A kit for measuring glycosylated hemoglobin content and a detection method thereof

Abstract

The present invention relates to the technical field of hemoglobin detection, in particular to a kit for determining the content of glycated hemoglobin, comprising a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a lysate, a luminescent substrate and a cleaning solution, wherein the anti-reagent is an alkaline phosphatase-labeled glycated hemoglobin antibody, the magnetic particle reagent is a non-glycated hemoglobin antibody-coated carboxyl magnetic bead, the lysate comprises a buffer and a surfactant, and the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent, the lysate, the luminescent substrate and the cleaning solution are independently packaged. The present invention also relates to a method for detecting the content of glycated hemoglobin in whole blood, comprising the following steps: step one: sample lysis; step two: water bath; step three: magnetic sleeve de-washing; step four: chemiluminescence instrument detection.

Description

Technical Field

[0001] The present invention relates to the technical field of hemoglobin detection, in particular to a kit for determining the content of glycosylated hemoglobin and a detection method thereof. Background Art

[0002] Studies have found that glycosylated hemoglobin (HbA1c) is a good indicator of the degree of disease control in diabetic patients. It reflects the average blood sugar level of patients within 120 days before the test. HbA1c is the gold standard for blood sugar control. All diabetic patients, no matter what method they use to reflect changes in blood sugar, must ultimately use changes in HbA1c as an effective test indicator for the final evaluation of a drug or a treatment plan. Therefore, the clinical significance of HbA1c testing is very important.

[0003] The commonly used HbA1c detection methods are mainly immunoassay and HPLC. Immunoassay is mainly divided into immunoturbidimetry, immunoagglutination, ion capture, chemiluminescence immunoluminescence, etc. Among them, chemiluminescence immunoluminescence is a new method developed in recent years. The chemiluminescence analysis system uses the catalysis of chemiluminescent substances and the oxidation of oxidants to form an excited intermediate. When this excited intermediate returns to a stable ground state, it simultaneously emits photons (hν) and uses a luminescence signal measuring instrument to measure the light quantum yield. The immunoreaction system directly labels the luminescent substance (excited intermediate generated under the excitation of the reactant) on the antigen or antibody, or the enzyme acts on the luminescent substrate. The chemical detection system detects the luminescence intensity through a photoelectric signal detector, and finally determines the content of the analyte through the relationship between the chemiluminescent marker and the luminescence intensity. It has the characteristics of high degree of automation, good specificity, high accuracy, wide detection range, simple operation, low cost, less sampling and high throughput.

[0004] In addition, the existing test kit containing detection reagents cannot be well fixed when placed on the desktop, and the reagents are taken out one by one. When taking out the reagents, the test kit is easy to move, which is inconvenient to take out the reagents. Moreover, the test kit is easy to fall due to unexpected external force, causing damage to the reagents. Summary of the invention

[0005] The object of the present invention is to provide a kit for determining the content of glycosylated hemoglobin and a detection method thereof, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A kit for measuring the content of glycated hemoglobin comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a lysate, a luminescent substrate and a cleaning solution. The anti-reagent is a glycated hemoglobin antibody labeled with alkaline phosphatase, the magnetic particle reagent is a carboxyl magnetic bead coated with a non-glycated hemoglobin antibody, the lysate comprises a buffer and a surfactant, and the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent, the lysate, the luminescent substrate and the cleaning solution are packaged independently.

[0008] Preferably, the alkaline phosphatase-labeled glycated hemoglobin antibody is formed by connecting alkaline phosphatase and glycated hemoglobin antibody through a cross-linking agent disuccinimidyl suberate, and the solution concentration of the alkaline phosphatase-labeled glycated hemoglobin antibody is 0.8-1.5 μg / mL, pH 7-9, and the concentration of the magnetic particle reagent is 1-2 mg / mL. The preparation method of the magnetic particle reagent is: the magnetic bead concentrate is fully mixed, and after being fully mixed, it is placed in a magnetic field for 15 minutes, and the supernatant is removed after all the carboxyl magnetic beads are precipitated, and a phosphate buffer with a volume of 5 times that of the carboxyl magnetic beads is added. After mixing for 20-30 minutes, it is placed in a magnetic field and the supernatant is removed after all the magnetic beads are precipitated. The washing step is repeated three times, and the carboxyl magnetic bead solution is fixed to 10-50 mg / mL; According to the mass ratio of magnetic beads: antibody = 100:1, the treated antibody is added to the magnetic beads, and the reaction is maintained at 2-8°C for 18 hours in a mixed state, and the phosphate buffer is used to wash three times, and the volume is fixed to 1 mg / mL, stored at 2-8℃ until use, to prepare the magnetic particle reagent.

