Preparation method of serum amyloid protein A immunochromatography detection reagent strip

Abstract

The invention discloses a serum amyloid protein A immunochromatography detection reagent strip which comprises a reaction bottom plate, a sample pad, a combination pad, a chromatography membrane and absorbent paper, the chromatography membrane is arranged in the middle of the reaction bottom plate, the sample pad and the absorbent paper are arranged at the two ends of the reaction bottom plate respectively, the chromatography membrane is arranged in the middle of the reaction bottom plate, the combination pad is arranged between the chromatography membrane and the sample pad, the sample pad is partially overlapped on the combination pad, and the combination pad is partially overlapped on one side of the chromatography membrane. The absorbent paper is partially overlapped on the other side of the chromatography membrane; quantum dot fluorescent microspheres used for serum amyloid protein A antibody labeling and quantum dot fluorescent microspheres used for DNP-BSA labeling are solid-phase on the combination pad; the chromatographic membrane is provided with a detection line coated with a serum amyloid protein A detection antibody and a quality control line coated with a rabbit anti-DNP quality control antibody; and a detection line and a quality control line are sequentially arranged on the chromatography membrane in parallel at intervals along the chromatography direction. The invention further discloses a corresponding preparation method, a detection kit, a detection method and application.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay analysis, and in particular to a method for preparing a serum amyloid protein A immunochromatographic detection reagent strip. Background Art

[0002] Serum amyloid A protein (SAA) is an acute phase protein secreted by the liver. It comes from the apolipoprotein family and is a precursor of tissue amyloid A. Its plasma concentration increases significantly in cases of inflammation, trauma, myocardial infarction, infection, tumors, etc. It can serve as a chemotactic factor for immune cells such as lymphocytes; and upregulates the expression of multiple inflammatory factors (such as TNF, IL-6 and IL-8, etc.). SAA increases significantly during viral infection, 4-6 hours after infection, disappears faster during the recovery period, and the increase in patients with acute infection reflects the severity of the disease. SAA elevation has been detected in infectious diseases, tumors, transplant rejection and other diseases, which can provide better clinical diagnosis and treatment basis.

[0003] Currently known methods for determining human serum amyloid A include: enzyme-linked immunosorbent assay (ELISA), colloidal gold chromatography, immunoturbidimetry and fluorescent microsphere chromatography. Immunoturbidimetry has high requirements for instruments, is easily affected by blood lipids, and cannot detect whole blood. Colloidal gold chromatography can be used for clinical testing, but has low sensitivity. Enzyme-linked immunosorbent assay (ELISA) has high sensitivity, but the operation process is cumbersome, and a standard curve needs to be drawn for each measurement. The accuracy error is large, the measurement time is long, the instrument is expensive and requires professionally trained personnel to operate, which does not meet the needs of clinical rapid testing.

[0004] Therefore, developing a product of human serum amyloid A that is simple to operate, has low instrument cost, and is suitable for on-site instant detection is a technical problem that needs to be solved by technicians in this field. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and to provide a quantitative detection human serum amyloid A immunochromatography reagent strip based on quantum dot fluorescent microspheres and a preparation method thereof in response to the technical defects in the related art. The sensitivity of quantum dot fluorescent microsphere immunochromatography is utilized in combination with a fluorescent immunoassay analyzer to achieve high sensitivity, quickness and convenience, and accurate quantitative detection of human serum amyloid A reagent strip and preparation method.

[0006] The first aspect of the present invention is to provide a serum amyloid protein A immunochromatographic detection reagent strip, comprising a reaction base plate, a sample pad, a conjugation pad, a chromatographic membrane and absorbent paper; the chromatographic membrane is arranged in the middle of the reaction base plate, the sample pad and the absorbent paper are respectively arranged at both ends of the reaction base plate, the middle of the reaction base plate is the chromatographic membrane, the middle of the chromatographic membrane and the sample pad is the conjugation pad, the sample pad is partially overlapped on the conjugation pad, the conjugation pad is partially overlapped on one side of the chromatographic membrane, and the absorbent paper is partially overlapped on the other side of the chromatographic membrane;

[0007] The conjugate pad is solid-phased with quantum dot fluorescent microspheres for labeling human serum amyloid A antibody and quantum dot fluorescent microspheres for labeling DNP-DNP-BSA;

[0008] The chromatographic membrane is provided with a detection line coated with human serum amyloid protein A antibody and a quality control line coated with rabbit anti-DNP quality control antibody; the detection line and the quality control line are arranged in parallel and at intervals in the chromatographic direction on the chromatographic membrane.

