Serum amyloid protein A homogeneous chemiluminescence kit and its preparation and application

Abstract

The invention provides a serum amyloid A homogeneous chemiluminescence kit and its preparation and application, comprising a biotin-labeled anti-SAA first antibody freeze-dried ball, an anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, a streptavidin-coated donor microsphere freeze-dried ball, a reaction buffer and a detection card; the acceptor microsphere is filled with a substance that can react with singlet oxygen; the donor microsphere is filled with a substance that can generate singlet oxygen under the excitation of excitation light; the detection card comprises a sample addition hole, a detection hole and a blood filter membrane; the sample addition hole is connected to the detection hole; the blood filter membrane is arranged in the sample addition hole, and the blood filter membrane is coated with freeze-dried free SAA antibody and a blocking agent; the biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball and the streptavidin-coated donor microsphere freeze-dried ball are all placed in the detection hole.

Description

Technical Field

[0001] The invention relates to a serum amyloid A homogeneous chemiluminescence kit and a preparation and application thereof, belonging to the technical field of serum amyloid A detection. Background Art

[0002] Serum amyloid A (SAA) is a precursor of tissue amyloid A. Similar to C-reactive protein, it is an acute phase protein mainly derived from liver cells. SAA exists in a constant amount in the serum of normal people, but when inflammation occurs in the human body or within 48-72 hours of the acute phase of infection, SAA can increase hundreds to thousands of times, and rapidly degrades during the recovery period of the disease. It is a more sensitive marker for diagnosing bacterial or viral infections than CRP. Among several inflammatory factors, SAA elevation is seen in viral, mycoplasma, and bacterial infections, and its sensitivity is higher than CRP; CRP elevation is seen in bacterial infection, and viral and mycoplasma pathogens such as infection do not increase or increase slightly. The two can be used complementary and can be combined with CRP to determine whether it is a viral infection. At present, elevated serum SAA has been detected in bacterial and viral infections, atherosclerosis, coronary heart disease, acute transplant rejection, tumors and other diseases, which can provide better reference value for clinical practice.

[0003] However, the current SAA detection method has problems such as narrow linear range, poor high value discrimination, and susceptibility to interference from various interfering substances. Summary of the invention

[0004] The present invention provides a serum amyloid protein A homogeneous chemiluminescence kit and its preparation and application, which can effectively solve the above problems.

[0005] The present invention is achieved in that:

[0006] A serum amyloid A homogeneous chemiluminescence kit, comprising a biotin-labeled anti-SAA first antibody freeze-dried ball, an anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, a streptavidin-coated donor microsphere freeze-dried ball, a reaction buffer and a detection card;

[0007] The acceptor microspheres are filled with a substance that can react with singlet oxygen; the donor microspheres are filled with a substance that can generate singlet oxygen under the excitation of excitation light;

[0008] The test card comprises a sample addition hole, a test hole and a blood filter membrane; the sample addition hole and the test hole are connected; the blood filter membrane is arranged in the sample addition hole, and the blood filter membrane is coated with freeze-dried free SAA antibodies and blocking agents;

[0009] The biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, and the streptavidin-coated donor microsphere freeze-dried ball are all placed in the detection well.

[0010] In some embodiments, the preparation of the biotin-labeled anti-SAA first antibody freeze-dried ball includes: adding the anti-SAA first antibody and long-chain biotin to PBS buffer at a molar ratio of 1:1 to 40, shaking at 24 to 26°C for 2h to 8h, adding glycine after the reaction is completed, and then shaking and blocking at 24 to 26°C for 10min to 120min, and then dialyzing in PBS buffer for 22 to 26h to obtain the product, and finally preparing the product to the use concentration, controlling the droplet size to 10 to 20μL, dropping it into liquid nitrogen to form a frozen form, and then freeze-drying it to prepare it into the form of freeze-dried balls, and the preparation is completed.

[0011] In some embodiments, the preparation of the anti-SAA secondary antibody-coated receptor microsphere lyophilized beads comprises:

[0012] (1) placing the receptor microsphere suspension in a container, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding two volumes of MES solution at pH 4.5-6.5 to wash twice, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding MES solution at pH 4.5-6.5, and ultrasonically dispersing to prepare a primary microsphere suspension I;

[0013] (2) Add 0.005-0.2 mg of EDC and 0.005-0.2 mg of NHS to the primary microsphere suspension I prepared in step (1), dissolve them in MES solution with pH 4.5-6.5 to 10 mg / mL, mix well, shake on a shaker at 37°C for 0.5-2 h, centrifuge after the reaction to remove the supernatant, add HEPES solution with pH 7.0-8.0 to wash, centrifuge at 14000 rpm for 15-30 min, remove the supernatant, add HEPES solution with pH 7.0-8.5, and disperse by ultrasonication to prepare secondary microsphere suspension II;

[0014] (3) adding anti-SAA secondary antibody and Tween20 to the secondary microsphere suspension II prepared in step (2), mixing, shaking on a shaker at 37°C for 4 hours, adding bovine serum albumin after the reaction, mixing, blocking on a shaker at 37°C for 30 minutes, centrifuging at 14000 rpm for 15-30 minutes, removing the supernatant, adding two volumes of pH 7.0-8.5 HEPES solution to wash twice, and obtaining receptor microspheres coated with anti-SAA secondary antibody;

[0015] (4) The receptor microspheres coated with the anti-SAA secondary antibody are prepared to a working concentration, the droplet size is controlled to 10 to 20 μL, and the microspheres are dropped into liquid nitrogen to form a frozen form, and then freeze-dried to form a freeze-dried microsphere. The preparation is completed.

