Preparation method and application of prealbumin immunochromatography detection reagent strip

Abstract

The prealbumin immunochromatography detection reagent strip 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 respectively arranged at two ends of the reaction bottom plate, 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 and overlapped on the combination pad, and the combination pad and the absorbent paper are respectively partially overlapped and overlapped on one side and the other side of the chromatography membrane; time-resolved microspheres used for prealbumin antibody labeling and quantum dot 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 prealbumin 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 also discloses a corresponding preparation method, a detection kit, a detection method and application. High sensitivity, rapidness, convenience and accurate quantification are realized by utilizing the sensitivity of time-resolved microsphere immunochromatography.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay analysis, and in particular to a preparation method and application of a prealbumin immunochromatographic detection reagent strip. Background Art

[0002] Prealbumin (PA) is a serum protein synthesized by hepatocytes. It is a tetramer composed of four identical subunits with a relative molecular weight of 61,000 and a short biological half-life (1.9 days). At present, it is mainly used in the clinic to evaluate liver damage and malnutrition. It is also a sensitive indicator of liver damage caused by drug poisoning. Prealbumin is a nonspecific host defense substance that can clear some toxic metabolites released into the circulation during infection. It has been confirmed that serum prealbumin levels decrease during inflammation and bacterial infection, so it can be used as an indicator to evaluate the changes in the condition and treatment effects of infectious diseases. In addition, prealbumin can enhance immunity by promoting lymphocyte maturation, which is of great value in the early prediction of tumors.

[0003] Currently known methods for measuring prealbumin include: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunoturbidimetry, etc. These methods all have the disadvantages of complicated operation, special equipment, and sample pretreatment. For example, immunoturbidimetry is a detection method that has been used more in recent years, such as patent documents CN101000349A and CN107490697A, but it requires a fully automatic biochemical analyzer, the instrument cost is very high, and it can only test serum or plasma samples, requires sample pretreatment, and has high professional requirements for personnel. The quantitative detection of prealbumin using time-resolved microsphere immunochromatography technology has the advantages of delicate and light detection instruments, simple and rapid operation, and accurate results, and can test whole blood samples, serum samples, and plasma samples. However, there is no detection kit for this technology on the market.

[0004] Therefore, developing a prealbumin product that is simple to operate, has low instrument cost, and is suitable for on-site instant testing 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. In view of the technical defects in the related art, a preparation method and application of a prealbumin immunochromatographic detection reagent strip is provided. The prealbumin immunochromatographic detection reagent strip is an immunochromatographic reagent strip for quantitatively detecting prealbumin based on time-resolved microspheres. The sensitivity of time-resolved microsphere immunochromatography is utilized in combination with a fluorescent immunoassay analyzer to achieve a prealbumin detection reagent strip and preparation method with high sensitivity, quickness, convenience and accurate quantification.

[0006] The first aspect of the present invention is to provide a prealbumin immunochromatographic detection reagent strip for quantitative detection of prealbumin based on time-resolved microspheres, the prealbumin 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 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;

[0007] The conjugate pad is solid-phased with time-resolved microspheres for prealbumin antibody labeling and quantum dot microspheres for DNP-BSA labeling;

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

[0009] Preferably, the prealbumin antibody is obtained by immunizing mice with human prealbumin; 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.

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

[0011] S1, using time-resolved microspheres to label prealbumin antibodies to form a time-resolved microsphere-labeled prealbumin antibody solution; the time-resolved 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 microspheres to label DNP-BSA to form a quantum dot microsphere labeled DNP-BSA solution;

[0013] S3, solid phase treatment of the time-resolved microsphere-labeled prealbumin antibody solution;

[0014] S4, performing the coating of the prealbumin detection antibody and the rabbit anti-DNP quality control antibody;

[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, 0.1%-2% S9 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 keeping an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the prealbumin immunochromatography detection reagent strip.