[0009] Preferably, the human serum amyloid A antibody is obtained by immunizing mice with human serum amyloid A; the chromatographic membrane is a nitrocellulose membrane; and the absorbent paper is made of cotton and linen, so that the sample is chromatographed from the sample pad to the chromatographic membrane under the capillary action of the absorbent paper and an antibody-antigen reaction occurs.

[0010] The second aspect of the present invention is to provide a method for preparing a serum amyloid A immunochromatographic detection reagent strip according to the first aspect, comprising:

[0011] S1, using quantum dot fluorescent microspheres to label human serum amyloid A antibody to form a quantum dot fluorescent microsphere labeled human serum amyloid A antibody solution; the quantum dot fluorescent microspheres have a diameter of 200-400 nm, an excitation light of 340 nm, an emission light of 610 nm, and a Stokes shift greater than 150 nm;

[0012] S2, using quantum dot fluorescent microspheres to label DNP-BSA to form a quantum dot fluorescent microsphere labeled DNP-BSA solution;

[0013] S3, solid phase treatment of the quantum dot fluorescent microsphere labeled serum amyloid A antibody solution;

[0014] S4, performing the serum amyloid A detection antibody coating and the rabbit anti-DNP quality control antibody coating;

[0015] S5, pretreating the sample pad based on a sample pad pretreatment solution; the sample pad pretreatment solution comprises: 10-50 mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA and 0.1-2% Tween 20, and the pH value of the sample pad pretreatment solution is 7.0-8.0;

[0016] S6, assembling the reagent strip, including: overlapping and pasting the sample pad, the conjugate pad, the chromatography pad and the absorbent paper on the reaction base plate in sequence, and maintaining an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the serum amyloid A immunochromatography detection reagent strip.

[0017] Preferably, the S1 comprises:

[0018] S11, using EDC and NHS to activate the carboxyl groups on the surface of the quantum dot fluorescent microspheres;

[0019] S12, add human serum amyloid A antibody and couple at room temperature for 1-3 hours;

[0020] S13, adding a microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to human serum amyloid A antibody;

[0021] S14, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled human serum amyloid A antibody solution.

[0022] Preferably, S2 includes:

[0023] S21, using EDC and NHS to activate the carboxyl groups on the surface of quantum dot fluorescent microspheres;

[0024] S22, add DNP-BSA and couple at room temperature for 1-3 hours;

[0025] S23, adding microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to DNP-BSA;

[0026] S24, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled DNP-BSA solution.

[0027] Preferably, the centrifugal speed in S1 and S2 is 10000-18000rpm, and the centrifugation time is 10-20 minutes; S1 and S2 react on a room temperature shaker, and the shaker speed is controlled at 100-200rpm; the addition of microsphere sealant for 1-2 hours in S13 and S23 includes: adding the microsphere sealant and reacting for 1-2 hours at a room temperature shaker speed of 100-200rpm; wherein the microsphere sealant is a synthetic non-protein and non-serum matrix high molecular water-soluble polymer containing an amino terminal, which can be fixed to the surface of the microsphere by chemical bonds with carboxyl groups, thereby achieving a blocking effect, and the blocking effect includes: blocking non-specific sites after microsphere coupling protein and antibody, thereby enhancing the activity of the conjugate protein or antibody, and can replace BSA to achieve a high signal-to-noise ratio.

[0028] Preferably, S3 includes:

[0029] S31, diluting the quantum dot fluorescent microsphere-labeled serum amyloid A antibody solution by 4 times with a microsphere storage solution to form a first dilution solution;

[0030] S32, mixing the first dilution solution and the quantum dot fluorescent microsphere labeled DNP-BSA solution in a ratio of 100:1 to form a first mixed solution;

[0031] S33, spraying the first mixed solution on the conjugate pad at 2-6 μ l / cm, and drying and pre-treating the conjugate pad to obtain the conjugate pad; the drying condition is 30-45° C. for 12-24 hours;

[0032] The conjugate pad pretreatment is carried out using a prepared conjugate pad pretreatment solution, which includes: 10-50 mmol / L PBS, 1%-20% sucrose, 0.5%-10% trehalose, 0.5%-5% BSA, 0.05%-0.2% PVP, 0.05%-0.2% PEG8000, 0.5%-5% SDS-L and 0.1-5% Tween 20, and the pH value of the conjugate pad pretreatment solution is 7.4; the conjugate pad pretreatment includes: laying the conjugate pad flat in the conjugate pad pretreatment solution and letting it stand for 1-5 minutes, and then drying it with air at a temperature range of 40-60°C for 1-2 hours.