[0016] In some embodiments, the preparation of the streptavidin-coated donor microsphere lyophilized beads comprises:

[0017] (1) placing the donor microsphere suspension in a container, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding two volumes of pH 4.5-6.5 MES solution to wash twice, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding pH 4.5-6.5 MES solution, and ultrasonically dispersing to prepare a primary microsphere suspension I;

[0018] (2) Add streptavidin and EDC-MES solution to the primary microsphere suspension I prepared in step (1), mix well, shake on a shaker at 37°C for 2-4 hours, add 10% bovine serum albumin solution after the reaction, mix well, place on a shaker at 37°C for 30-60 minutes, centrifuge at 14000 rpm for 15-30 minutes, remove the supernatant, add two volumes of pH 4.5-6.5 MES solution and wash twice to obtain streptavidin-coated donor microspheres;

[0019] (3) The donor microspheres coated with streptavidin are prepared to a working concentration, the droplet size is controlled to 10 to 20 μL, and the microspheres are dropped into liquid nitrogen to form a frozen form, and then freeze-dried to form freeze-dried microspheres. The preparation is completed.

[0020] In some embodiments, the preparation of the blood filter membrane coated with lyophilized free SAA antibody and blocking agent comprises:

[0021] (1) Soak the blood filter membrane in PBS buffer with a concentration of 1 to 10 ug / mL free SAA antibody for 2 to 8 hours, then take it out and transfer the blood filter membrane to a plate and lay it flat to ensure that there is no overlap between the blood filter membranes. Use non-woven fabric to remove excess water on the surface of the blood filter membrane;

[0022] (2) Spray TBS buffer containing 1 to 100 ug / mL of blocking agent evenly onto the blood filter membrane;

[0023] (3) Freeze the blood filter membrane at a temperature below -40°C for 1 hour, and then dry it at 37°C for 2 to 6 hours. After drying, remove the blood filter membrane and seal it for storage.

[0024] In some embodiments, the reaction buffer has a formula of: 0.9% sodium chloride solution.

[0025] A detection method for the above serum amyloid A homogeneous chemiluminescence kit for non-disease diagnosis purposes comprises the following steps:

[0026] S1, dissolve the sample 180-220 times with 0.9% sodium chloride solution;

[0027] S2, adding the reaction buffer and the diluted sample to the blood filter membrane in the sample well of the test card;

[0028] S3, placing the test card into the detection instrument, the detection instrument draws negative pressure on the sample addition hole, and the reaction buffer and the sample enter the detection hole with the pressure and mix with the biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball and the streptavidin-coated donor microsphere freeze-dried ball, incubate, read the light signal, and calculate the concentration of serum amyloid A according to the intensity of the light signal.

[0029] In some embodiments, the sample is a whole blood sample or a plasma sample.

[0030] The beneficial effects of the present invention are:

[0031] (1) The kit provided by the present invention adopts a dry homogeneous immunochemiluminescence method, which does not require cleaning, is simple and fast to operate, and has excellent precision and sensitivity. The reagents are stored in the form of freeze-dried balls, and the reaction buffer can be stored at room temperature, which greatly improves the stability of reagent storage and transportation, and is easy to carry, solving the problem that some components of some detection reagents need to be separated and stored at low temperatures and have poor stability.

[0032] (2) The freeze-dried storage method of the free SAA antibody mentioned in the present invention solves the problem of protein instability in liquid storage, can flexibly adjust the linear range, and solves the problems of narrow linear range and low hook point concentration shown by existing SAA detection reagents. This processing method is simple, and the number of blood filter membranes processed in each batch is large, and the batch difference is stable.

[0033] (3) The method of freeze-drying the blocking agent in the blood filter membrane mentioned in the present invention can neutralize the interfering substances in the sample in advance before the sample reacts with the reagent, thereby reducing the concentration of the blocking agent and improving the blocking effect. The freeze-drying treatment allows the blocking agent to be stored at room temperature, solving the problem of placing the blocking agent in the sample diluent or buffer solution and storing the buffer solution components mainly at low temperatures.

[0034] (4) The receptor microspheres of the present invention are activated. After activation, the microspheres can shield the interference of dry whole blood samples and produce detection signals close to those of plasma samples, thereby improving the sensitivity, precision and correlation of the detection, etc., so that the kit of the present invention can be applied to the detection of whole blood samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 Schematic diagram of the structure of the detection card of the embodiment of the present invention.