[0017] Preferably, the S1 comprises:

[0018] S11, activating the carboxyl groups on the surface of the time-resolved microspheres using EDC and NHS;

[0019] S12, add prealbumin antibody and couple at room temperature for 1-3 hours;

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

[0021] S14, centrifuging the blocked solution to remove the supernatant, and adding microsphere storage solution to form a time-resolved microsphere-labeled prealbumin antibody solution.

[0022] Preferably, S2 includes:

[0023] S21, using EDC and NHS to activate the carboxyl groups on the surface of quantum dot 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 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 time-resolved microsphere-labeled prealbumin antibody solution 4 times with a microsphere storage solution to form a first dilution solution;

[0030] S32, mixing the first dilution solution and the quantum dot microsphere labeled DNP-BSA solution in a ratio of 100:(0.5-2) 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 performed 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% PEG2000, 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.

[0033] Preferably, S4 includes:

[0034] S41, diluting the prealbumin 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, spraying the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and drying to obtain a prealbumin detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying condition is drying at 30-45°C for 12-24 hours.

[0036] The third aspect of the present invention is to provide a detection kit for the prealbumin immunochromatographic detection reagent strip of the first aspect, comprising:

[0037] A test card, a sample addition hole and a sample diluent; wherein the test card comprises the prealbumin immunochromatography reagent strip and a card shell loaded with the prealbumin 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 prealbumin 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] Based on the test result, the quantitative test result of the prealbumin and the judgment result of whether the prealbumin is in the normal reference interval are obtained, wherein the content range of the prealbumin in the normal reference interval is: 200-400 mg / L.

[0042] The fifth aspect of the present invention is to provide the use of time-resolved microspheres as markers in the preparation of the prealbumin immunochromatographic detection reagent strip described in the first aspect for quantitative detection of prealbumin; wherein: the diameter of the time-resolved microspheres is 200-400nm, the excitation light is 340nm, the emission light is 610nm, and the Stokes shift is greater than 150nm.

[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 time-resolved microspheres to label prealbumin antibodies for application in immunochromatographic detection. On the one hand, as a special functional microsphere, the time-resolved microspheres can encapsulate tens of thousands of fluorescent molecules in each microsphere, which greatly improves the fluorescent labeling efficiency and effectively improves the analysis sensitivity. On the other hand, the surface of the time-resolved 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 prealbumin immunochromatographic detection reagent strip provided according to an embodiment of the present invention;

[0048] Figure 2 This is the standard curve of the prealbumin standard 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 prealbumin immunochromatographic detection reagent strip for quantitative detection of prealbumin based on time-resolved microspheres, the prealbumin 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 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 time-resolved microspheres for prealbumin antibody labeling and quantum dot microspheres for DNP-BSA labeling;

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

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

[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 prealbumin immunochromatographic detection reagent strip according to the first aspect, comprising:

[0058] S1, using time-resolved microspheres to label prealbumin antibodies to form a time-resolved microsphere-labeled prealbumin antibody solution;

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

[0060] S11, activating the carboxyl groups on the surface of the time-resolved microspheres using EDC and NHS;

[0061] Specifically, the time-resolved 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 time-resolved 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 prealbumin antibody and couple at room temperature for 1-3 hours;

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

[0064] S14, centrifuging the blocked solution to remove the supernatant, and adding microsphere storage solution to form a time-resolved microsphere-labeled prealbumin antibody solution.

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

[0066] As a preferred embodiment, S2 includes:

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

[0068] Specifically, the quantum dot microspheres are washed and resuspended using a MES buffer having a pH value of 6.0; then, activation reagents EDC and NHS are added to the surface of the time-resolved microspheres, reacted at room temperature for 20-60 minutes, centrifuged to remove the supernatant, and resuspended using 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 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 prealbumin antibody and the time-resolved microspheres under certain conditions, and the DNP-BSA and the quantum dot 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 time-resolved microsphere-labeled prealbumin antibody solution;

[0076] S31, diluting the time-resolved microsphere-labeled prealbumin antibody solution 4 times with a microsphere storage solution to form a first dilution solution;

[0077] S32, mixing the first dilution solution and the quantum dot microsphere labeled DNP-BSA solution in a ratio of 100:(0.5-2) 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% PEG2000, 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 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 coating of the prealbumin detection antibody and the rabbit anti-DNP quality control antibody;

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

[0082] S41, diluting the prealbumin 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, spraying the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and drying to obtain a prealbumin detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying condition is 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, 0.1%-2% S9 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 keeping an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the prealbumin immunochromatography detection reagent strip.