[0033] Preferably, S4 includes:

[0034] S41, diluting the serum amyloid A detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml to form a second dilution solution and a third dilution solution respectively;

[0035] S42, spray the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and dry them to obtain a serum amyloid A detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying conditions are drying at 30-45°C for 12-24 hours.

[0036] The third aspect of the present invention is to provide a test kit for the serum amyloid A immunochromatographic test strip of the first aspect, comprising:

[0037] A test card, a sample addition hole and a sample diluent; wherein the test card includes the serum amyloid A immunochromatography reagent strip and a card shell loaded with the serum amyloid A immunochromatography reagent strip; the sample diluent is used to dilute whole blood or serum samples; and the sample addition hole is used to add diluted whole blood or serum samples.

[0038] The fourth aspect of the present invention provides a method for quantitatively detecting serum amyloid A using the detection kit of the third aspect, comprising:

[0039] Aspirating a quantitative whole blood or serum sample and diluting it with the sample diluent and then mixing it to form a diluted sample;

[0040] After adding the diluted sample to the sample well for a certain period of time to react, insert the test card into the card slot of the adapter instrument to obtain the test result;

[0041] The quantitative detection result of the serum amyloid A is obtained based on the detection result.

[0042] The fifth aspect of the present invention is to provide the use of quantum dot fluorescent microspheres as markers in the preparation of the serum amyloid A immunochromatographic detection reagent strip described in the first aspect for quantitatively detecting serum amyloid A; on the one hand, the quantum dot fluorescent microspheres are prepared by using amphiphilic macromolecules as carriers and embedding a large number of quantum dots in the microspheres by self-assembly, which improves the biocompatibility and coupling efficiency of the quantum dots while being simpler to use, and improves the fluorescent labeling efficiency, thereby effectively improving the analytical sensitivity; on the other hand, the surface of the quantum dot fluorescent microspheres is modified with suitable carboxyl groups or other functional groups for covalent coupling with proteins or antibodies, thereby improving the stability of the marker.

[0043] Beneficial effects of the method and system of the present invention:

[0044] It can effectively complete the evaluation of clinical patients' nutrition and metabolic status, and help clinical physicians judge the changes in critical illness and prognosis;

[0045] The present invention utilizes quantum dot fluorescent microspheres to label serum amyloid A antibodies for application in immunochromatographic detection. On the one hand, the quantum dot fluorescent microspheres are prepared by using amphiphilic macromolecules as carriers and embedding a large number of quantum dots in the microspheres by self-assembly. While improving the biocompatibility and coupling efficiency of the quantum dots, the use is simpler and the fluorescence labeling efficiency is improved, thereby effectively improving the analysis sensitivity. On the other hand, the surface of the quantum dot fluorescent microspheres is modified with suitable carboxyl groups or other functional groups for covalent coupling with proteins or antibodies, thereby improving the stability of the markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 A schematic diagram of the structure of a serum amyloid A immunochromatographic detection reagent strip provided according to an embodiment of the present invention;

[0048] Figure 2 This is the standard curve of the serum amyloid A standard substance in detection example 1 provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. 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.

[0050] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0051] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0052] The first aspect of the present invention is to provide a serum amyloid A immunochromatographic detection reagent strip for quantitatively detecting serum amyloid A based on quantum dot fluorescent microspheres, the serum amyloid A immunochromatographic detection reagent strip comprising a reaction base, a sample pad, a conjugation pad, a chromatographic membrane and absorbent paper; the chromatographic membrane is arranged in the middle of the reaction base, the sample pad and the absorbent paper are respectively arranged at both ends of the reaction base, the middle of the reaction base is the chromatographic membrane, the middle of the chromatographic membrane and the sample pad is the conjugation pad, the sample pad is partially overlapped on the conjugation pad, the conjugation pad is partially overlapped on one side of the chromatographic membrane, and the absorbent paper is partially overlapped on the other side of the chromatographic membrane;

[0053] The conjugate pad is solid-phased with quantum dot fluorescent microspheres for labeling serum amyloid A antibody and quantum dot fluorescent microspheres for labeling DNP-BSA;

[0054] The chromatographic membrane is provided with a detection line coated with serum amyloid A detection antibody and a quality control line coated with rabbit anti-DNP quality control antibody; the detection line and the quality control line are arranged in parallel and at intervals in the chromatographic direction on the chromatographic membrane.