[0009] Preferably, the buffer solution of the calibrator and the quality control product is prepared by adding 50-70 g bovine serum albumin, 1-3 mL PC-300 and 10-15 g HEPES to 1 L of purified water, and the components of the calibrator and the quality control product are the same, and are obtained by diluting the buffer solution of the glycated hemoglobin antigen with a buffer solution containing bovine serum albumin.

[0010] Preferably, the lysate is prepared by adding 50-70mM Tris-Hcl pH 7.4, 0.3-0.5% TritonX-100, 0.2-0.4% Tween-80, and 0.1% SDS to 1L of purified water, the luminescent substrate is prepared by diluting the ALPS luminescent substrate in a ratio of 1:4-1:10 to a buffer solution of Tris 0.1-1M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, bovine serum albumin 0.15%, and pH 9.5, and the cleaning solution is a Tris buffer containing about 1% Tween20, with a pH of 8.0±0.1.

[0011] Preferably, the test kit also includes a test kit body containing the above-mentioned reagents, a reagent cavity is provided inside the test kit body, a sponge base is provided in the reagent cavity, a placement hole is provided on the sponge base, limiting protrusions are symmetrically fixedly provided on both sides of one end of the test kit body, a first locking hole is provided at the lower end of the limiting protrusion, and installation slides are symmetrically provided on both sides of the test kit body, and supporting protrusions are symmetrically fixedly provided on both sides of one end of the test kit body away from the limiting protrusion, a second locking hole is provided at the lower end of the supporting protrusion, and a limiting lower convex plate is also fixedly provided at the lower end of the supporting protrusion, a bearing inner cavity is also provided on the test kit body, supporting slides are symmetrically provided on both sides of the inner side of the bearing inner cavity, and a connecting through hole is provided at the bottom of the bearing inner cavity, a through groove is provided between the reagent cavity and the bearing inner cavity, a fixing cavity hole is provided at the lower end of the test kit body, the fixing cavity hole is connected with the connecting through hole, and a convenient box fixing mechanism is provided on the test kit body.

[0012] Preferably, the convenient fixing mechanism of the box body includes a sliding cover, a movable plate strip, a movable matching strip and a load-bearing movable plate. The sliding cover covers the reagent box body, and limiting top blocks are symmetrically fixed on both sides of one end of the sliding cover, and side load-bearing plates are symmetrically fixed on both sides of the sliding cover.

[0013] Preferably, a plug-in sliding plate is fixedly provided at the lower end of the lateral bearing plate, the plug-in sliding plate is plugged into the installation slide, and matching plug-in holes are symmetrically opened on both sides of one end of the lateral bearing plate close to the limiting top block, the matching plug-in holes penetrate the plug-in sliding plate, and a push-up contact block and a limiting anti-movement strip are fixedly provided on the lower end surface of the sliding cover, and a movable plate strip is installed on the sliding cover.

[0014] Preferably, an elastic connecting strip is fixedly provided on the movable slat, the lower end of the elastic connecting strip is fixed on the sliding cover, and supporting convex plates are symmetrically fixedly provided at both ends of the movable slat, a matching plug rod is fixedly provided at the lower end of the supporting convex plate, the matching plug rod is inserted in the matching plug hole, and a matching bottom plate is fixedly provided at the lower end of the matching plug rod, a first locking column is fixedly provided at one end of the matching bottom plate, and a second locking column is fixedly provided at the other end.

