Heparin binding protein homogeneous chemiluminescence detection kit and preparation method thereof

Abstract

The heparin binding protein homogeneous chemiluminescence detection kit comprises a first reagent, a second reagent and a third reagent, and the first reagent comprises a biotin-labeled first heparin binding protein antibody; the second reagent comprises receptor microspheres coupled with a second heparin binding protein antibody; the third reagent comprises donor microspheres coupled with streptavidin; the first reagent, the second reagent and the third reagent are all freeze-dried reagents. The kit is high in test sensitivity, wide in linear range, good in repeatability and high in thermal stability.

Description

Technical Field

[0001] The invention relates to a heparin-binding protein homogeneous chemiluminescence detection kit and a preparation method thereof, belonging to the technical field of heparin-binding protein detection. Background Art

[0002] Heparin-binding protein (HBP), also known as Azurocidin (AZU) and cationic antimicrobial protein 37 (CAP37), is a granule protein secreted by neutrophils. HBP is a single-chain glycoprotein with a structure of 222 amino acids and a relative molecular weight of 24,000. It is mainly present in the secretory granules and azurocidin granules of neutrophils. Heparin-binding protein plays an important role in clinical diagnosis and early identification of infectious diseases, especially in the early diagnosis of bacterial infections and the severity assessment of sepsis.

[0003] However, the current heparin-binding protein detection methods on the market are mainly limited to lateral flow immunofluorescence detection and traditional magnetic microparticle chemiluminescence detection. Different detection methods have different clinical application shortcomings. Lateral flow immunofluorescence analysis is limited by the bottleneck of lateral flow methodology, and the reagent performance parameters are difficult to break through and stably control. The magnetic microparticle chemiluminescence method will have greatly improved performance compared to the lateral flow immunofluorescence method, but the magnetic microparticle chemiluminescence method requires a corresponding cleaning module to clean the magnetic particles, which has high requirements for the supporting instruments and will also increase the imprecision of the test.

[0004] Homogeneous chemiluminescent immunoassay is an immunoassay technology based on nanospheres. This method uses two different types of microspheres: donor microspheres and acceptor microspheres. Specific antibodies are cross-linked on the surfaces of donor microspheres and acceptor microspheres, respectively. After mixed incubation with the analyte, the two antibodies form a double antibody sandwich immune complex with the antigen, thereby keeping the distance between the two microspheres within 200nm. When the donor microspheres are excited by laser, the donor microspheres produce singlet oxygen and diffuse to the acceptor microspheres, and the acceptor microspheres generate emission light after receiving energy. The light signal is collected by a photosensitive element, and the concentration of the protein to be tested in the analyte is calculated by mathematical fitting. Homogeneous chemiluminescent immunoassay has the advantages of high sensitivity, high throughput, and high precision. Homogeneous chemiluminescent immunoassay has the prospect of being applied in the detection of heparin-binding proteins. However, there is no reported method for homogeneous chemiluminescent immunoassay of heparin-binding proteins in the prior art.

[0005] Lyophilized reagents are reagent products prepared by freeze-drying technology. This technology rapidly freezes liquid reagents at low temperatures, then sublimates under vacuum conditions to remove water, and finally obtains solid reagents. Lyophilized reagents can be matched with more convenient testing equipment and can be transported and stored at room temperature.

[0006] If the homogeneous chemiluminescent immunoassay and lyophilized reagents can be used together to detect heparin-binding proteins, it will be of great significance. However, the existing lyophilized reagents also have the following disadvantages: there is a lyophilization step in the preparation process, which often loses some of the performance of the detection reagents; the lyophilized reagents need to be reconstituted before use. For antibody reagents, during the reconstitution operation, if high-temperature reconstitution is used, it often leads to protein denaturation and reduced reagent activity. If low-temperature reconstitution is used, the dissolution is often incomplete, the reagent agglomerates, and the reaction sensitivity decreases; the general thermal stability of lyophilized reagents is not high, and they generally need to be stored at low or room temperature, and are difficult to be used in high-temperature environments. Summary of the invention

[0007] The present invention provides a heparin binding protein homogeneous chemiluminescence detection kit and a preparation method thereof, which can effectively solve the above problems.