[0055] As a preferred embodiment, the serum amyloid A antibody is obtained by immunizing mice with human serum amyloid A.

[0056] As a preferred embodiment, the chromatographic membrane is a nitrocellulose membrane, which has good chromatographic performance and strong protein adsorption capacity; the absorbent paper is made of cotton and linen, which can enable the sample to be chromatographed from the sample pad to the chromatographic membrane under the capillary action of the absorbent paper and produce an antibody-antigen reaction.

[0057] The second aspect of the present invention is to provide a method for preparing a serum amyloid A immunochromatographic detection reagent strip according to the first aspect, comprising:

[0058] S1, using quantum dot fluorescent microspheres to label serum amyloid A antibody to form a quantum dot fluorescent microsphere labeled serum amyloid A antibody solution;

[0059] As a preferred embodiment, the S1 includes:

[0060] S11, using EDC and NHS to activate the carboxyl groups on the surface of the quantum dot fluorescent microspheres;

[0061] Specifically, the quantum dot fluorescent microspheres are washed and resuspended with a boric acid buffer having a pH value in the range of 6.0-8.2; then, activation reagents EDC and NHS are added to the surface of the quantum dot fluorescent microspheres, reacted at room temperature for 20-60 minutes, centrifuged to remove the supernatant, and then resuspended with a boric acid buffer;

[0062] S12, add serum amyloid A antibody and couple at room temperature for 1-3 hours;

[0063] S13, adding a microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to the serum amyloid A antibody;

[0064] S14, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled serum amyloid A antibody solution.

[0065] S2, using quantum dot fluorescent microspheres to label DNP-BSA to form a quantum dot fluorescent microsphere labeled DNP-BSA solution;

[0066] As a preferred embodiment, the S2 includes:

[0067] S21, using EDC and NHS to activate the carboxyl groups on the surface of quantum dot fluorescent microspheres;

[0068] Specifically, the quantum dot fluorescent microspheres are washed and resuspended with a MES buffer having a pH value of 6.0; then, activation reagents EDC and NHS are added to the surface of the quantum dot fluorescent microspheres, reacted at room temperature for 20-60 minutes, centrifuged to remove the supernatant, and then resuspended with a boric acid buffer;

[0069] S22, add DNP-BSA and couple at room temperature for 1-3 hours;

[0070] S23, adding microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to DNP-BSA;

[0071] S24, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled DNP-BSA solution.

[0072] As a preferred embodiment, the centrifugal speed in S1 and S2 is 10000-18000 rpm, and the centrifugal time is 10-20 minutes. The use of this centrifugal technical parameter scheme can reduce the loss rate of raw materials.

[0073] As a preferred embodiment, the S1 and the S2 are reacted on a shaker at room temperature, and the rotation speed of the shaker is controlled at 100-200 rpm; using this technical solution, the present invention fully mixes the serum amyloid A antibody and the quantum dot fluorescent microspheres under certain conditions, and the DNP-BSA and the quantum dot fluorescent microspheres are also fully mixed, thereby improving the coupling efficiency of the antibody and DNP-BSA.

[0074] As a preferred embodiment, the addition of the microsphere sealant for 1-2 hours in S13 and S23 includes: adding the microsphere sealant and reacting for 1-2 hours at a room temperature shaker speed of 100-200rpm. The purpose of adopting this technical solution is to reduce the non-specific binding of antibodies; wherein the microsphere sealant is a synthetic non-protein and non-serum matrix high molecular water-soluble polymer containing an amino terminal, which can be fixed to the surface of the microsphere by chemical bonds with carboxyl groups, thereby achieving a blocking effect, and the blocking effect includes: blocking non-specific sites after microsphere coupling protein and antibody, thereby enhancing the activity of the conjugate (protein or antibody), and can replace BSA to achieve a high signal-to-noise ratio.