[0037] Figure 2 It is a schematic diagram of the detection principle of the kit according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] The embodiment of the present invention provides a serum amyloid A homogeneous chemiluminescence kit, comprising a biotin-labeled anti-SAA first antibody freeze-dried ball, an anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, a streptavidin-coated donor microsphere freeze-dried ball, a reaction buffer and a detection card;

[0040] The acceptor microspheres are filled with a substance that can react with singlet oxygen; the donor microspheres are filled with a substance that can generate singlet oxygen under the excitation of excitation light;

[0041] The test card comprises a sample addition hole, a test hole and a blood filter membrane; the sample addition hole and the test hole are connected; the blood filter membrane is arranged in the sample addition hole, and the blood filter membrane is coated with freeze-dried free SAA antibodies and blocking agents;

[0042] The biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, and the streptavidin-coated donor microsphere freeze-dried ball are all placed in the detection well.

[0043] The detection principle of the kit of the embodiment of the present invention is: under the excitation of excitation light of about 680nm, the donor microspheres coated with avidin and filled with phthalocyanine derivatives can generate singlet oxygen. When the donor microspheres are connected to the biotin-labeled anti-SAA first antibody, and further connected to the detection object SAA and the acceptor microspheres coated with the anti-SAA second antibody, the singlet oxygen can be transferred to the acceptor microspheres, and react chemically with the dimethylthiophene filled on the acceptor microspheres to generate ultraviolet light, and the ultraviolet light further excites the europium chelate filled on the acceptor microspheres to generate photons; if the acceptor and the donor microspheres are not combined through the detection object SAA, the singlet oxygen cannot reach the acceptor microspheres due to the long transmission distance to make it emit light. The intensity of the light signal is positively correlated with the concentration of the detected SAA. The concentration of SAA is calculated according to the intensity of the detected light signal to complete the qualitative or quantitative detection of the concentration of SAA.

[0044] In some embodiments, the preparation of the biotin-labeled anti-SAA first antibody freeze-dried ball includes: adding the anti-SAA first antibody and long-chain biotin to PBS buffer at a molar ratio of 1:1 to 40, shaking at 24 to 26°C for 2h to 8h, adding glycine after the reaction is completed, and then shaking and blocking at 24 to 26°C for 10min to 120min, and then dialyzing in PBS buffer for 22 to 26h to obtain the product, and finally preparing the product to the use concentration, controlling the droplet size to 10 to 20μL, dropping it into liquid nitrogen to form a frozen form, and then freeze-drying it to prepare it into the form of freeze-dried balls, and the preparation is completed.

[0045] In some embodiments, the preparation of the anti-SAA secondary antibody-coated receptor microsphere lyophilized beads comprises:

[0046] (1) Place the receptor microsphere suspension in a container, centrifuge at 14000 rpm for 15-30 min, remove the supernatant, add two volumes of MES solution with a pH of 4.5-6.5 to wash twice, centrifuge at 14000 rpm for 15-30 min, remove the supernatant, add MES solution with a pH of 4.5-6.5, and disperse by ultrasonication to prepare a primary microsphere suspension I.

[0047] (2) Add 0.005-0.2 mg of EDC and 0.005-0.2 mg of NHS to the primary microsphere suspension I prepared in step (1), dissolve them in MES solution with pH 4.5-6.5 to 10 mg / mL, mix well, shake on a shaker at 37°C for 0.5-2 h, centrifuge to remove the supernatant after the reaction, add pH 7.0-8.0 HEPES solution for washing, centrifuge at 14000 rpm for 15-30 min, remove the supernatant, add pH 7.0-8.5 HEPES solution, and disperse by ultrasound to prepare secondary microsphere suspension II. This step is to activate the receptor microspheres. After activation, the microspheres can shield the interference of dry whole blood samples and produce detection signals close to plasma samples, thereby improving the sensitivity, precision and correlation of the detection, so that the embodiment of the present invention can be applied to the detection of whole blood samples.

[0048] (3) Add anti-SAA secondary antibody and Tween20 to the secondary microsphere suspension II prepared in step (2) in sequence, mix well, and shake on a shaker at 37°C for 4 hours. After the reaction is completed, add bovine serum albumin, mix well, and place on a shaker at 37°C for 30 minutes. Centrifuge at 14000 rpm for 15 to 30 minutes, remove the supernatant, and add twice the volume of pH 7.0 to 8.5 HEPES solution to wash twice to obtain receptor microspheres coated with anti-SAA secondary antibody.

[0049] (4) The receptor microspheres coated with the anti-SAA secondary antibody are prepared to a working concentration, the droplet size is controlled to 10 to 20 μL, and the microspheres are dropped into liquid nitrogen to form a frozen form, and then freeze-dried to form a freeze-dried microsphere. The preparation is completed.