[0008] The present invention is achieved in that:

[0009] A heparin binding protein homogeneous chemiluminescence detection kit comprises a first reagent, a second reagent and a third reagent.

[0010] The first reagent includes a first heparin-binding protein antibody labeled with biotin;

[0011] The second reagent includes receptor microspheres coupled to a second heparin binding protein antibody;

[0012] The third reagent includes donor microspheres coupled with streptavidin;

[0013] The first reagent, the second reagent and the third reagent are all freeze-dried reagents;

[0014] The freeze-drying parameters of the freeze-drying process of the freeze-dried reagent are as follows: -45°C, 6h; -45°C, 2h; -40°C, 2h; -35°C, 2h; -30°C, 20h; -25°C, 2h; -20°C, 2h; -15°C, 2h; -10°C, 2h; -5°C, 2h; 0°C, 2h; 10°C, 2h; 20°C, 2h; 30°C, 2h; 30°C, 2h; 30°C, 20h.

[0015] In some embodiments, in the first reagent, the molar ratio of biotin to the first heparin-binding protein antibody is 5-15:1.

[0016] In some embodiments, in the second reagent, the ratio of the second heparin binding protein antibody to the receptor microspheres is 5-150 μg:1 mg.

[0017] In some embodiments, in the third reagent, the ratio of streptavidin to donor microspheres is 5-150 μg:1 mg.

[0018] A method for preparing a heparin binding protein homogeneous chemiluminescence detection kit comprises the following steps:

[0019] S1, labeling the first heparin-binding protein antibody with biotin to prepare a first reagent;

[0020] S2, coupling a second heparin-binding protein antibody to the receptor microspheres to prepare a second reagent;

[0021] S3, coupling streptavidin to the donor microspheres to prepare the third reagent;

[0022] S4, prepare the first reagent, the second reagent and the third reagent into freeze-dried reagents; the freeze-drying parameters are as follows: -45℃, 6h; -45℃, 2h; -40℃, 2h; -35℃, 2h; -30℃, 20h; -25℃, 2h; -20℃, 2h; -15℃, 2h; -10℃, 2h; -5℃, 2h; 0℃, 2h; 10℃, 2h; 20℃, 2h; 30℃, 2h; 30℃, 2h; 30℃, 20h.

[0023] In some embodiments, step S1 specifically includes: adding NHS-biotin solution dropwise into the first heparin binding protein antibody solution, incubating at room temperature for 50-80 minutes, and removing unreacted biotin to obtain the first reagent.

[0024] In some embodiments, step S2 specifically includes: washing the acceptor microspheres; resuspending the acceptor microspheres in MES buffer, adding EDC and NHS, and incubating at room temperature for 15-30 minutes to activate the acceptor microspheres; removing the supernatant and washing the acceptor microspheres, adding the second heparin-binding protein antibody to the activated acceptor microspheres, and incubating at 37°C for 3.5-4.5 hours; blocking and washing to obtain the second reagent.

[0025] In some embodiments, step S3 is specifically as follows: washing the donor microspheres; resuspending the donor microspheres in MES buffer, adding EDC and NHS, and incubating at room temperature for 15-30 minutes to activate the donor microspheres; removing the supernatant and washing the donor microspheres, adding streptavidin to the activated donor microspheres, and incubating at 37°C for 3.5-4.5 hours; blocking and washing to obtain the third reagent.

[0026] In some embodiments, the EDC and NHS are used at a concentration of 45-55 mg / mL.

[0027] In some embodiments, the lyophilized reagent is a lyophilized pellet, and the diameter of the lyophilized pellet is 0.5-5 mm.