[0075] S3, solid phase treatment of the quantum dot fluorescent microsphere labeled serum amyloid A antibody solution;

[0076] S31, diluting the quantum dot fluorescent microsphere-labeled serum amyloid A antibody solution by 4 times with a microsphere storage solution to form a first dilution solution;

[0077] S32, mixing the first dilution solution and the quantum dot fluorescent microsphere labeled DNP-BSA solution in a ratio of 100:1 to form a first mixed solution;

[0078] S33, spraying the first mixed solution on the conjugate pad at 2-6 μ l / cm, and drying and pre-treating the conjugate pad to obtain the conjugate pad; the drying condition is 30-45° C. for 12-24 hours.

[0079] As a preferred embodiment, the conjugate pad pretreatment is performed using a prepared conjugate pad pretreatment solution, the conjugate pad pretreatment solution comprising: 10-50 mmol / L PBS, 1%-20% sucrose, 0.5%-10% trehalose, 0.5%-5% BSA, 0.05%-0.2% PVP, 0.05%-0.2% PEG8000 and 0.1-5% Tween 20, and the pH value of the conjugate pad pretreatment solution is 7.4; the conjugate pad pretreatment comprises: laying the conjugate pad flat in the conjugate pad pretreatment solution and letting it stand for 1-5 minutes, and then air drying it at a temperature range of 40-60°C for 1-2 hours.

[0080] S4, performing the serum amyloid A detection antibody coating and the rabbit anti-DNP quality control antibody coating;

[0081] As a preferred implementation, the S4 includes:

[0082] S41, diluting the serum amyloid A detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml to form a second dilution solution and a third dilution solution respectively;

[0083] S42, spray the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and dry them to obtain a serum amyloid A detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying conditions are drying at 30-45°C for 12-24 hours.

[0084] S5, pretreating the sample pad based on a sample pad pretreatment solution; the sample pad pretreatment solution comprises: 10-50 mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA and 0.1-2% Tween 20, and the pH value of the sample pad pretreatment solution is 7.0-8.0.

[0085] S6, assembling the reagent strip, including: overlapping and pasting the sample pad, the conjugate pad, the chromatography pad and the absorbent paper on the reaction base plate in sequence, and maintaining an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the serum amyloid A immunochromatography detection reagent strip.

[0086] The third aspect of the present invention is to provide a test kit for the serum amyloid A immunochromatographic test strip of the first aspect, comprising:

[0087] A test card, a sample addition hole and a sample diluent; wherein the test card includes the serum amyloid A immunochromatography reagent strip and a card shell loaded with the serum amyloid A immunochromatography reagent strip; the sample diluent is used to dilute whole blood or serum samples; and the sample addition hole is used to add diluted whole blood or serum samples.

[0088] The fourth aspect of the present invention provides a method for quantitatively detecting serum amyloid A using the serum amyloid A immunochromatography reagent strip, comprising:

[0089] Aspirating a quantitative whole blood or serum sample and diluting it with the sample diluent and then mixing it to form a diluted sample;

[0090] After adding the diluted sample to the sample well for a certain period of time to react, insert the test card into the card slot of the adapter instrument to obtain the test result;

[0091] The quantitative detection result of the serum amyloid A is obtained based on the detection result.

[0092] In this embodiment, 5 μl of whole blood or serum sample is taken and added to sample diluent 1. After mixing, 50 μl is added to sample diluent 2. After mixing, 100 μl of sample diluent 2 is added to the sample well. After 20 minutes, the test card is inserted into the card slot of the adapter instrument to obtain the quantitative test results of serum amyloid A.

[0093] The fifth aspect of the present invention is to provide the use of quantum dot fluorescent microspheres as markers in the preparation of the serum amyloid A immunochromatographic detection reagent strip described in the first aspect for quantitative detection of serum amyloid A. On the one hand, the quantum dot fluorescent microspheres are prepared by embedding a large number of quantum dots in the microspheres by self-assembly using amphiphilic macromolecules as carriers, which improves the biocompatibility and coupling efficiency of the quantum dots, is simpler to use, improves the fluorescent labeling efficiency, and effectively improves the analytical sensitivity. On the other hand, the surface of the quantum dot fluorescent microspheres is modified with suitable carboxyl groups or other functional groups for covalent coupling with proteins or antibodies, thereby improving the stability of the marker.

[0094] The present invention discloses a quantitative detection serum amyloid A immunochromatographic reagent strip based on quantum dot fluorescent microspheres prepared by the following example, and the raw materials used are as follows:

[0095] The serum amyloid A marker antibody of the present application is provided by Nanjing Hanrui Boco Biotechnology Co., Ltd., with the product number of Med ix 100279-A;

[0096] The serum amyloid A detection antibody of the present application is provided by Nanjing Hanruibaiko Biotechnology Co., Ltd., with the product number of Medix 100289-A;

[0097] The DNP-BSA of the present application is produced by Beijing InnoWorld Biotechnology Co., Ltd., with the product number of Inno681;

[0098] The rabbit anti-DNP antibody of the present application is produced by Beijing InnoWorld Biotechnology Co., Ltd., with the product number Inno0532.