[0015] Preferably, a movable matching strip is installed in the bearing inner cavity, and both ends of the movable matching strip are inserted in the supporting slide, an upper end of the movable matching strip is fixedly provided with an upper convex driving block, and the lower end of the movable matching strip is hinged with a movable hinged rod, and the lower end of the movable hinged rod is hinged with a bearing movable plate, and the lower end of the bearing movable plate is fixedly provided with a connecting matching rod, the connecting matching rod passes through the connecting through hole and is inserted in the fixed cavity hole, and the lower end of the connecting matching rod is fixedly provided with a cavity hole matching plug plate.

[0016] A method for detecting the content of glycosylated hemoglobin in whole blood comprises the following steps:

[0017] Step 1: Sample lysis: Add 200 μL of lysis agent to lyse the sample to be tested;

[0018] Step 2: Water bath: Based on step 1, add 80 μL of anti-reagent and 80 μL of magnetic particle reagent, and then bathe in 37°C water for 15 minutes;

[0019] Step 3: Magnetic elution: Based on step 2, add 300 μL of cleaning solution and repeat the magnetic elution three times;

[0020] Step 4: Chemiluminescence detection: Based on step 3, add 180 μL of luminescent substrate and mix well, then use a chemiluminescence instrument to detect the luminescence intensity.

[0021] Compared with the prior art, the invention has the following advantages:

[0022] 1. The present invention discloses a kit for determining the content of glycated hemoglobin in anticoagulated venous whole blood by magnetic particle chemiluminescence method. The reagent components include calibrators, quality control products, anti-reagents, magnetic particle reagents, lysates and luminescent substrates, etc., and the kit has good stability and a shelf life of more than one year. The kit has complete operating specifications, and the results are stable and reliable. Alkaline phosphatase (AP) is used as a labeling enzyme, antibodies are labeled by chemical reactions, and gel chromatography is used to separate unreacted enzymes, antibodies or antigens to improve the sensitivity of the reaction. Second, immunomagnetic beads are used as a solid phase to directly connect glycated hemoglobin antibodies to form magnetic beads-immune complexes. The complex has strong magnetic responsiveness and moves in a directed manner under the action of magnetic force, so that the complex is separated from other substances in the liquid, thereby achieving the purpose of separating, concentrating and purifying specific proteins. It not only makes it easier to mix and separate the immune response, but also greatly improves the reaction speed. In addition, the new chemiluminescent substrate ALPS is used as the substrate. This substrate is a luminescent substrate and quickly reaches a plateau phase, which is beneficial to signal detection and improves the sensitivity and specificity of the final test kit; and the chemiluminescence enhancement system is further optimized to ensure that the final product has high signal sensitivity, good stability and small variation.

[0023] 2. In addition, when taking the reagent, place the test kit body on a flat table, then push the movable strip downward and then push the sliding cover to open the test kit body. At the same time, the movement of the sliding cover can drive the movable matching strip to move, and then the movable supporting plate can be driven to move under the action of the movable matching strip, so that the cavity hole on the supporting movable plate can move upward with the plug plate. In this way, negative pressure can be formed in the fixed cavity hole under the action of the cavity hole and plug plate, which plays a certain fixing role on the test kit body, ensures its placement stability, facilitates the taking of reagents, and also avoids the risk of it falling due to external force. The operation is simple and convenient, and the test kit can be reused many times to avoid waste of resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the first-person view of the kit assembly.

[0025] Figure 2 A second perspective schematic diagram of the kit assembly.

[0026] Figure 3 It is a schematic diagram of the assembly inside the bearing cavity.

[0027] Figure 4 It is a schematic diagram of the structure of the kit body from the first perspective.

[0028] Figure 5 This is a schematic diagram of the second perspective structure of the test kit body.

[0029] Figure 6 It is a schematic diagram of the first-person perspective structure of the sliding cover.

[0030] Figure 7 It is a schematic structural diagram of the sliding cover from a second perspective.

[0031] Figure 8 It is a structural schematic diagram of the movable slats.

[0032] Fig. 9 It is a schematic diagram of the assembly of the movable matching strip and the load-bearing movable plate.