[0028] The beneficial effects of the present invention are:

[0029] In the preparation process of the heparin-binding protein homogeneous chemiluminescence detection kit involved in the present invention, a special freeze-drying process technology is used to produce a high-quality freeze-dried reagent. During the preparation process of the freeze-dried reagent, the freeze-drying procedure is optimized to ensure that its detection performance will not decline due to the freeze-drying step, thereby ensuring the efficiency and reliability of the reagent. During the reconstitution process, the reagent shows extremely high stability and will not have any adverse effects on the performance of the reagent, ensuring the accuracy and consistency of the test results. In addition, the freeze-dried reagent also has excellent thermal stability, and can be stably stored for more than 28 days even at a temperature of up to 45°C. This feature makes it very suitable for transportation, storage and use in high temperature environments, greatly reducing the cost of transportation and storage and improving the convenience of use.

[0030] The heparin-binding protein homogeneous chemiluminescence detection kit of the present invention has excellent detection performance, has extremely high detection sensitivity, and can accurately capture trace amounts of heparin-binding protein; its linear range is wide, and can cover a wide range of detection concentrations to meet the detection needs of different samples; at the same time, the kit has excellent repeatability, and the coefficient of variation of multiple test results is extremely low, which ensures the reliability and consistency of the test data and provides strong support for clinical diagnosis and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is a diagram showing the preferred coating ratio of the donor microspheres in Example 1.

[0033] Figure 2 This is a diagram showing the optimal coating ratio of the receptor microspheres of Example 2.

[0034] Figure 3 This is a diagram showing the preferred working concentration of donor microspheres in Example 3.

[0035] Figure 4This is the preferred concentration diagram of the receptor microsphere working solution in Example 4.

[0036] Figure 5 This is a comparison diagram of the effects of the amplification system of Example 5.

[0037] Figure 6 The effect of different freeze-drying processes on reagent repeatability in Example 6

[0038] Figure 7 The figure is a performance comparison chart of the freeze-dried reagent and the liquid reagent of Example 7.

[0039] Figure 8 This is the linear range verification diagram of Example 8. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] The reagents used in the embodiments of the present invention are as follows:

[0042] Anti-HBP antibody was purchased from Chilue Biotechnology, catalog number: Ab <hbp>05,Ab <hbp>06;

[0043] Donor microspheres were purchased from Weidu Biotechnology, catalog number: 67500100;

[0044] The receptor microspheres were purchased from Weidu Biotechnology, catalog number: 67700100.

[0045] Example 1 Optimization of the ratio of HBP antibody-coated donor microspheres

[0046] 1. Microsphere cleaning:

[0047] Carboxyl microspheres (manufacturer: Weidu Biotechnology, catalog number: 67500100) were suspended in MES buffer, the microspheres were precipitated by low-speed centrifugation and the supernatant was removed, and the washing was repeated 2-3 times to remove the storage buffer.

[0048] 2. Activate carboxyl group:

[0049] Resuspend the microspheres in MES buffer. Add EDC and NHS to the suspension at a concentration of 50 mg / mL. Gently stir the reaction and incubate at room temperature for 15-30 minutes. This step converts the carboxyl groups to highly reactive ester groups.

[0050] 3. Antibody Binding:

[0051] After the activation step, the supernatant was removed and the microspheres were quickly washed with MES buffer. <hbp>05) was added to the activated microspheres. The antibody and microsphere feed ratios were divided into 4 groups and incubated at 37°C for 4 hours to promote the binding of the antibody to the microsphere surface.

[0052] Group 1: Microspheres: Antibody = 1mg:5ug

[0053] Group 2: Microspheres: Antibody = 1mg:25ug

[0054] Group 3: Microspheres: Antibody = 1mg:75ug

[0055] Group 4: Microspheres: Antibody = 1mg: 150ug

[0056] 4. Blocking reaction:

[0057] The microspheres were treated with a buffer containing 5% BSA to block unreacted active sites and prevent nonspecific binding. Incubate at 37°C for 1 hour.

[0058] 5. Washing and storage:

[0059] Wash the microspheres several times with PBS buffer to remove unbound antibody and blocking agent. Resuspend the coated microspheres in storage buffer and store at 4°C, protected from light.