[0050] In some embodiments, the preparation of the streptavidin-coated donor microsphere lyophilized beads comprises:

[0051] (1) placing the donor microsphere suspension in a container, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding two volumes of pH 4.5-6.5 MES solution to wash twice, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding pH 4.5-6.5 MES solution, and ultrasonically dispersing to prepare a primary microsphere suspension I;

[0052] (2) Add streptavidin and EDC-MES solution to the primary microsphere suspension I prepared in step (1), mix well, shake on a shaker at 37°C for 2-4 hours, add 10% bovine serum albumin solution after the reaction, mix well, place on a shaker at 37°C for 30-60 minutes, centrifuge at 14000 rpm for 15-30 minutes, remove the supernatant, add two volumes of pH 4.5-6.5 MES solution and wash twice to obtain streptavidin-coated donor microspheres;

[0053] (3) The donor microspheres coated with streptavidin are prepared to a working concentration, the droplet size is controlled to 10 to 20 μL, and the microspheres are dropped into liquid nitrogen to form a frozen form, and then freeze-dried to form freeze-dried microspheres. The preparation is completed.

[0054] In some embodiments, the preparation of the blood filter membrane coated with lyophilized free SAA antibody and blocking agent comprises:

[0055] (1) Soak the blood filter membrane in PBS buffer with a concentration of 1 to 10 ug / mL free SAA antibody for 2 to 8 hours, then take it out and transfer the blood filter membrane to a plate and lay it flat to ensure that there is no overlap between the blood filter membranes. Use non-woven fabric to remove excess water on the surface of the blood filter membrane;

[0056] (2) Spray TBS buffer containing 1 to 100 ug / mL of blocking agent evenly onto the blood filter membrane;

[0057] (3) Freeze the blood filter membrane at a temperature below -40°C for 1 hour, and then dry it at 37°C for 2 to 6 hours. After drying, remove the blood filter membrane and seal it for storage.

[0058] In the embodiment of the present invention, the free SAA antibody is freeze-dried on the blood filter membrane, which solves the problem of protein instability during liquid storage. The free SAA antibody can flexibly adjust the linear range, which solves the problems of narrow linear range and low hook point concentration shown by existing SAA test reagents. In addition, this processing method is simple and easy to operate, and the number of blood filter membranes processed in each batch is large, and the batch difference is stable.

[0059] The method of freeze-drying the blocking agent in the blood filter membrane implemented in the present invention can neutralize the interfering substances in the sample in advance before the sample reacts with the reagent, reduce the concentration of the blocking agent, improve the blocking effect, and overcome the problem that the blocking agent in the traditional reagent only starts to take effect when the sample reacts with the reagent, the effect is weak, and the amount required is large. In addition, the blocking agent is a protein and is unstable in liquid, but the blocking agent in the embodiment of the present invention can be stored at room temperature after freeze-drying.

[0060] In some embodiments, the reaction buffer has a formula of: 0.9% sodium chloride solution.

[0061] The embodiment of the present invention provides a detection method for the above-mentioned serum amyloid A homogeneous chemiluminescence kit for non-disease diagnosis purposes, comprising the following steps:

[0062] S1, dissolve the sample 180 to 220 times with 0.9% sodium chloride solution; after the sample is diluted, the problems of narrow linear range and poor high value discrimination of the test can be improved.

[0063] S2, add the reaction buffer and diluted sample to the blood filter membrane in the sample well of the test card; the free SAA antibody on the blood filter membrane can improve the problem of the hook point being too close to the front; the blocking agent freeze-dried on the blood filter membrane reacts with the sample in advance, and the blocking effect is timely.

[0064] S3, placing the test card into the detection instrument, the detection instrument draws negative pressure on the sample addition hole, and the reaction buffer and the sample enter the detection hole with the pressure and mix with the biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball and the streptavidin-coated donor microsphere freeze-dried ball, incubate, read the light signal, and calculate the concentration of serum amyloid A according to the intensity of the light signal.

[0065] The above detection method can be used for non-disease diagnosis purposes such as scientific research experiments.

[0066] In some embodiments, the sample is a whole blood sample or a plasma sample.

[0067] Example 1 Preparation of serum amyloid A homogeneous chemiluminescence kit

[0068] The preparation process of the anti-SAA antibody-coated receptor microspheres is as follows:

[0069] (1) 1 mg of a 10 mg / mL receptor microsphere suspension was placed in a test tube, centrifuged at 14,000 rpm for 20 min, the supernatant was removed, and two volumes of a pH 6.5 MES solution were added to wash twice, and the tube was centrifuged at 14,000 rpm for 20 min, the supernatant was removed, 200 μL of a pH 5 MES solution was added, and ultrasonic dispersion was performed to prepare a primary microsphere suspension I;

[0070] (2) Add 5 μL of 10 mg / mL EDC-MES solution and NHS-MES solution to the primary microsphere suspension I prepared in step (1), mix well, shake on a shaker at 37°C for 0.5 to 2 h, centrifuge after the reaction to remove the supernatant, add 200 μL of pH 7.5 HEPES solution for washing, centrifuge at 14,000 rpm for 20 min, remove the supernatant, add 100 μL of pH 7.5 HEPES solution, and disperse by ultrasonication to prepare a secondary microsphere suspension II;

[0071] (3) To the secondary microsphere suspension II prepared in step (2), 50 μg of anti-SAA antibody (purchased from Chongqing Tansheng Technology, model: FAB-B004-C4) and 0.02 g of Tween20 were added in sequence. Finally, the suspension II was supplemented with pH 7.5 HEPES solution to a total volume of 200 uL, mixed, and shaken on a shaker at 30°C for 3 h. After the reaction was completed, 20 μL of 10% bovine serum albumin solution was added, mixed, and placed on a shaker at 37°C for 1.5 h. Centrifuged at 14000 rpm for 20 min, the supernatant was removed, and two volumes of pH 6.5-8.0 Tris solution were added to wash twice, and 50 μL of pH 7.5 Tris solution was added for storage to obtain receptor microspheres coated with anti-SAA antibodies.