[0033] Fig.10 This is a schematic diagram of the correlation analysis of the results obtained by measuring whole blood samples in Example 1.

[0034] In the figure: 1, reagent box body; 11, reagent chamber; 12, sponge base; 13, placement socket; 14, limiting convex block; 15, first locking socket; 16, mounting slide; 17, supporting convex block; 171, second locking socket; 172, limiting lower convex plate; 18, bearing inner cavity; 181, supporting slide; 182, connecting through hole; 183, passing through groove; 19, fixed cavity hole; 2, sliding cover; 21, limiting top block; 22, lateral Carrying plate; 23, plug-in sliding plate; 24, matching plug-in hole; 25, push-up contact block; 26, limiting anti-movement strip; 3, movable plate strip; 31, elastic connecting strip; 32, supporting convex plate; 33, matching plug rod; 34, matching bottom plate; 35, first locking column; 36, second locking column; 4, movable matching strip; 41, upper convex driving block; 42, movable hinged rod; 5, carrying movable plate; 51, connecting matching rod; 52, cavity matching plug plate. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] See also Figures 1 to 10 , the present invention provides a technical solution:

[0037] Embodiment 1, a detection kit for glycated hemoglobin content, comprising a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a lysate, a luminescent substrate and a cleaning solution, wherein the anti-reagent is a glycated hemoglobin antibody labeled with alkaline phosphatase, the magnetic particle reagent is a carboxyl magnetic bead coated with a non-glycated hemoglobin antibody, the lysate comprises a buffer and a surfactant, and the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent, the lysate, the luminescent substrate and the cleaning solution are packaged independently.

[0038] The concentration of the alkaline phosphatase-labeled HbA1c antibody solution was 1 μg / mL.

[0039] The concentration of the non-glycated hemoglobin antibody-coated magnetic microparticle reagent was 1 mg / mL.

[0040] The buffer for calibrators and quality controls was prepared by adding 60 g bovine serum albumin, 1 mL PC-300, and 10 g HEPES to 1 L of purified water.

[0041] The components of the calibrator and the quality control product are the same. The glycated hemoglobin antigen is diluted with the above buffer to different concentration points, and the calibrator and the quality control product are obtained by calibration.

[0042] The lysate was prepared by adding 50 mM Tris pH 7.4, 0.3% Triton X-100, 0.2% Tween-80, and 0.1% SDS to 1 L of purified water.

[0043] The luminescent substrate is prepared by the following steps: diluting the ALPS luminescent substrate into a buffer solution containing the following components at pH 9.5 at a ratio of 1:4: Tris 0.1 M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, and bovine serum albumin 0.15%.

[0044] A method for detecting the content of glycosylated hemoglobin in whole blood comprises the following steps:

[0045] Step 1: Sample lysis: Add 200 μL of lysis agent to lyse the sample to be tested;

[0046] Step 2: Water bath: Based on step 1, add 80 μL of anti-reagent and 80 μL of magnetic particle reagent, and then bathe in 37°C water for 15 minutes;

[0047] Step 3: Magnetic elution: Based on step 2, add 300 μL of cleaning solution and repeat the magnetic elution three times;

[0048] Step 4: Chemiluminescence detection: Based on step 3, add 180 μL of luminescent substrate and mix well, then use a chemiluminescence instrument to detect the luminescence intensity.

[0049] The HbA1c in the control product was detected using the kit of Example 1. Control products of different concentrations were taken and numbered as control product 1 and control product 2. The control products were re-dissolved to obtain control product solutions. The control solutions were stored in a refrigerator at 2°C to 8°C and the HbA1c was detected on the 3rd and 7th days. The test results are as follows:

[0050]

[0051] As can be seen from the above table, as the storage time goes by, when the quality control product is detected using the kit of Example 1 and the detection method of this example, the result of HbA1c does not change substantially, indicating that the stability of the kit is good.