[0099] The microsphere sealant of this application is produced by Beijing InnoWorld Biotechnology Co., Ltd., with the product number Inno5150

[0100] The nitrocellulose membrane used in this application was purchased from Runhe Biomedical Technology (Shantou) Co., Ltd. It has a white appearance, uniform texture, a flat and flawless membrane surface, and neat and non-serrated membrane edges.

[0101] The glass fiber used in this application was purchased from Shanghai Jinbiao Biotechnology Co., Ltd., and its fiber structure is evenly distributed, dense, and free of dirt, dust, and stains.

[0102] Example 1 Immunochromatographic reagent strip for quantitative detection of serum amyloid protein A

[0103] Part I: Composition of immunochromatographic reagent strips for quantitative detection of serum amyloid A based on quantum dot fluorescent microspheres

[0104] The composition and structure of the immunochromatographic reagent strip for quantitative detection of serum amyloid A in this embodiment are as follows: Figure 1 As shown, specifically, the detection reagent strip is composed of a reaction base plate 7, a sample pad 1, a conjugation pad 2, a chromatography membrane 5 and absorbent paper 6, wherein the sample pad 1 and the absorbent paper 6 are respectively pasted on both ends of the reaction base plate; the middle part of the reaction base plate is the chromatography membrane 5, and the middle part of the chromatography membrane and the sample pad is the conjugation pad 2, wherein the sample pad 1 is partially superimposed on the conjugation pad 2, the conjugation pad is partially superimposed on the chromatography membrane 5, and the absorbent paper 6 is partially superimposed on the chromatography membrane 5; the detection line 3 and the quality control line 4 are arranged in parallel and at intervals along the chromatography direction on the chromatography membrane, the detection line is coated with serum amyloid A antibody, and the quality control line is coated with rabbit anti-DNP antibody.

[0105] Part II: Preparation of immunochromatographic reagent strips for quantitative detection of serum amyloid A based on quantum dot fluorescent microspheres

[0106] The following steps are involved:

[0107] Method for labeling serum amyloid A antibody with quantum dot fluorescent microspheres:

[0108] S1, Activation

[0109] Take 50ul of quantum dot fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 450ul of boric acid buffer, mix well, add 20ugEDC and 20ugNHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 500ul of boric acid buffer and mix well.

[0110] S2, coupling

[0111] Add 50ug serum amyloid A antibody to the microsphere solution activated in S1, mix well, mark on a shaker at room temperature for 2 hours, and disperse the microspheres by ultrasound to avoid microsphere aggregation;

[0112] S3, closed

[0113] Add 50 μl of microsphere blocking agent to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10,000 rpm for 10 minutes, discard the supernatant, add 2,000 μl of microsphere preservation solution, disperse the microspheres, and obtain a serum amyloid A microsphere solution;

[0114] Method for labeling DNP-BSA with quantum dot fluorescent microspheres:

[0115] S1, Activation

[0116] Take 20ul of quantum dot fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 180ul of boric acid buffer, mix well, add 5ugEDC and 5ugNHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 200ul of MES buffer and mix well.

[0117] S2, coupling

[0118] Add 20ug DNP-BSA to the microsphere solution activated in S1, mix well, and place on a shaker at room temperature in the dark for 1.5 hours. Disperse the microspheres with ultrasound to prevent aggregation of the microspheres.

[0119] S3, closed

[0120] Add 20 μl of microsphere blocking agent to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10,000 rpm for 10 minutes, discard the supernatant, add 200 μl of microsphere preservation solution, disperse the microspheres, and obtain a DNP-BSA microsphere solution;

[0121] The serum amyloid A binding pad solid phase method is:

[0122] Add 20ul DNP-BSA microsphere solution to 2000ul serum amyloid A microsphere solution, mix well, spray on the pretreated conjugate pad at 4ul / cm, and dry at 37°C for 24 hours to obtain a serum amyloid A conjugate pad;

[0123] Coating method of serum amyloid A antibody:

[0124] Serum amyloid A antibody was diluted to 1.0 mg / ml, and rabbit anti-DNP antibody was diluted to 1.0 mg / ml, and coated on the chromatographic membrane with a spray volume of 1 ul / cm to form a serum amyloid A detection line and a rabbit anti-DNP quality control line, which were placed at 37°C to dry for 24 hours to obtain a serum amyloid A chromatographic membrane.

