBS solution, shake it for 3 - 5 minutes to dissolve, and then use the kit for detection. After waiting for 10 - 15 minutes for detection chromatography, a dry fluorescence chromatography analyzer is used for quantitative analysis, and the detected concentrations of human hemoglobin and human MPO protein by the kit are calculated respectively according to the above-prepared standard curves. If human hemoglobin is not detected, i.e., the detected concentration < 1 ng / mL, it is a negative sample and can be excluded as human bloodstain; if the detected concentration value of human hemoglobin ≧ 1 ng / mL, it can be judged as a suspected human bloodstain; if it is judged as a suspected human bloodstain and human MPO protein is not detected at the same time, i.e., MPO < 0.1 ng / mL, it means that there are too few white blood cells in the sample to support DNA detection, and the DNA detection may show a negative result; if it is judged as a suspected human bloodstain and human MPO protein is detected at the same time, i.e., the detected concentration value of MPO ≧ 0.1 ng / mL, it is determined as a human bloodstain, indicating that the DNA detection should show a positive result, and the sample sampling amount for DNA detection can be adjusted according to the value of MPO. Finally, the DNA information of the person with the bloodstain is obtained through DNA detection.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present application targets the specific abundant protein MPO in nucleated blood cells, develops a specific monoclonal antibody against human MPO, and invents a human blood stain detection kit based on the dual detection of hHb / hMPO. MPO, Myeloperoxidase, is a heme protein that is rich in neutrophils and monocytes, synthesized in the bone marrow before granulocytes enter the circulation and stored in azurophilic granules; under normal circumstances, neutrophils account for 40-75% of human blood cells, so MPO is the second most abundant protein in the blood after hemoglobin. Hemoglobin exists in red blood cells, and mature human red blood cells have no nucleus and cannot be used for DNA detection. Therefore, developing a kit containing the MPO quantitative detection index can specifically detect blood stains and determine whether there are nucleated white blood cells, providing a basis for DNA detection (only nucleated cells have DNA, while mature human red blood cells have no nucleus), and the sample sampling amount for DNA detection can be adjusted according to the content of MPO. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic structural diagram of a human blood stain detection kit based on the dual detection of hHb / hMPO;

[0019] Figure 2 Standard curve for the determination of hHb content;

[0020] Figure 3 Standard curve for the determination of hMPO content. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The present invention will be described in detail below with reference to the drawings and specific embodiments:

[0022] The present invention discloses a human blood stain detection kit based on the dual detection of hHb / hMPO. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are regarded as included in the present invention. The preparation method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the preparation method and application described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0023] Explanation of some terms and abbreviations:

[0024] hMPO: Human Myeloperoxidase, human myeloperoxidase;

[0025] hHb: Human Hemoglobin, human hemoglobin;

[0026] DNP: Dinitrophenyl, dinitrophenol; The antigen DNP-BSA obtained by coupling DNP with BSA, and the anti-DNP antibody are often used as the independent quality control line C line of the chromatographic test strip.

[0027] The recombinant human MPO protein antigen, recombinant human Hb antigen, and carboxyl quantum dot microspheres used in the embodiments of the present invention are all commercially available products, among which:

[0028] Source of the human MPO protein antigen (i.e., hMPO): Aimeijie Technology Co., Ltd., product number: ENZ-074, https: / / www.amyjet.com / products / ENZ-074.shtml ;

[0029] Source of the recombinant human Hb antigen (i.e., hHb): Shanghai Qiming Biotechnology Co., Ltd., product number: PT3748, https: / / www.biomart.cn / infosupply / 104685491.htm.

[0030] Source of the carboxyl quantum dot microspheres: Hangzhou Boyin Biotechnology Co., Ltd., product number: QM605-300C, see website https: / / www.biogenome.cn / 343.

[0031] Preparation of quantum dot fluorescently labeled proteins Q-hHb-01, Q-hMPO-01, and Q-DNP in Example 1

[0032] Immunize mice with the human MPO protein antigen and prepare monoclonal antibodies against the human MPO protein using the mouse monoclonal antibody technique. In antibody screening, blood samples from different animals such as human blood, as well as pig, cow, sheep, dog, cat, rabbit, and mouse blood are used for testing to obtain a pair of anti-human MPO protein specific monoclonal antibodies that do not react with the blood of different animals such as pig, cow, sheep, dog, cat, rabbit, and mouse. One of the antibodies is labeled as hMPO-01 and is labeled with quantum dot fluorescent microspheres as Q-hMPO-01, and the other is used as the coating antibody and is labeled as C-hMPO-02.