[0052] Example 2: The kit of Example 1 was used to detect HbA1c in the sample in the same manner, wherein the sample was EDTA anticoagulated venous whole blood, stored at 2°C to 8°C for not less than 7 days. There were 24 samples in total, 20 μL of each sample was taken, and the lysis solution of Example 1 was added at a ratio of 1:100 for lysis. The detection results are as follows:

[0053]

[0054] It can be seen from the two test results that the results obtained by measuring the whole blood samples stored at 2°C to 8°C for 7 days using the kit of Example 1 and the detection method of Example 1 have excellent correlation. Therefore, the kit and the detection method of the present invention have high clinical application value.

[0055] Example 3, a detection kit for glycated hemoglobin content, including a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a lysate, a luminescent substrate and a cleaning solution, the anti-reagent is a glycated hemoglobin antibody labeled with alkaline phosphatase, the magnetic particle reagent is a non-glycated hemoglobin antibody coated with carboxyl magnetic beads, the lysate includes a buffer and a surfactant, and the calibrator, quality control product, anti-reagent, magnetic particle reagent, lysate, luminescent substrate and cleaning solution are packaged independently.

[0056] The concentration of the alkaline phosphatase-labeled HbA1c antibody solution was 0.8 μg / mL.

[0057] The concentration of the non-glycated hemoglobin antibody-coated magnetic microparticle reagent was 1.2 mg / mL.

[0058] The buffer for calibrators and quality controls was prepared by adding 50 g bovine serum albumin, 2 mL PC-300, and 12 g HEPES to 1 L of purified water.

[0059] The calibrator and the quality control have the same composition and are both prepared by an operation comprising the following steps: diluting the glycated hemoglobin antigen with the above buffer to obtain a lyophilized product.

[0060] The lysate was prepared by adding 60 mM Tris pH 7.4, 0.4% Triton X-100, 0.3% Tween-80, and 0.1% SDS to 1 L of purified water.

[0061] The luminescent substrate was prepared by an operation including the following steps: diluting the ALPS luminescent substrate into a buffer solution containing the following components at pH 9.5 at a ratio of 1:6: Tris 0.5 M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, and bovine serum albumin 0.15%.

[0062] Example 4, a detection kit for glycated hemoglobin content, including calibrators, quality control products, anti-reagents, magnetic particle reagents, lysates, luminescent substrates and cleaning solutions, the anti-reagents are glycated hemoglobin antibodies labeled with alkaline phosphatase, the magnetic particle reagents are carboxyl magnetic beads coated with non-glycated hemoglobin antibodies, the lysates include buffer and surfactants, and the calibrators, quality control products, anti-reagents, magnetic particle reagents, lysates, luminescent substrates and cleaning solutions are packaged independently.

[0063] The concentration of the alkaline phosphatase-labeled HbA1c antibody solution was 1.5 μg / mL.

[0064] The concentration of the non-glycated hemoglobin antibody-coated magnetic microparticle reagent was 2 mg / mL.

[0065] The buffer for calibrators and quality controls was prepared by adding 70 g bovine serum albumin, 3 mL PC-300, and 15 g HEPES to 1 L of purified water.

[0066] The calibrator and the quality control have the same composition and are both prepared by an operation comprising the following steps: diluting the glycated hemoglobin antigen with the above buffer to obtain a lyophilized product.

[0067] The lysate was prepared by adding 70 mM Tris pH 7.4, 0.5% Triton X-100, 0.4% Tween-80, and 0.1% SDS to 1 L of purified water.

[0068] The luminescent substrate was prepared by an operation including the following steps: diluting the ALPS luminescent substrate into a buffer solution containing the following components at pH 9.5 at a ratio of 1:10: Tris 1M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, and bovine serum albumin 0.15%.