[0125] Reagent strip assembly method:

[0126] The sample pad, the above-mentioned binding pad, the chromatography pad and the absorbent paper are overlapped and pasted on the reaction bottom plate in sequence, and the overlap is maintained at 1-2 mm, and the reagent strips are cut into 4 mm width using a strip cutter.

[0127] Test Example 1

[0128] The quantitative detection of serum amyloid A immunochromatography reagent strip based on quantum dot fluorescent microspheres prepared in Example 1 was used to detect standard samples of different concentrations (seven different concentrations, namely 2.5, 5, 10, 25, 50, 100, and 200 mg / L serum amyloid A standards), and 5 repeated tests were performed for each concentration to obtain the average value. 5 μl of sample was drawn and added to the sample diluent. After mixing, 100 μl of sample diluent was added to the sample well. After 15 minutes, the test card was inserted into the card slot of the adapter instrument to read the results. The ratio of the fluorescence intensity of the test line and the quality control line and the concentration of the calibration solution were used to draw the serum amyloid A standard curve, as shown in FIG. Figure 2 As shown; the standard curve is a 4-parameter equation, the signal value is the independent variable, the concentration value is the dependent variable, and the data is shown in Table 1. Standard curve data of serum amyloid A in detection example 1.

[0129] Standard curve equation correlation coefficient R 2 The standard curve has a good fit and can be used as a calibration formula.

[0130] Table 1

[0131]

[0132]

[0133] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A serum amyloid protein A immunochromatographic detection reagent strip, characterized in that: It comprises a reaction bottom plate, a sample pad, a conjugation pad, a chromatography membrane and absorbent paper; the chromatography membrane is arranged in the middle of the reaction bottom plate, the sample pad and the absorbent paper are arranged at both ends of the reaction bottom plate respectively, the chromatography membrane is in the middle of the reaction bottom plate, the conjugation pad is between the chromatography membrane and the sample pad, the sample pad is partially overlapped on the conjugation pad, the conjugation pad is partially overlapped on one side of the chromatography membrane, and the absorbent paper is partially overlapped on the other side of the chromatography membrane; The conjugate pad is solid-phased with quantum dot fluorescent microspheres for labeling serum amyloid A antibody and quantum dot fluorescent microspheres for labeling DNP-BSA; The chromatographic membrane is provided with a detection line coated with serum amyloid A detection antibody and a quality control line coated with rabbit anti-DNP quality control antibody; the detection line and the quality control line are arranged in parallel and at intervals in the chromatographic direction on the chromatographic membrane.

2. The serum amyloid A immunochromatographic detection reagent strip according to claim 1, characterized in that: The serum amyloid A antibody is obtained by immunizing mice with human serum amyloid A; the chromatographic membrane is a nitrocellulose membrane; the absorbent paper is made of cotton and linen, so that the sample is chromatographed from the sample pad to the chromatographic membrane under the capillary action of the absorbent paper and an antibody-antigen reaction occurs.

3. A method for preparing a serum amyloid A immunochromatographic detection reagent strip according to any one of claims 1 to 2, characterized in that: include: S1, using quantum dot fluorescent microspheres to label serum amyloid A antibody to form a quantum dot fluorescent microsphere labeled serum amyloid A antibody solution; S2, using quantum dot fluorescent microspheres to label DNP-BSA to form a quantum dot fluorescent microsphere labeled DNP-BSA solution; S3, solid phase treatment of the quantum dot fluorescent microsphere labeled serum amyloid A antibody solution; S4, performing the serum amyloid A detection antibody coating and the rabbit anti-DNP quality control antibody coating; S5, pretreating the sample pad based on a sample pad pretreatment solution; The sample pad pretreatment solution comprises: 10-50 mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA and 0.1-2% Tween 20, and the pH value of the sample pad pretreatment solution is 7.0-8.0; S6, assembling the reagent strip, including: overlapping and pasting the sample pad, the conjugate pad, the chromatography pad and the absorbent paper on the reaction base plate in sequence, and maintaining an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the serum amyloid A immunochromatography detection reagent strip.