[0072] The manufacturer of the receptor microspheres used above is Suzhou Weidu Biotechnology Co., Ltd., and the product number is 67700100.

[0073] The preparation process of biotin-labeled anti-SAA antibody is as follows:

[0074] 50 μg of anti-SAA antibody (purchased from Chongqing Tansheng Technology, model: FAB-B004-2C5) and NHS-biotin were added to PBS buffer at a molar ratio of 1:10, and shaken at 25°C for 1.5 h. After the reaction, 8 μL of 10% glycine solution was added, and then shaken and blocked at 25°C for 40 min. The biotin-labeled anti-SAA antibody solution was dialyzed in 0.01 mol / L, pH 7.4 PBS buffer for 24 to 48 h.

[0075] The preparation process of streptavidin-coated donor microspheres is as follows:

[0076] (1) 100 μL of a 10 mg / mL donor microsphere suspension was placed in a test tube, centrifuged at 14,000 rpm for 20 min, the supernatant was removed, and two volumes of a pH 5.5 MES solution were added to wash twice, and the tube was centrifuged at 14,000 rpm for 20 min, the supernatant was removed, 200 μL of a pH 5.5 MES solution was added, and ultrasonic dispersion was performed to prepare a primary microsphere suspension I;

[0077] (2) To the primary microsphere suspension I prepared in step (1), 50 μg of avidin and 2 μL of 10 mg / mL EDC-MES solution were added, respectively, and the mixture was mixed and shaken on a shaker at 37°C for 2 to 4 h. After the reaction was completed, 30 μL of 10% bovine serum albumin solution was added, mixed, and sealed on a shaker at 37°C for 40 min. The mixture was centrifuged at 14,000 rpm for 30 min, the supernatant was removed, and two volumes of buffer ① were added to wash twice. 50 μL of Tris solution at pH 7.5 was added for storage to obtain donor microspheres coated with streptavidin.

[0078] The manufacturer of the donor microspheres used above is Suzhou Weidu Biotechnology Co., Ltd., with the product number being 67500100.

[0079] Liquid nitrogen cryospheres were prepared as follows:

[0080] Turn on the bead dropper and adjust the volume of the liquid ball dropped by the bead dropper to 10 μL. Prepare liquid nitrogen, biotin-labeled anti-SAA antibody working solution, anti-SAA-coated acceptor microsphere working solution, and streptavidin-coated donor microsphere working solution. Pre-cool the container containing liquid nitrogen and frozen balls with liquid nitrogen. The three-component working solution is divided and dripped into liquid nitrogen through the bead dropper. The dripping droplets are frozen in liquid nitrogen and become spherical. Transfer the frozen balls to a freeze dryer pre-frozen to -40°C and start freeze drying. After freeze drying, the three-component freeze-dried balls are obtained.

[0081] Preparation of the blood filter membrane coated with freeze-dried free SAA antibody and blocking agent:

[0082] Take 1-10 mg of free SAA antibody and add it to 1 L of 10 mM / L PBS buffer so that the concentration of free SAA antibody in the PBS buffer is 5 ug / mL to obtain a blood filter membrane pretreatment solution; soak the blood filter membrane in the buffer to avoid overlapping the blood filter membranes, soak for 2-8 hours, take it out, and then transfer the blood filter membrane to a plate and lay it flat to ensure that there is no overlapping between the blood filter membranes, and use non-woven fabric to remove excess moisture on the surface of the blood filter membrane.

[0083] Take 100 mg of the blocker, which is HBR1 from Scantibodies, and add it to 1 L of 10 mM / L TBS buffer, so that the concentration of the blocker in the TBS buffer is 100 ug / mL, to obtain a blood filter membrane pretreatment solution; spray the pretreatment solution evenly onto each blood filter membrane, with 20 μL of the pretreatment solution sprayed on each blood filter membrane.

[0084] After spraying, the blood filter membrane was frozen at below -40°C for 1 hour and placed in a freeze dryer for freeze drying. At the end of the freeze drying process, the temperature was set to 37°C. After drying at 37°C for 4 hours, the blood filter membrane was taken out and placed in a sealed aluminum foil bag for standby use. The pretreatment of the blood filter membrane was now completed.

[0085] The structure of the test card is as follows Figure 1 As shown, the test card includes a test hole 1 and a sample addition hole 2, the test hole 1 and the sample addition hole 2 are connected, a blood filter membrane 21 is arranged at the bottom of the sample addition hole 2, and a filter element 22 is arranged on the blood filter membrane 21. The blood filter membrane 21 has been processed as above, and a three-component freeze-dried ball reagent is placed in the test hole 1. The test card is sealed with aluminum foil, and the test card processing is completed.