[0060] 6. Detection coating results:

[0061] The prepared donor microspheres bound to anti-HBP antibodies were made into working solution and measured on a chemiluminescent immunoassay analyzer (Beijing Bovitec Biotechnology Co., Ltd.). The results are shown in Table 1 and Figure 1 shown.

[0062] Table 1 Optimization of photosensitive microsphere coating ratio

[0063]

[0064] From the above experimental data, it can be seen that when the microsphere: antibody ratio is 1mg:75ug, the signal-to-background ratio is the highest; when the antibody dosage is low, the signal value will decrease; when the antibody dosage is too high, the background signal value will increase and the signal-to-background ratio will decrease.

[0065] Example 2 Optimization of the ratio of HBP antibody-coated receptor microspheres

[0066] 1. Microsphere cleaning:

[0067] Carboxyl microspheres (manufacturer: Weidu Biotechnology, catalog number: 67700100) were suspended in MES buffer, the microspheres were precipitated by low-speed centrifugation and the supernatant was removed, and the washing was repeated 2-3 times to remove the storage buffer.

[0068] 2. Activate carboxyl group:

[0069] Resuspend the microspheres in MES buffer. Add EDC and NHS to the suspension at a concentration of 50 mg / mL. Gently stir the reaction and incubate at room temperature for 15-30 minutes. This step converts the carboxyl groups to highly reactive ester groups.

[0070] 3. Antibody Binding:

[0071] After the activation step, the supernatant was removed and the microspheres were quickly washed with MES buffer. <hbp>06) was added to the activated microspheres. The antibody and microsphere feed ratios were divided into 4 groups and incubated at 37°C for 4 hours to promote the binding of the antibody to the microsphere surface.

[0072] Group 1: Microspheres: Antibody = 1mg:5ug

[0073] Group 2: Microspheres: Antibody = 1mg: 25ug

[0074] Group 3: Microspheres: Antibody = 1mg:75ug

[0075] Group 4: Microspheres: Antibody = 1mg: 150ug

[0076] 4. Blocking reaction:

[0077] The microspheres were treated with a buffer containing 5% BSA to block unreacted active sites and prevent nonspecific binding. Incubate at 37°C for 1 hour.

[0078] 5. Washing and storage:

[0079] Wash the microspheres several times with PBS buffer to remove unbound antibody and blocking agent. Resuspend the coated microspheres in storage buffer and store at 4°C, protected from light.

[0080] 6. Detection coating results:

[0081] The prepared donor microspheres bound to anti-HBP antibodies were made into working solution and measured on a chemiluminescent immunoassay analyzer (Beijing Bovitec Biotechnology Co., Ltd.); the results are shown in Table 2 and Figure 2 shown.

[0082] Table 2 Optimization of receptor microsphere coating ratio

[0083]

[0084]

[0085] From the above experimental data, it can be seen that when the receptor microspheres: antibody is 1mg:75ug, the signal-to-background ratio is the highest; when the antibody dosage is low, the signal value will decrease; when the antibody dosage is too high, the background signal value will increase and the signal-to-background ratio will decrease.

[0086] Example 3 Optimization of the concentration of donor microspheres bound to HBP antibodies

[0087] The feed ratio of antibody to microspheres was microspheres: antibody = 1 mg: 75 ug, and the mixture was incubated at 37°C for 4 hours to promote the binding of the antibody to the surface of the microspheres. The prepared donor microspheres bound to the anti-HBP antibody were made into working solutions, and the concentrations of the donor microsphere working solutions were set to three gradients of 2 ug / mL, 20 ug / mL, and 100 ug / mL. Other operations were the same as in Example 1. The measurement results are shown in Tables 3 and Figure 3 shown.

[0088] Table 3 Optimization of donor microsphere working solution concentration

[0089]

[0090] From the above experimental data, it can be seen that when the concentration of donor microspheres is 20ug / mL, the reagent signal-to-background ratio is the highest; when the concentration is lower, the signal value will decrease, and when the concentration is too high, the background value of the experimental results will be too high and the signal-to-background ratio will decrease.