4. The method for preparing the serum amyloid A immunochromatographic detection reagent strip according to claim 3, characterized in that: The S1 includes: S11, using EDC and NHS to activate the carboxyl groups on the surface of the quantum dot fluorescent microspheres; S12, add serum amyloid A antibody and couple at room temperature for 1-3 hours; S13, adding a microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to the serum amyloid A antibody; S14, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled serum amyloid A antibody solution.

5. The method for preparing the serum amyloid A immunochromatographic detection reagent strip according to claim 4, characterized in that: The S2 includes: S21, using EDC and NHS to activate the carboxyl groups on the surface of quantum dot fluorescent microspheres; S22, add DNP-BSA and couple at room temperature for 1-3 hours; S23, adding microsphere blocking agent to block for 1-2 hours, thereby blocking the carboxyl groups that are not coupled to DNP-BSA; S24, centrifuging the blocked solution to remove the supernatant, and adding microsphere preservation solution to form a quantum dot fluorescent microsphere labeled DNP-BSA solution.

6. The method for preparing the serum amyloid A immunochromatographic detection reagent strip according to claim 5, characterized in that: The centrifugal speed in S1 and S2 is 10000-18000rpm, and the centrifugation time is 10-20 minutes; S1 and S2 react on a room temperature shaker, and the shaker speed is controlled at 100-200rpm; the addition of microsphere sealant for 1-2 hours in S13 and S23 includes: adding the microsphere sealant and reacting for 1-2 hours at a room temperature shaker speed of 100-200rpm; wherein the microsphere sealant is a synthetic non-protein and non-serum matrix high molecular water-soluble polymer containing an amino terminal, which can be fixed to the surface of the microsphere by chemical bonds with the carboxyl group, thereby achieving a blocking effect, and the blocking effect includes: blocking non-specific sites after microsphere coupling protein and antibody, thereby enhancing the activity of the conjugate protein or antibody, and can replace BSA to achieve a high signal-to-noise ratio.

7. The method for preparing the serum amyloid A immunochromatographic detection reagent strip according to claim 6, characterized in that: The S3 includes: S31, diluting the quantum dot fluorescent microsphere-labeled serum amyloid A antibody solution by 4 times with a microsphere storage solution to form a first dilution solution; S32, mixing the first dilution solution and the quantum dot fluorescent microsphere labeled DNP-BSA solution in a ratio of 100:1 to form a first mixed solution; S33, spraying the first mixed solution onto the conjugate pad at 2-6 ul / cm, and drying and pre-treating the conjugate pad to obtain the conjugate pad; the drying condition is 30-45° C. for 12-24 hours; The conjugate pad pretreatment is carried out using a prepared conjugate pad pretreatment solution, which includes: 10-50mmol / L PBS, 1%-20% sucrose, 0.5%-10% trehalose, 0.5%-5% BSA, 0.05%-0.2% PVP, 0.05%-0.2% PEG8000, 0.5%-5% SDS-L and 0.1-5% Tween 20, and the pH value of the conjugate pad pretreatment solution is 7.4; the conjugate pad pretreatment includes: laying the conjugate pad flat in the conjugate pad pretreatment solution and letting it stand for 1-5 minutes, and then air drying it at a temperature range of 40-60°C for 1-2 hours.

8. The method for preparing the serum amyloid A immunochromatographic detection reagent strip according to claim 7, characterized in that: The S4 includes: S41, diluting the serum amyloid A detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml to form a second dilution solution and a third dilution solution respectively; S42, spray the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and dry them to obtain a serum amyloid A detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying conditions are drying at 30-45°C for 12-24 hours.

9. A detection kit based on the serum amyloid A immunochromatographic detection reagent strip according to any one of claims 1-2, characterized in that: include: A test card, a sample addition hole and a sample diluent; wherein the test card includes the serum amyloid A immunochromatography reagent strip and a card shell loaded with the serum amyloid A immunochromatography reagent strip; the sample diluent is used to dilute whole blood or serum samples; and the sample addition hole is used to add diluted whole blood or serum samples.

10. A method for quantitatively detecting serum amyloid A based on the detection kit according to claim 9, characterized in that: include: Aspirating a quantitative whole blood or serum sample and diluting it with the sample diluent and then mixing it to form a diluted sample; After adding the diluted sample to the sample well for a certain period of time to react, insert the test card into the card slot of the adapter instrument to obtain the test result; The quantitative detection result of the serum amyloid A is obtained based on the detection result.

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