[0086] Example 2 Precision Test

[0087] The kit prepared in Example 1 was used to test clinical samples, which were whole blood samples from a comprehensive tertiary-level Class A hospital. First, the sample was dissolved 200 times with a 0.9% sodium chloride solution, and after oscillation and mixing, 10 μL of the sample and 150 μL of the reaction buffer were added to the sample addition well 2, and the sample was placed in a multi-channel chemiluminescence analyzer for testing. At this time, the multi-channel chemiluminescence analyzer subjected the test card to negative pressure treatment from the detection well 1, and the sample and the reaction buffer flowed into the detection well 1 to re-dissolve the three-component freeze-dried ball reagent, and the reaction began.

[0088] Three samples with low, medium and high concentrations were taken, and each sample was tested 10 times to examine the precision. The precision results of the SAA reagent test are shown in Table 1 below.

[0089] Table 1

[0090] Sample concentration mg / L 4.27 56.91 184.38 Repeat 1 4.18 52.25 177.15 Repeat 2 4.49 58.01 173.75 Repeat 3 4.32 54.23 192.92 Repeat 4 4.39 56.31 188.11 Repeat 5 4.14 56.55 191.36 Repeat 6 4.33 57.10 184.39 Repeat 7 4.12 55.38 186.21 Repeat 8 4.27 56.01 185.70 Repeat 9 4.40 56.56 177.84 Repeat 10 4.33 58.76 184.37 Coefficient of variation 4.15％ 3.31％ 3.38％

[0091] From the data in Table 1, it can be seen that the coefficient of variation is less than 5%, the precision is excellent, and it is suitable for testing whole blood samples.

[0092] Example 3 Hook point test

[0093] Prepare gradient antigen reference and test with the kit prepared in Example 1. First, dissolve the sample 200 times with 0.9% sodium chloride solution, shake and mix, take 10μL sample and 150μL reaction buffer and add them to the sample well, put it into the multi-channel chemiluminescence analyzer for testing, at this time, the multi-channel chemiluminescence analyzer performs negative pressure treatment on the test card, the sample and reaction buffer flow into the test well to re-dissolve the small ball, and the reaction starts at this time. The measured hook situation is shown in Table 2:

[0094] Table 2

[0095] Concentration pg / mL Test signal value 10000 4030878 5000 4195209 3000 4034327 2000 3748689 1000 3176675 500 2369945 200 1139043 100 580280 50 279196 20 94780 10 43331 5 19784

[0096] The data of the conformity with clinical samples measured by the kit prepared in Example 1 are shown in Table 3. The third-party reagent is Siemens serum amyloid A assay kit (nephelometry) N Latex SAA, product number: OQMP11.

[0097] Table 3

[0098]

[0099]

[0100] Example 4 Anti-interference test

[0101] A HAMA interference sample (SAA concentration of 5.23 mg / L measured by a third-party reagent) and a RF interference sample (SAA concentration of 4.48 mg / L measured by a third-party reagent) were prepared and tested with the kit prepared in Example 1, and the results are shown in Table 4. The third-party reagent was Siemens serum amyloid A assay kit (nephelometry) N Latex SAA, product number: OQMP11.

[0102] Table 4 Anti-interference test of the kit prepared in Example 1

[0103] Test samples Test signal value AV Test concentrationAV mg / L HAMA interference sample 20543 5.16 RF Interference Sample 17804 4.72

[0104] The test results show that HAMA and RF do not interfere with the reagent prepared in Example 1.

[0105] Comparative Example 1

[0106] The experiment was divided into two groups:

[0107] Experimental group 1: free SAA antibody was added and the blood filter membrane was pretreated. Other operations were the same as in Example 1.

[0108] Experimental group 2: No free SAA antibody was added, and the blood filter membrane was pretreated. Other operations were the same as in Example 1.

[0109] Gradient antigen reference was prepared and the hook conditions were measured as follows:

[0110] The Hook point inspection experimental data is shown in Table 5.

[0111] Table 5 Comparison of Hook conditions between experimental group 1 and experimental group 2 in comparative example 1

[0112]

[0113] The data of the compliance with clinical samples measured by the kits used for the variables set in experimental group 1 and experimental group 2 are shown in Table 6. The third-party reagent is the serum amyloid A assay kit (nephelometry) N Latex SAA of Siemens, product number: OQMP11.

[0114] Table 6 Comparative Example 1 Clinical Sample Conformity between Experimental Group 1 and Experimental Group 2

[0115]

[0116]

[0117] Note: Deviation 1 is the deviation between the group without free SAA antibody and the measured concentration and the concentration of the third-party reagent. Deviation 2 is the deviation between the group with free SAA antibody added and the measured concentration and the concentration of the third-party reagent.

[0118] From the data in Tables 2 and 3, it can be seen that after adding free SAA antibodies, the improvement in the front of the hook point is obvious, and the addition of free SAA antibodies has no obvious effect on the test results.