[0091] Example 4 Optimization of the concentration of receptor microspheres binding to HBP antibody

[0092] The feed ratio of antibody to microspheres was microspheres: antibody = 1 mg: 75 ug, and the mixture was incubated at 37°C for 4 hours to promote the binding of the antibody to the surface of the microspheres. The prepared acceptor microspheres bound to the anti-HBP antibody were made into working solution, and the concentration of the donor microsphere working solution was set to 2 ug / mL, 20 ug / mL, and 100 ug / mL. Other operations were the same as in Example 2. The measurement results are shown in Tables 4 and Figure 4 shown.

[0093] Table 4 Optimization of the concentration of receptor microsphere working solution

[0094]

[0095] From the above experimental data, it can be seen that when the concentration of the receptor microspheres is 20ug / mL, the reagent signal-to-background ratio is the highest; when the concentration is lower, the signal value will decrease, and when the concentration is too high, the background value of the experimental results will be too high and the signal-to-background ratio will decrease.

[0096] Example 5: Verification of Streptavidin-Biotin Amplification System

[0097] 1. Receptor microspheres coupled to HBP antibody

[0098] 1.1. Microsphere cleaning:

[0099] The carboxylated microspheres were suspended in MES buffer, pelleted by low-speed centrifugation and the supernatant removed, and the wash was repeated 2-3 times to remove the storage buffer.

[0100] 1.2. Activation of carboxyl groups:

[0101] Resuspend the microspheres in MES buffer. Add EDC and NHS to the suspension at a concentration of 50 mg / mL. Gently stir the reaction and incubate at room temperature for 15-30 minutes. This step converts the carboxyl groups to highly reactive ester groups.

[0102] 1.3. Antibody Binding:

[0103] After the activation step, the supernatant was removed and the microspheres were quickly washed with MES buffer. Anti-HBP antibody was added to the activated microspheres. The ratio of antibody to microspheres was microspheres: antibody = 1 mg: 75 ug, and incubated at 37°C for 4 hours to promote the binding of the antibody to the microsphere surface.

[0104] 1.4. Blocking reaction:

[0105] The microspheres were treated with a buffer containing 5% BSA to block unreacted active sites and prevent nonspecific binding. Incubate at 37°C for 1 hour.

[0106] 1.5. Washing and storage:

[0107] Wash the microspheres several times with PBS buffer to remove unbound antibody and blocking agent. Resuspend the coated microspheres in storage buffer and store at 4°C, protected from light.

[0108] 2. Donor microspheres coupled to streptavidin

[0109] 2.1. Microsphere cleaning:

[0110] The carboxylated microspheres were suspended in MES buffer, pelleted by low-speed centrifugation and the supernatant removed, and the wash was repeated 2-3 times to remove the storage buffer.

[0111] 2.2. Activation of carboxyl groups:

[0112] Resuspend the microspheres in MES buffer. Add EDC and NHS to the suspension at a concentration of 50 mg / mL. Gently stir the reaction and incubate at room temperature for 15-30 minutes. This step converts the carboxyl groups to highly reactive ester groups.

[0113] 2.3. Protein binding:

[0114] After the activation step, the supernatant was removed and the microspheres were quickly washed with MES buffer.

[0115] Add streptavidin to the activated microspheres. The ratio of streptavidin to microspheres is microspheres: streptavidin = 1 mg: 75 ug, and incubate at 37°C for 4 hours to promote the binding of the antibody to the surface of the microspheres.

[0116] 2.4. Blocking reaction:

[0117] The microspheres were treated with a buffer containing 5% BSA to block unreacted active sites and prevent nonspecific binding. Incubate at 37°C for 1 hour.

[0118] 2.5. Washing and storage:

[0119] Wash the microspheres several times with PBS buffer to remove unbound antibody and blocking agent. Resuspend the coated microspheres in storage buffer and store at 4°C, protected from light.