[0086] The third aspect of the present invention is to provide a detection kit for the prealbumin immunochromatographic detection reagent strip of the first aspect, comprising:

[0087] A test card, a sample addition hole and a sample diluent; wherein the test card comprises the prealbumin immunochromatography reagent strip and a card shell loaded with the prealbumin 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 prealbumin using the prealbumin immunochromatographic 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] Based on the test result, the quantitative test result of the prealbumin and the judgment result of whether the prealbumin is in the normal reference interval are obtained, wherein the content range of the prealbumin in the normal reference interval is: 200-400 mg / L.

[0092] In this embodiment, 5 μl of whole blood or serum sample is taken and added to sample diluent 1, mixed, 50 μl is added to sample diluent 2, mixed, 100 μl of sample diluent 2 is added to the sample well, and after 20 minutes, the test card is inserted into the card slot of the adapter instrument to obtain the quantitative test results of prealbumin and the judgment result of whether prealbumin is in the normal reference range. Normal reference range: prealbumin 200-400 mg / L.

[0093] The fifth aspect of the present invention is to provide the use of time-resolved microspheres as markers in the preparation of the prealbumin immunochromatographic detection reagent strip described in the first aspect for quantitative detection of prealbumin; wherein: the diameter of the time-resolved microspheres is 200-400nm, the excitation light is 340nm, the emission light is 610nm, the Stokes shift is greater than 150nm, the signal interference between the absorption spectrum and the emission spectrum is small, and the detection instrument can more accurately monitor the fluorescence signal.

[0094] The present invention discloses a quantitative detection of prealbumin immunochromatographic reagent strip based on time-resolved microspheres prepared by the following example, and the raw materials used are as follows:

[0095] The prealbumin antibody of the present application is provided by Shanghai Medis Medical Technology Co., Ltd., with the catalog number Med ix100830;

[0096] The prealbumin detection antibody of the present application is provided by Shanghai Medix Medical Technology Co., Ltd., with the catalog number Medix100828;

[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 prealbumin

[0103] Part I: Composition of the immunochromatographic strip for quantitative detection of prealbumin based on time-resolved microspheres

[0104] The composition and structure of the immunochromatographic reagent strip for quantitative detection of prealbumin 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 attached to 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 prealbumin 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 prealbumin based on time-resolved microspheres

[0106] The following steps are involved:

[0107] 1. Method of time-resolved microsphere labeling of prealbumin antibody:

[0108] S1, Activation

[0109] Take 50ul of time-resolved microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 450ul of boric acid buffer, mix well, add 50ugEDC and 50ugNHS, activate for 40min, 15000rpm, 15 minutes, discard the supernatant, add 500ul of boric acid buffer and mix well.

[0110] S2, coupling

[0111] Add 50ug of prealbumin 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 prealbumin microsphere solution;

[0114] 2. Method of labeling DNP-BSA with quantum dot microspheres:

[0115] S1, Activation

[0116] Take 20ul of quantum dot microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 180ul of boric acid buffer, mix well, add 10ugEDC and 10ugNHS, 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] 3. The solid phase method of prealbumin binding pad is:

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

[0123] 4. Coating method of prealbumin antibody:

[0124] Prealbumin antibody was diluted to 1.5 mg / ml, and rabbit anti-DNP antibody was diluted to 1.0 mg / ml. The prealbumin antibody and rabbit anti-DNP antibody were coated on the chromatographic membrane at a spray volume of 1 u l / cm to form a prealbumin detection line and a rabbit anti-DNP quality control line. The prealbumin chromatographic membrane was dried at 37°C for 24 hours to obtain a prealbumin chromatographic membrane.