[0119] Comparative Example 2

[0120] The experiment was divided into the following groups: the blocking agent was added to the three-component reagent prepared in Example 1, the sample diluent (0.9% sodium chloride solution), the reaction buffer, and the blood filter membrane to test the blocking effect of the blocking agent on RF and HAMA interference samples.

[0121] A HAMA interference sample (SAA concentration of 5.23 mg / L measured by a third-party reagent) and a RF interference sample (SAA concentration of 4.48 mg / L measured by a third-party reagent) were tested, and gradient concentrations of blockers were added. The test results are shown in Tables 7 and 8: The third-party reagent is Siemens Serum Amyloid A Assay Kit (Nephelometry) N Latex SAA, product number: OQMP11.

[0122] Table 7 Signal value AV of HAMA interference samples tested when different blocker concentrations were added to different locations

[0123]

[0124]

[0125] Table 8 Signal values ​​of RF interference samples tested when different blocker concentrations were added to different locations

[0126]

[0127] As shown in Tables 7 and 8, when added to the reaction buffer and blood filter membrane, the blocking effect is timely due to the early reaction between the blocker and the sample, and HAMA and RF interference can be eliminated at about 1.5μg / test and 2μg / test respectively; while the blocker added to the three-component reagent is basically in contact with the reagent and the blocker at the same time, and the blocking effect is weak, and an amount of about 16μg / test is required to achieve the required effect. When added to the sample diluent, since the interfering substances in the sample stock solution need to be directly blocked, the amount of the blocker is about 99.5μg / test (for HAMA interference) and 199μg / test (for RF interference), which is much greater than other solutions.

[0128] The blocking agent was added to the sample diluent, the buffer solution, and the blood filter membrane. After being thermally accelerated for different times, the blocking effect of the blocking agent on the RF and HAMA interference samples was tested, as shown in Table 9.

[0129] Table 9 Signal value AV of RF interference sample tested after thermal acceleration when blocker is added to different positions

[0130]

[0131] As shown in Table 9, during the thermal acceleration process, if the blocker is added to the reaction buffer (10 μg / mL), the effect of the blocker will be weakened after one day of acceleration; if the blocker is added to the sample diluent (100 μg / mL), the effect of the blocker will be weakened after three days of acceleration; if the blocker is freeze-dried on the blood filter membrane and the blood filter membrane is accelerated, the effect of the blocker is maximally protected, and there is no significant change in the effect after 28 days of thermal acceleration.

[0132] Comparative Example 3

[0133] The experiment was divided into the following groups: in the process of preparing the freeze-dried beads of acceptor microspheres coated with the anti-SAA secondary antibody, an inactivated microsphere group and an activated microsphere group were set up.

[0134] To test the precision performance, two whole blood samples, high and low, were tested respectively and repeated 10 times.

[0135] Table 10 Precision test of whole blood samples in the activated microsphere group

[0136]

[0137]

[0138] Table 11 Precision test of whole blood samples in the unactivated microsphere group

[0139] Sample concentration mg / L 4.27 56.91 184.38 Repeat 1 3.98 48.44 164.1 Repeat 2 3.39 48.49 195 Repeat 3 3.42 46.85 194.8 Repeat 4 3.27 45.59 169 Repeat 5 3.35 50.35 198.4 Repeat 6 3.41 48.63 150.1 Repeat 7 3.67 43.94 199.6 Repeat 8 3.56 49.69 181.9 Repeat 9 3.72 44.14 177.4 Repeat 10 3.3 44.32 150.2 Coefficient of variation 6.40％ 5.12％ 10.75％

[0140] As shown in Tables 10 and 11, the coefficient of variation of the activated microsphere group was less than 5%, and the precision was excellent.

[0141] Multiple whole blood samples were tested to compare the sensitivity and correlation before and after activation. The third-party reagent was Siemens serum amyloid A assay kit (nephelometry) N Latex SAA, product number: OQMP11.

[0142] Table 12 Whole blood sample test before and after activation of microspheres

[0143]

[0144]

[0145] As can be seen from Table 12, the low value sensitivity of the activated microsphere group is high, the concentration value is close to the third-party manufacturer's value, and the correlation is good.

[0146] 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.