[0033] Immunize mice with the recombinant human Hb as the antigen and prepare anti-human Hb antibodies using the mouse monoclonal antibody technique. In antibody screening, blood samples from different animals such as human blood, as well as pig, cow, sheep, dog, cat, rabbit, and mouse blood are used for testing to obtain a pair of anti-human Hb specific monoclonal antibodies that do not react with the blood of different animals such as pig, cow, sheep, dog, cat, rabbit, and mouse; One of the antibodies is labeled as hHb-01 and is labeled with quantum dot fluorescent microspheres as Q-hHb-01, and the other is used as the coating antibody and is labeled as C-hHb-02.

[0034] Anti-human MPO monoclonal antibody hMPO-01, anti-human Hb monoclonal antibody hHb-01, and DNP-BSA were conjugated to carboxyl quantum dot microspheres respectively. The specific steps are as follows:

[0035] 1) Adjustment of the carboxyl quantum dot microsphere buffer system: Dilute 3 ml of 1 μmol / L carboxyl quantum dot microspheres with 3 ml of 20 mmol / L pH 6 MES to approximately 0.5 μmol / L. The final buffer system is 10 mmol / L pH 6 MES.

[0036] 2) Microsphere activation: Add 7.5 μl of cross-linking agent EDC (76.7 mg / ml) and 7.5 μl of NHS (69.1 mg / ml) in sequence. After rapid mixing, react at 37 °C for 15 min. After the reaction, centrifuge at 15000 g for 15 min; resuspend the precipitate with a solution of 10 mmol / L MES pH 6.0 to a 3 ml volume solution (to make the microsphere concentration approximately 1 μmol / L), mix well, and ultrasonically disperse if necessary. If precipitation occurs during the reaction, it can be ultrasonically dispersed and mixed well.

[0037] 3) Aliquot the activated microspheres: 1 mL in tube A, 1 mL in tube B, and 1 mL in tube C.

[0038] 4) Antibody-conjugated microspheres: Add 600 μg of hMPO-01, 600 μg of hHb-01, and 600 μg of DNP-BSA to tubes A, B, and C respectively; react at 37 °C for 1 h. If precipitation occurs during the reaction, try to ultrasonically disperse and mix well.

[0039] 5) Washing and blocking: Add 200 μL of blocking agent (100 mM glycine solution containing 1% BSA) to tubes A, B, and C respectively; react at 37 °C for 0.5 h; centrifuge at 13000 g for 40 min and remove the supernatant; resuspend and disperse each tube with 200 μL of PB buffer (pH 7.0, 1% BSA, 8% sucrose, 0.05% NaN3), and ultrasonically disperse to obtain quantum dot fluorescently labeled protein Q-hMPO-01 solution, Q-hHb-01 solution, and Q-DNP solution, and store at 4 °C for standby.

[0040] Example 2 Preparation of a human blood stain detection kit based on the hHb / hMPO dual detection method

[0041] Refer to the traditional immunochromatographic test strip preparation process to prepare a quantum dot fluorescence-based human blood stain detection chromatographic test strip for hHb / hMPO dual detection.

[0042] 1) Take 5 μL each of the Q-hMPO-01 solution and Q-hHb-01 solution prepared in Example 1 above, and take 1 μL of the Q-DNP solution. Add them all to 49 μL of the gold-spraying diluent (0.1 M PB solution + 1% BSA + 4% sucrose + 0.05% NaN3). After mixing, use a gold-spraying instrument to coat it on the fluorescence release pad at a spraying volume of 60 μL / 30 cm, and dry it with forced air at 37 °C for 12 h. Seal it in a bag for later use;

[0043] 2) Coat the anti-human Hb monoclonal antibody C-hHb-02 and the anti-human MPO monoclonal antibody C-hMPO-02 on the detection line area T1 line area and T2 line area of the NC membrane respectively with the coating diluent (150 mM PB, pH 7.4), and coat the rabbit anti-DNP polyclonal antibody (i.e., Figure 1 the anti-DNP in