[0069] The reagent kit also includes a reagent kit body 1 containing the above reagents, a reagent chamber 11 is provided inside the reagent kit body 1, a sponge base 12 is provided in the reagent chamber 11, a placement socket 13 is provided on the sponge base 12, a reagent bottle containing the reagent is plugged into the placement socket 13, a limiting protrusion 14 is symmetrically fixedly provided on both sides of one end of the reagent kit body 1, a first locking socket 15 is provided at the lower end of the limiting protrusion 14, and installation slideways 16 are symmetrically provided on both sides of the reagent kit body 1, and a support protrusion 16 is symmetrically fixedly provided on both sides of the end of the reagent kit body 1 away from the limiting protrusion 14 7. A second locking socket 171 is provided at the lower end of the supporting protrusion 17, and a limiting lower protrusion 172 is fixedly provided at the lower end of the supporting protrusion 17. A carrying cavity 18 is also provided on the reagent box body 1, and supporting slideways 181 are symmetrically provided on both sides of the inner part of the carrying cavity 18, and a connecting through hole 182 is provided at the bottom of the carrying cavity 18, a through groove 183 is provided between the reagent cavity 11 and the carrying cavity 18, a fixing cavity hole 19 is provided at the lower end of the reagent box body 1, and the fixing cavity hole 19 is connected with the connecting through hole 182, and a convenient box fixing mechanism is provided on the reagent box body 1.

[0070] The convenient fixing mechanism of the box body includes a sliding cover 2, a movable plate strip 3, a movable matching strip 4 and a supporting movable plate 5. The sliding cover 2 covers the reagent box body 1. Limiting top blocks 21 are symmetrically fixed on both sides of one end of the sliding cover 2, and side supporting plates 22 are symmetrically fixed on both sides of the sliding cover 2.

[0071] A plug-in sliding plate 23 is fixedly provided at the lower end of the lateral bearing plate 22, and the plug-in sliding plate 23 is plugged into the installation slide 16, and matching plug-in holes 24 are symmetrically opened on both sides of one end of the lateral bearing plate 22 close to the limiting top block 21, and the matching plug-in holes 24 penetrate the plug-in sliding plate 23. A push-up contact block 25 and a limiting anti-movement strip 26 are fixedly provided on the lower end surface of the sliding cover 2, and a movable plate strip 3 is installed on the sliding cover 2.

[0072] An elastic connecting strip 31 is fixedly provided on the movable slat 3, and the lower end of the elastic connecting strip 31 is fixed on the sliding cover 2, and supporting convex plates 32 are symmetrically fixedly provided at both ends of the movable slat 3, and a matching plug rod 33 is fixedly provided at the lower end of the supporting convex plate 32, and the matching plug rod 33 is inserted in the matching plug hole 24, and a matching bottom plate 34 is fixedly provided at the lower end of the matching plug rod 33, and a first locking column 35 is fixedly provided at one end of the matching bottom plate 34, and a second locking column 36 is fixedly provided at the other end, which limits the top block 21 to be below the movable slat 3, and plays a role of limiting it, so as to avoid the movable slat 3 from moving down too much and causing damage to the elastic connecting strip 31. When the sliding cover 2 seals the reagent chamber, the first locking column 35 is inserted in the first locking hole 15.

[0073] A movable matching strip 4 is installed in the bearing inner cavity 18, and the two ends of the movable matching strip 4 are inserted in the supporting slide 181. The upper end of the movable matching strip 4 is fixedly provided with an upper convex driving block 41, and the lower end of the movable matching strip 4 is hinged with a movable hinged rod 42, and the lower end of the movable hinged rod 42 is hinged with a bearing movable plate 5, and the lower end of the bearing movable plate 5 is fixedly provided with a connecting matching rod 51, and the connecting matching rod 51 passes through the connecting through hole 182 and is inserted in the fixed cavity hole 19, and the lower end of the connecting matching rod 51 is fixedly provided with a cavity hole matching plug plate 52, and the cavity hole matching plug plate 52 cooperates with the fixed cavity hole 19 to ensure the formation of negative pressure. In addition, when the sliding cover 2 seals the reagent chamber, the upper convex driving block 41 at this time contacts with the limiting anti-movement strip 26 to prevent the movable matching strip 4 from moving away from the reagent chamber 11, and the movable matching strip 4 at this time contacts with the inner wall of the supporting slide 181 close to the reagent chamber 11.