Claims

1. A serum amyloid A homogeneous chemiluminescence kit, characterized in that: It includes a biotin-labeled anti-SAA first antibody freeze-dried ball, an anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, a streptavidin-coated donor microsphere freeze-dried ball, a reaction buffer and a detection card; The acceptor microspheres are filled with a substance that can react with singlet oxygen; the donor microspheres are filled with a substance that can generate singlet oxygen under the excitation of excitation light; The test card comprises a sample addition hole, a test hole and a blood filter membrane; the sample addition hole and the test hole are connected; the blood filter membrane is arranged in the sample addition hole, and the blood filter membrane is coated with freeze-dried free SAA antibodies and blocking agents; The biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball, and the streptavidin-coated donor microsphere freeze-dried ball are all placed in the detection well; The preparation of the anti-SAA second antibody-coated receptor microsphere lyophilized ball comprises: (1) placing the receptor microsphere suspension in a container, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding two volumes of MES solution at pH 4.5-6.5 to wash twice, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding MES solution at pH 4.5-6.5, and ultrasonically dispersing to prepare a primary microsphere suspension I; (2) Add 0.005-0.2 mg of EDC and 0.005-0.2 mg of NHS to the primary microsphere suspension I prepared in step (1), dissolve them in MES solution with pH 4.5-6.5 to 10 mg / mL, mix well, shake on a shaker at 37°C for 0.5-2 h, centrifuge after the reaction to remove the supernatant, add HEPES solution with pH 7.0-8.0 to wash, centrifuge at 14000 rpm for 15-30 min, remove the supernatant, add HEPES solution with pH 7.0-8.5, and disperse by ultrasonication to prepare secondary microsphere suspension II; (3) adding anti-SAA secondary antibody and Tween20 to the secondary microsphere suspension II prepared in step (2), mixing, shaking on a shaker at 37°C for 4 hours, adding bovine serum albumin after the reaction, mixing, blocking on a shaker at 37°C for 30 minutes, centrifuging at 14000 rpm for 15-30 minutes, removing the supernatant, adding two volumes of pH 7.0-8.5 HEPES solution to wash twice, and obtaining receptor microspheres coated with anti-SAA secondary antibody; (4) preparing the receptor microspheres coated with the anti-SAA secondary antibody to a use concentration, controlling the droplet size to 10 to 20 μL, dropping them into liquid nitrogen to form a frozen form, and then freeze-drying them to form freeze-dried microspheres, and the preparation is completed; The preparation of the blood filter membrane coated with freeze-dried free SAA antibody and blocking agent includes: (1) Soak the blood filter membrane in PBS buffer with a concentration of 1 to 10 ug / mL free SAA antibody for 2 to 8 hours, then take it out and transfer the blood filter membrane to a plate and lay it flat to ensure that there is no overlap between the blood filter membranes. Use non-woven fabric to remove excess water on the surface of the blood filter membrane; (2) Spray TBS buffer containing 1 to 100 ug / mL of blocking agent evenly onto the blood filter membrane; (3) Freeze the blood filter membrane at a temperature below -40°C for 1 hour, and then dry it at 37°C for 2 to 6 hours. After drying, remove the blood filter membrane and seal it for storage; The blocker is HBR1.

2. The serum amyloid A homogeneous chemiluminescence kit according to claim 1, characterized in that: The preparation of the biotin-labeled anti-SAA first antibody freeze-dried ball comprises: adding the anti-SAA first antibody and long-chain biotin into a PBS buffer at a molar ratio of 1:1-40, shaking at 24-26°C for 2h-8h, adding glycine after the reaction is completed, shaking and blocking at 24-26°C for 10min-120min, and then dialyzing in a PBS buffer for 22-26h to obtain a product, and finally preparing the product to a use concentration, controlling the droplet size to 10-20μL, dropping it into liquid nitrogen to form a frozen form, and then freeze-drying it to prepare it into the form of freeze-dried beads, and the preparation is completed.

3. The serum amyloid A homogeneous chemiluminescence kit according to claim 1, characterized in that: The preparation of the streptavidin-coated donor microsphere lyophilized ball comprises: (1) placing the donor microsphere suspension in a container, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding two volumes of pH 4.5-6.5 MES solution to wash twice, centrifuging at 14000 rpm for 15-30 min, removing the supernatant, adding pH 4.5-6.5 MES solution, and ultrasonically dispersing to prepare a primary microsphere suspension I; (2) Add streptavidin and EDC-MES solution to the primary microsphere suspension I prepared in step (1), mix well, shake on a shaker at 37°C for 2-4 hours, add 10% bovine serum albumin solution after the reaction, mix well, place on a shaker at 37°C for 30-60 minutes, centrifuge at 14000 rpm for 15-30 minutes, remove the supernatant, add two volumes of pH 4.5-6.5 MES solution and wash twice to obtain streptavidin-coated donor microspheres; (3) The donor microspheres coated with streptavidin are prepared to a working concentration, the droplet size is controlled to 10 to 20 μL, and the microspheres are dropped into liquid nitrogen to form a frozen form, and then freeze-dried to form freeze-dried microspheres. The preparation is completed.

4. The serum amyloid A homogeneous chemiluminescence kit according to claim 1, characterized in that: The formula of the reaction buffer is: 0.9% sodium chloride solution.

5. A detection method for non-disease diagnosis purposes of the serum amyloid A homogeneous chemiluminescence kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, dissolve the sample 180-220 times with 0.9% sodium chloride solution; S2, adding the reaction buffer and the diluted sample to the blood filter membrane in the sample well of the test card; S3, placing the test card into the detection instrument, the detection instrument draws negative pressure on the sample addition hole, and the reaction buffer and the sample enter the detection hole with the pressure and mix with the biotin-labeled anti-SAA first antibody freeze-dried ball, the anti-SAA second antibody-coated acceptor microsphere freeze-dried ball and the streptavidin-coated donor microsphere freeze-dried ball, incubate, read the light signal, and calculate the concentration of serum amyloid A according to the intensity of the light signal.

6. The detection method according to claim 5, characterized in that: The sample is a whole blood sample or a plasma sample.

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