[0120] 3. Biotinylated HBP antibody

[0121] 3.1. Add NHS-biotin solution dropwise into the antibody solution at a biotin:antibody molar ratio of 10:1 and mix slowly to avoid antibody denaturation.

[0122] 3.2. Reaction incubation: Incubate at room temperature for 1 hour, gently shake or stir to ensure sufficient reaction.

[0123] 3.3. Remove unreacted biotin: Remove free NHS-biotin by dialysis. The dialysis should be performed at 4°C, using a large amount of buffer, and changing it at least 3 times.

[0124] 3.4. Aliquot the labeled antibodies and store at 4°C to avoid multiple freezing and thawing.

[0125] 4. Detection coating results:

[0126] The prepared acceptor microspheres bound to anti-HBP antibody, the donor microspheres bound to streptavidin, and the HBP antibody bound to biotin were made into working solution, and the assay was performed on a chemiluminescent immunoassay analyzer (Beijing Bovitec Biotechnology Co., Ltd.); the effect of the streptavidin-biotin amplification system was compared. The test results are shown in Figure 5 and Figure 5 shown.

[0127] Table 5 Samples Sample concentration Direct package antibody SA-Biotin amplification system

[0128]

[0129] From the above experimental data, it can be seen that when the streptavidin-biotin amplification system is used, the signal value is higher, the signal-to-background ratio is also higher, and the reagent sensitivity is better.

[0130] Example 6 Comparison of performance of different freeze-drying processes

[0131] The acceptor microspheres bound to the anti-HBP antibody, the donor microspheres bound to streptavidin and the HBP antibody bound to biotin prepared according to the method of Example 5 were made into working solutions, and the above three working solutions were lyophilized at 10uL per drop to prepare freeze-dried beads of the acceptor microspheres bound to the anti-HBP antibody, freeze-dried beads of the donor microspheres bound to streptavidin and freeze-dried beads of the HBP antibody bound to biotin; the freeze-drying process is shown in Table 6.

[0132] Table 6 Freeze-drying process

[0133]

[0134] The freeze-dried beads prepared by different freeze-drying processes were assembled into test cards. After adding 150ul 0.9% saline (reconstitution reagent) and 10uL of the sample to be tested to the test card, the test was performed on a chemiluminescent immunoassay analyzer (CLi-1600, Biotech Bio). The test results are shown in Tables 7, 8 and Figure 6 shown.

[0135] Table 7

[0136]

[0137]

[0138] Table 8

[0139]

[0140]

[0141] The test cards assembled from the freeze-dried beads produced by the above-mentioned different freeze-drying processes were placed in a 45°C incubator for 28 days, and then the high and low value quality control products were measured on a chemiluminescence immunoassay analyzer (CLi-1600, Biotech). The test results were compared and analyzed with the test results before 45°C thermal acceleration. The results are shown in Table 9.

[0142] Table 9

[0143]

[0144] From the above data, we can see that the CV of the reagents in freeze-drying process group B is significantly better than those in freeze-drying process group A and freeze-drying process group C. The results of the 45°C thermal acceleration test also show that the reagents corresponding to freeze-drying process group B have the smallest drop in amplitude, which can better meet the reagent performance requirements.

[0145] Example 7 Comparison of performance between freeze-dried pellet reagent system and liquid reagent

[0146] The acceptor microspheres bound to the anti-HBP antibody prepared according to the method of Example 5, the donor microspheres bound to streptavidin, and the HBP antibody bound to biotin were made into a working solution, which was a liquid reagent.

[0147] At the same time, some liquid reagents were taken and freeze-dried according to the freeze-drying process B of Example 6, and the above three working solutions were freeze-dried at 10 uL per drop to prepare freeze-dried beads of acceptor microspheres bound to anti-HBP antibodies, freeze-dried beads of donor microspheres bound to streptavidin, and freeze-dried beads of HBP antibodies bound to biotin.