[0074] When taking out the reagent, place the reagent box body 1 on a flat table so that the cavity of the fixed cavity hole 19 is sealed under the action of the table, then fix the reagent box body 1 with your hands, push the movable strip 3 downward to make the first locking column 35 withdraw from the first locking hole 15, so that the sliding cover 2 is in an active state, and then push the sliding cover to make the matching bottom plate 34 contact with the lower limiting convex plate 172, and then release the movable strip 3 to make the movable strip 3 move upward under the action of the elastic connecting strip 31, thereby driving the second locking column 36 to be inserted into the second locking hole 171, so as to fix the sliding cover 2. In the process of moving the sliding cover 2, the push-up contact block 25 will pass through the penetration The groove 183 contacts the upper convex driving block 41, so that as the sliding cover 2 moves, the movable matching strip 4 will be pushed to move under the action of the push-up contact block 25, and as the movable matching strip 4 moves, the movable supporting plate 5 will be driven to move upward under the action of the movable hinge rod 42, thereby making the cavity matching plug plate 52 move upward, and negative pressure can be formed in the fixed cavity 19 under the action of the cavity matching plug plate 52, thereby realizing the fixation of the reagent box body 1, ensuring its stability, facilitating taking and avoiding falling. After use, the movable plate strip 3 is also pushed downward so that the second locking column 36 exits the second locking socket 171, and the sliding cover 2 can be moved in the opposite direction, so that the negative pressure disappears, and the reagent box can be recycled.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A kit for measuring the content of glycosylated hemoglobin, characterized in that: The invention comprises a reagent box body (1), wherein a reagent chamber (11) is provided inside the reagent box body (1), a sponge base (12) is provided in the reagent chamber (11), a placement plug hole (13) is provided on the sponge base (12), a limiting protrusion (14) is symmetrically fixedly provided on both sides of one end of the reagent box body (1), a first locking plug hole (15) is provided at the lower end of the limiting protrusion (14), and a mounting slideway (16) is symmetrically provided on both sides of the reagent box body (1), and a supporting protrusion (17) is symmetrically fixedly provided on both sides of one end of the reagent box body (1) away from the limiting protrusion (14), and a first locking plug hole (15) is provided at the lower end of the supporting protrusion (17). The reagent box body (1) further comprises a plurality of locking jacks (171), and a limiting lower convex plate (172) is fixedly provided at the lower end of the supporting convex block (17). The reagent box body (1) further comprises a bearing inner cavity (18), and supporting slideways (181) are symmetrically provided on both sides of the inner cavity of the bearing inner cavity (18). A connecting through hole (182) is provided at the bottom of the bearing inner cavity (18). A through groove (183) is provided between the reagent cavity (11) and the bearing inner cavity (18). A fixing cavity hole (19) is provided at the lower end of the reagent box body (1), and the fixing cavity hole (19) is connected to the connecting through hole (182). A convenient box fixing mechanism is provided on the reagent box body (1); The box body convenient fixing mechanism comprises a sliding cover (2), a movable plate strip (3), a movable matching strip (4) and a bearing movable plate (5); the sliding cover (2) covers the reagent box body (1); limiting top blocks (21) are symmetrically fixedly arranged on both sides of one end of the sliding cover (2); and lateral bearing plates (22) are symmetrically fixedly arranged on both sides of the sliding cover (2); A plug-in sliding plate (23) is fixedly provided at the lower end of the lateral bearing plate (22), the plug-in sliding plate (23) is plugged into the mounting slideway (16), and matching plug-in holes (24) are symmetrically provided on both sides of one end of the lateral bearing plate (22) close to the limiting top block (21), the matching plug-in holes (24) penetrate the plug-in sliding plate (23), a push-up contact block (25) and a limiting anti-movement strip (26) are fixedly provided on the lower end surface of the sliding cover (2), and a movable plate strip (3) is installed on the sliding cover (2); An elastic connecting strip (31) is fixedly provided on the movable slat (3), the lower end of the elastic connecting strip (31) is fixed on the sliding cover (2), and supporting convex plates (32) are symmetrically fixedly provided at both ends of the movable slat (3), a matching plug rod (33) is fixedly provided at the lower end of the supporting convex plate (32), the matching plug rod (33) is inserted into the matching plug hole (24), and a matching bottom plate (34) is fixedly provided at the lower end of the matching plug rod (33), a first locking column (35) is fixedly provided at one end of the matching bottom plate (34), and a second locking column (36) is fixedly provided at the other end; A movable matching strip (4) is installed in the bearing inner cavity (18), and the two ends of the movable matching strip (4) are inserted into the supporting slideway (181). The upper end of the movable matching strip (4) is fixedly provided with an upper convex driving block (41), and the lower end of the movable matching strip (4) is hinged with a movable hinge rod (42), and the lower end of the movable hinge rod (42) is hinged with a bearing movable plate (5), and the lower end of the bearing movable plate (5) is fixedly provided with a connecting matching rod (51), and the connecting matching rod (51) passes through the connecting through hole (182) and is inserted into the fixed cavity hole (19), and the lower end of the connecting matching rod (51) is fixedly provided with a cavity hole matching plug plate (52); It also includes calibration products, quality control products, anti-reagents, magnetic particle reagents, lysis solutions, luminescent substrates and cleaning solutions. The anti-reagents are alkaline phosphatase-labeled glycated hemoglobin antibodies, the magnetic particle reagents are non-glycated hemoglobin antibodies coated with carboxyl magnetic beads, the lysis solutions include buffer and surfactants, and the calibration products, quality control products, anti-reagents, magnetic particle reagents, lysis solutions, luminescent substrates and cleaning solutions are packaged independently.