[0044] ) on the control line area C line area. The coating and scribing concentration is 1 mg / mL, and the scribing liquid volume is 30 μL / 30 cm. Dry it with forced air at 37 °C for 12 h. Seal it in a bag for later use; Figure 1 ) on the control line area C line area. The coating and scribing concentration is 1 mg / mL, and the scribing liquid volume is 30 μL / 30 cm. Dry it with forced air at 37 °C for 12 h. Seal it in a bag for later use; Figure 1 as shown in Figure 1 . Refer to

[0045] Example 3 Application of the Human Blood Stain Detection Kit Based on Dual Detection of hHb / hMPO

[0046] The human blood stain detection kit based on dual detection of hHb / hMPO developed based on the technical solution of the present invention can be applied to accurately and quickly identify human blood stains in criminal investigation.

[0047] 1) Standard curve 1: Use human MPO antigen as the standard product, and prepare concentrations of 1 ng / mL, 4 ng / mL, 16 ng / mL, 64 ng / mL, 256 ng / mL, and 1024 ng / mL respectively with PBS at pH 7.4. The blank is PBS at pH 7.4. Dot the test strips prepared in Example 2 of the present invention on the samples, 100 μl per well, and the chromatography time is 10 minutes. Use a dry fluorescence chromatography analyzer to measure the fluorescence value of the T1 line and the fluorescence value of the C line; set 5 replicates for each concentration, and draw Standard curve 1 with the average T1 / C fluorescence value ratio as the ordinate and the human MPO protein concentration as the abscissa. The results are shown in Figure 2 ; The curve equation is Y = 0.0198X + 0.8469, R2 = 0.9713.

[0048] 2) Standard curve 2: Using human Hb protein as the standard, prepare concentrations of 1 ng / mL, 4 ng / mL, 16 ng / mL, 64 ng / mL, 256 ng / mL, and 1024 ng / mL respectively with PBS at pH 7.4. The blank is PBS at pH 7.4. Spot the test strips prepared in Example 2 of the sample invention, 100 μL per well, with a chromatography time of 10 minutes. Measure the fluorescence values of the T2 line and the C line using a dry fluorescence chromatography analyzer; set 5 replicates for each concentration. Plot standard curve 2 with the average T2 / C fluorescence value ratio as the ordinate and the human Hb protein concentration as the abscissa. The results are shown in Figure 3 ; The curve equation is Y = 0.0284X + 2.6383, R 2 = 0.9105.

[0049] 3) Recruit 10 volunteers, 5 males and 5 females. Take 200 μL of fingertip blood from each person and drop it on a cotton cloth of about 1 cm × 1 cm size. At the same time, take 200 μL of fingertip blood and drop it on another cotton cloth of about 1 cm × 1 cm size. After naturally placing for 3 days at room temperature, cut a 0.5 cm × 0.5 cm cotton cloth sample from each and put it into 0.5 ml of PBS concentrate. After shaking and soaking for 3 minutes, take 100 μL of the sample solution and drop it into the sample addition hole of the test strip prepared in Example 5 of the present invention. Let it stand for a chromatography time of 10 minutes. Measure the fluorescence values of the T1 line, the T2 line, and the C line using a dry fluorescence chromatography analyzer. Substitute the T1 / C fluorescence value ratio into standard curve 1 to calculate the hMPO content, and substitute the T2 / C fluorescence value ratio into standard curve 2 to calculate the hHb content. The test results are shown in Table 1. The results show that all 10 bloodstain samples are positive, with an accuracy rate of 100%. Use the same method to detect 3 dog blood samples, 3 pig blood samples, 3 chicken blood samples, 3 rabbit blood samples, and 3 human urine stain samples respectively. The results are all negative, with a specificity of 100%.

[0050] Table 1 Detection results of 10 volunteer bloodstain samples by the kit of the present invention

[0051]