[0125] 5. 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 prealbumin immunochromatographic reagent strip based on time-resolved microspheres prepared in Example 1 was used to detect standard samples of different concentrations (7 different concentrations, 25.93, 49.3, 106.7, 195, 292, 400, 575 mg / L of prealbumin standard), and 5 repeated tests were performed for each concentration to obtain the average value. 5 μl of sample was taken and added to sample diluent 1. After mixing, 50 μl was added to sample diluent 2, mixed, and 100 μl of sample diluent 2 was added to the sample well. After 20 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 was used to draw the prealbumin 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 can be found in Table 1 for the standard curve data of prealbumin 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 prealbumin immunochromatographic detection reagent strip, characterized in that: The prealbumin immunochromatographic detection reagent strip comprises 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 arranged at both ends of the reaction base plate respectively, the middle of the reaction base plate is the chromatographic membrane, the conjugation pad is between the chromatographic 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 chromatographic membrane, and the absorbent paper is partially overlapped on the other side of the chromatographic membrane; The conjugate pad is solid-phased with time-resolved microspheres for prealbumin antibody labeling and quantum dot microspheres for DNP-BSA labeling; The chromatographic membrane is provided with a detection line coated with a prealbumin detection antibody and a quality control line coated with a rabbit anti-DNP quality control antibody; the detection line and the quality control line are sequentially arranged in parallel and at intervals along the chromatographic direction on the chromatographic membrane.

2. A prealbumin immunochromatographic detection reagent strip according to claim 1, characterized in that: The prealbumin antibody is obtained by immunizing mice with human prealbumin; 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 the prealbumin immunochromatographic detection reagent strip according to any one of claims 1 to 2, characterized in that: include: S1, using time-resolved microspheres to label prealbumin antibodies to form a time-resolved microsphere-labeled prealbumin antibody solution; the time-resolved 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; S2, using quantum dot microspheres to label DNP-BSA to form a quantum dot microsphere labeled DNP-BSA solution; S3, solid phase treatment of the time-resolved microsphere-labeled prealbumin antibody solution; S4, performing the coating of the prealbumin detection antibody and the rabbit anti-DNP quality control antibody; 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, 0.1%-2% S9 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 keeping an overlap of 1-2 mm, and using a strip cutter to cut into 4 mm width to form the prealbumin immunochromatography detection reagent strip.

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

5. The method for preparing the prealbumin 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 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 microsphere labeled DNP-BSA solution.

6. The method for preparing the prealbumin 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 prealbumin immunochromatographic detection reagent strip according to claim 6, characterized in that: The S3 includes: S31, diluting the time-resolved microsphere-labeled prealbumin antibody solution 4 times with a microsphere storage solution to form a first dilution solution; S32, mixing the first dilution solution and the quantum dot microsphere labeled DNP-BSA solution in a ratio of 100:(0.5-2) 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% PEG2000, 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 prealbumin immunochromatographic detection reagent strip according to claim 7, characterized in that: The S4 includes: S41, diluting the prealbumin 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, spraying the second diluent and the third diluent on the chromatographic membrane with a spray volume of 0.5-2.0ul / cm and drying to obtain a prealbumin detection line, a rabbit anti-DNP quality control line and the chromatographic membrane; the drying condition is drying at 30-45°C for 12-24 hours.

9. A detection kit based on the prealbumin 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 comprises the prealbumin immunochromatography reagent strip and a card shell loaded with the prealbumin 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 prealbumin 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; Based on the test result, the quantitative test result of the prealbumin and the judgment result of whether the prealbumin is in the normal reference interval are obtained, wherein the content range of the prealbumin in the normal reference interval is: 200-400 mg / L.

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