[0148] The test card assembled from the above freeze-dried beads and the liquid reagent were used to test the high and low value samples respectively. The results are shown in Table 10 and Figure 7 shown.

[0149] Table 10

[0150]

[0151] Repeatability test:

[0152] The test card assembled with the above freeze-dried beads and the liquid reagent were used to measure the quality control product 1 and the quality control product 2 10 times respectively, and the average concentration (Mean,) and standard deviation (SD) of the measurement results were calculated. The repeatability (CV) = (SD / ) × 100.0%, and the coefficient of variation CV was obtained according to the formula. The results are shown in Table 11.

[0153] Table 11

[0154]

[0155]

[0156] Thermal stability test: The test card assembled from the above freeze-dried beads and the liquid reagent were placed in a 45°C incubator for 28 days and then tested. The test results were compared and analyzed with the test results before 45°C thermal acceleration. The results are shown in Table 12.

[0157] Table 12

[0158]

[0159]

[0160] From the above experimental data, it can be seen that the signal value of the lyophilized reagent will be slightly lower than that of the liquid reagent, but there is no significant difference in the signal-to-background ratio and repeatability. In addition, the lyophilized reagent has better performance in thermal stability and can be suitable for transportation and storage at room temperature. At the same time, it can be matched with more convenient POCT instruments and will have a wider application market.

[0161] Example 8: Performance Verification of Freeze-Dried Pellets Reagent System

[0162] The acceptor microspheres bound to the anti-HBP antibody, the donor microspheres bound to streptavidin, and the HBP antibody bound to biotin prepared according to the method of Example 5 were made into working solutions. According to the freeze-drying process B of Example 6, the above three working solutions were freeze-dried at 10 uL per drop to prepare freeze-dried beads of the acceptor microspheres bound to the anti-HBP antibody, freeze-dried beads of the donor microspheres bound to streptavidin, and freeze-dried beads of the HBP antibody bound to biotin.

[0163] Blank detection limit test

[0164] The blank limit reference substance B0 (provided by Xiamen Biotech Biotechnology Co., Ltd.) was measured on the chemiluminescence immunoassay (Xiamen Biotech Biotechnology Co., Ltd.) using the test card assembled with the above-mentioned freeze-dried beads. The measurement was repeated 20 times to obtain the RLU values ​​(relative luminescence values) of the 20 measurement results. The mean value () and standard deviation (SD) of the measurement results were calculated to obtain +2SD. The concentration value of +2SD calculated according to the calibration curve of the kit was the blank limit.

[0165] Table 13

[0166]

[0167]

[0168] Linear range test:

[0169] HBP linear reference products L1 to L6 were mixed at a ratio of 1:0, 4:1, 3:2, 2:3, 1:4, and 0:1 to form 6 dilution concentrations (xi). Each concentration of the sample was tested twice, and the average value was calculated. The results are shown in Table 14. The average value of the result and the dilution ratio were fitted with a linear line using the least squares method, and the correlation coefficient r of the linear regression was calculated according to the following formula. The fitted straight line is shown in Figure 8 shown.

[0170]

[0171] The formula is:

[0172] x i - dilution concentration;

[0173] ——average value of dilution concentration;

[0174] y i ——mean value of test results;

[0175] ——the average of the mean values ​​of the test results;

[0176] n——Number of measurements

[0177] Table 14

[0178]

[0179]

[0180] Thermal stability test: The test card assembled with the above-mentioned freeze-dried beads was placed in a 45°C incubator for 7 days, 14 days, and 28 days, respectively. Then, it was measured on a chemiluminescent immunoassay (Xiamen Biotech Co., Ltd.), and the test results were compared with the test results before 45°C thermal acceleration, as shown in Table 15.