Claims

1. A human blood stain detection kit based on the hHb / hMPO dual detection method, characterized in that The kit also includes an immunochromatographic test strip, which includes a backing, an NC membrane, a sample pad, a release pad and absorbent paper; the sample pad, the release pad, the NC membrane and the absorbent paper are overlapped and assembled on the backing, the release pad and the absorbent paper are respectively superimposed on the two ends of the NC membrane, and a detection area is formed on the surface of the NC membrane, and the sample pad is superimposed on the release pad; the NC membrane in the detection area is provided with a detection line area T1 line and a T2 line close to the release pad and a control line area C line close to the absorbent paper; DNP-BSA is coupled to carboxyl quantum dot microspheres and marked as Q-DNP; Q-hHb-01, Q-hMPO-01, and Q-DNP are mixed and sprayed on the release pad, and the T1 line area on the NC membrane is coated with C-hHb-02, the T2 line area is coated with C-hMPO-02, and the C line area is coated with rabbit anti-DNP polyclonal antibody; Among them, one of the antibodies of a pair of anti-human blood MPO specific monoclonal antibodies was labeled with quantum dot fluorescent microspheres and labeled as Q-hMPO-01, and the other was used as a coating antibody and labeled as C-hMPO-02; One of the antibodies of a pair of anti-human hemoglobin specific monoclonal antibodies was labeled with quantum dot fluorescent microspheres and labeled as Q-hHb-01, and the other was used as a coating antibody and labeled as C-hHb-02.

2. A human blood stain detection kit based on the hHb / hMPO dual detection method as claimed in claim 1, characterized in that The preparation method of the anti-human MPO specific monoclonal antibody is as follows: mice are immunized with human MPO antigen protein, and anti-human MPO monoclonal antibodies are prepared using mouse monoclonal antibody technology; in antibody screening, human blood and blood samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are used for testing to obtain a pair of anti-human blood MPO specific monoclonal antibodies that do not react with blood samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice.

3. A human blood stain detection kit based on the hHb / hMPO dual detection method as claimed in claim 1, characterized in that The preparation method of the anti-human hemoglobin specific monoclonal antibody is as follows: mice are immunized with human hemoglobin, and the anti-human hemoglobin monoclonal antibody is prepared using mouse monoclonal antibody technology; in the antibody screening, human blood and blood samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are respectively used for testing to obtain a pair of anti-human blood hemoglobin specific monoclonal antibodies that do not react with the blood of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice.

4. A detection method based on the hHb / hMPO dual detection method human blood stain detection kit as claimed in claim 1, characterized in that The following steps are involved: 1) The detection limit of the kit for human hemoglobin is set at 1 ng / mL, and the detection limit of human MPO protein is set at 0.1 ng / mL, and a dry fluorescence chromatography analyzer is used for quantitative analysis, and quantitative standard curves of human hemoglobin and human MPO protein are preset in the detection instrument, and then the corresponding protein content in the actual sample detection is calculated according to the corresponding standard curves; 2) Soak the suspected blood sample in PBS solution, shake for 3-5 minutes to dissolve, and then use the kit to detect it. After the detection chromatography is left for 10-15 minutes, use a dry fluorescence chromatography analyzer for quantitative analysis, and calculate the detection concentrations of human hemoglobin and human MPO protein by the kit according to the standard curve prepared in step 1). If human hemoglobin is not detected, that is, the detection concentration is <1ng / mL, it is a negative sample and can be excluded as human blood; if the concentration value of human hemoglobin detected is ≧1ng / mL, it can be judged as suspected human blood; if it is judged as suspected human blood and human MPO protein is not detected, that is, MPO<0.1ng / mL, it means that there are too few white blood cells in the sample to be tested, which is not enough to support DNA detection, and the DNA test may show a negative result; if it is judged as suspected human blood and human MPO protein is detected, that is, the concentration value of MPO detected is ≧0.1ng / mL, it is determined to be human blood, indicating that the DNA test should show a positive result.

5. The detection method according to claim 4, characterized in that The preparation of the standard curve in step 1) includes the following process: Step 1, preparing standard curve 1: using human MPO antigen protein as standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 1 ng / mL-1024 ng / mL, using pH 7.4 PBS buffer as blank control, respectively using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to measure the T1 line fluorescence value and the C line fluorescence value, using the ratio of the T1 line fluorescence value to the C line fluorescence value, i.e., the T1 / C fluorescence value ratio as the ordinate, and the concentration of the human MPO antigen protein as the abscissa to draw standard curve 1; Step 2, preparing standard curve 2: using human Hb protein as the standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 1 ng / mL-1024 ng / mL, using pH 7.4 PBS buffer as the blank control, and using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to determine the T2 line fluorescence value and the C line fluorescence value, and plotting standard curve 2 with the ratio of the T2 line fluorescence value to the C line fluorescence value, i.e., the T2 / C fluorescence value ratio, as the ordinate and the human Hb protein concentration as the abscissa.

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