2. A kit for measuring the content of glycosylated hemoglobin according to claim 1, characterized in that: The alkaline phosphatase-labeled glycated hemoglobin antibody is formed by connecting alkaline phosphatase and the glycated hemoglobin antibody through a cross-linking agent, disuccinimidyl suberate, and the solution concentration of the alkaline phosphatase-labeled glycated hemoglobin antibody is 0.8-1.5 μg / mL, pH 7-9, and the concentration of the magnetic particle reagent is 1-2 mg / mL. The preparation method of the magnetic particle reagent is: fully mixing the magnetic bead concentrate, placing it in a magnetic field for 15 minutes after fully mixing, absorbing the supernatant after the carboxyl magnetic beads are completely precipitated, adding a phosphate buffer solution 5 times the volume of the carboxyl magnetic beads, mixing for 20-30 minutes, placing it in a magnetic field after the magnetic beads are completely precipitated, absorbing the supernatant, repeating the washing step three times, and fixing the carboxyl magnetic bead solution to 10-50 mg / mL; according to the magnetic beads: antibody = The treated antibody was added to the magnetic beads at a mass ratio of 100:1, and the mixture was kept in a mixed state at 2-8°C for 18 hours. The beads were washed three times with phosphate buffer, the volume was fixed to 1 mg / mL, and the beads were stored at 2-8°C for later use to obtain the magnetic particle reagent.

3. A kit for measuring the content of glycosylated hemoglobin according to claim 2, characterized in that: The buffer solution of the calibrator and the quality control product is prepared by adding 50-70 g bovine serum albumin, 1-3 mL PC-300 and 10-15 g HEPES to 1 L of purified water. The components of the calibrator and the quality control product are the same, and are obtained by diluting the buffer solution of the glycated hemoglobin antigen with a buffer solution containing bovine serum albumin.

4. A kit for measuring the content of glycosylated hemoglobin according to claim 3, characterized in that: The lysate is prepared by adding 50-70mM Tris-Hcl pH 7.4, 0.3-0.5% TritonX-100, 0.2-0.4% Tween-80, and 0.1% SDS to 1L of purified water. The luminescent substrate is prepared by diluting the ALPS luminescent substrate at a ratio of 1:4-1:10 to a buffer solution of Tris 0.1-1M, sodium sulfite 0.1%, SDS 1%, lucigenin 0.3%, bovine serum albumin 0.15%, and pH 9.

5. The cleaning solution is a Tris buffer containing 1% Tween20, with a pH of 8.0±0.1.

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