[0181] Table 15

[0182]

[0183] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.< / hbp> < / hbp> < / hbp> < / hbp>

Claims

1. A heparin binding protein homogeneous chemiluminescence detection kit, characterized in that: comprising a first reagent, a second reagent and a third reagent, The first reagent includes a first heparin-binding protein antibody labeled with biotin; The second reagent includes receptor microspheres coupled to a second heparin binding protein antibody; The third reagent includes donor microspheres coupled with streptavidin; The first reagent, the second reagent and the third reagent are all freeze-dried reagents; The freeze-drying parameters of the freeze-drying process of the freeze-dried reagent are as follows: -45°C, 6h; -45°C, 2h; -40°C, 2h; -35°C, 2h; -30°C, 20h; -25°C, 2h; -20°C, 2h; -15°C, 2h; -10°C, 2h; -5°C, 2h; 0°C, 2h; 10°C, 2h; 20°C, 2h; 30°C, 2h; 30°C, 2h; 30°C, 20h.

2. The heparin binding protein homogeneous chemiluminescence detection kit according to claim 1, characterized in that: In the first reagent, the molar ratio of biotin to the first heparin-binding protein antibody is 5-15:

1.

3. The heparin binding protein homogeneous chemiluminescence detection kit according to claim 1, characterized in that: In the second reagent, the ratio of the second heparin binding protein antibody to the receptor microspheres is 5-150 μg:1 mg.

4. The heparin binding protein homogeneous chemiluminescence detection kit according to claim 1, characterized in that: In the third reagent, the ratio of streptavidin to donor microspheres is 5-150 μg:1 mg.

5. A method for preparing a homogeneous chemiluminescent detection kit for heparin binding protein, characterized in that: The following steps are involved: S1, labeling the first heparin-binding protein antibody with biotin to prepare a first reagent; S2, coupling a second heparin-binding protein antibody to the receptor microspheres to prepare a second reagent; S3, coupling streptavidin to the donor microspheres to prepare the third reagent; S4, prepare the first reagent, the second reagent and the third reagent into freeze-dried reagents; the freeze-drying parameters are as follows: -45℃, 6h; -45℃, 2h; -40℃, 2h; -35℃, 2h; -30℃, 20h; -25℃, 2h; -20℃, 2h; -15℃, 2h; -10℃, 2h; -5℃, 2h; 0℃, 2h; 10℃, 2h; 20℃, 2h; 30℃, 2h; 30℃, 2h; 30℃, 20h.

6. The method for preparing the heparin binding protein homogeneous chemiluminescence detection kit according to claim 5, characterized in that: Step S1 specifically includes: adding NHS-biotin solution dropwise into the first heparin-binding protein antibody solution, incubating at room temperature for 50-80 minutes, removing unreacted biotin, and obtaining the first reagent.

7. The method for preparing the heparin binding protein homogeneous chemiluminescence detection kit according to claim 5, characterized in that: Step S2 specifically comprises: washing the receptor microspheres; resuspending the receptor microspheres in MES buffer, adding EDC and NHS, and incubating at room temperature for 15-30 minutes to activate the receptor microspheres; The supernatant was removed and the acceptor microspheres were washed, and the second heparin-binding protein antibody was added to the activated acceptor microspheres and incubated at 37°C for 3.5-4.5h; After blocking and washing, the second reagent is obtained.

8. The method for preparing the heparin binding protein homogeneous chemiluminescence detection kit according to claim 5, characterized in that: Step S3 specifically comprises: washing the donor microspheres; The donor microspheres were resuspended in MES buffer, EDC and NHS were added, and incubated at room temperature for 15-30 min to activate the donor microspheres; The supernatant is removed and the donor microspheres are washed, streptavidin is added to the activated donor microspheres, and incubated at 37° C. for 3.5-4.5 hours; the microspheres are blocked and washed to obtain the third reagent.

9. The method for preparing the heparin binding protein homogeneous chemiluminescence detection kit according to claim 7 or 8, characterized in that: The EDC and NHS were both used at a concentration of 45-55 mg / mL.

10. The method for preparing the heparin binding protein homogeneous chemiluminescence detection kit according to claim 5, characterized in that: The freeze-dried reagent is a freeze-dried pellet, and the diameter of the freeze-dried pellet is 0.5-5 mm.

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