Prealbumin and C-reactive protein joint detection kit and preparation method thereof

Abstract

The prealbumin and C-reactive protein combined detection kit comprises a detection card, an IC card, a sample adding hole, a card reading window and a sample diluent, the detection card comprises a prealbumin detection reagent strip, a C-reactive protein detection reagent strip and a card shell for loading the prealbumin detection reagent strip and the C-reactive protein detection reagent strip; each of the prealbumin detection reagent strip and the C-reactive protein detection reagent strip comprises a reaction bottom plate, a sample pad, a combination pad, a chromatography membrane and absorbent paper, and a prealbumin monoclonal antibody marked by fluorescent microspheres or a C-reactive protein monoclonal antibody marked by fluorescent microspheres and DNP-BSA marked by fluorescent microspheres are correspondingly adopted; a quality control line and a detection line are arranged on the chromatography membrane; the sample adding holes comprise a prealbumin sample adding hole and a C reactive protein sample adding hole; the sample diluent is used for diluting a whole blood or serum sample and comprises a prealbumin sample diluent and a C-reactive protein sample diluent. Corresponding methods of preparation and methods of use are also disclosed.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a prealbumin and C-reactive protein combined detection kit and a preparation method thereof. Background Art

[0002] Prealbumin (PA) is a serum protein synthesized by liver cells. It is named because its electrophoretic mobility in a buffer solution with a pH of 8.2-8.6 is faster than that of albumin. It has a molecular weight of 5.5 and a sedimentation coefficient of 3.9. Its main physiological function is to transport thyroxine and vitamin A, and it has thymic hormone activity, which can enhance the body's immunity by promoting the maturation of lymphocytes. It has a small content in plasma and a short half-life (1.9 days), which can quickly reflect that the nutritional intake is in positive and negative balance. Therefore, this indicator can reflect slight changes in liver synthesis and catabolism, and the extent of its serum concentration reduction is closely related to the degree of liver parenchymal damage. PA is a very host defense substance, so in acute infection, the serum concentration of PA can decrease rapidly, especially in bacterial infection. It is also a very sensitive indicator for nutritional status detection.

[0003] C-reaction protein is an acute phase protein synthesized by the liver. Its concentration in the blood increases rapidly and significantly in acute myocardial infarction, trauma, infection, inflammation, and surgical operation, and can reach 2,000 times the normal level. Because it can bind to the C-polysaccharide of the pneumococcal cell wall, it is called CRP. CRP is a plasma protein, which is mainly produced in liver parenchymal cells induced by interleukin-6 (IL-6). It has the function of identifying foreign pathogens and destroying host cells. It is mainly used clinically for the differential diagnosis of bacterial and viral infections, the prediction of cardiovascular diseases, and the prediction of gynecological diseases.

[0004] At present, the clinical evaluation index for malnutrition of clinical patients is usually the detection of prealbumin. There is a prealbumin detection kit on the market, and the patent application number is 201710679344.3. However, the single index of prealbumin only reflects the loss of protein and its degree in clinical patients, and cannot reflect the metabolic status and organ function of patients. The guidance provided in the process of nutritional evaluation and prognosis is limited. At the same time, hospitalized patients, especially critically ill patients, are mostly in severe stress reactions such as severe infection, trauma and poisoning. In order to solve the problem of limited guidance basis of single indicators, it is necessary to jointly detect prealbumin and C-reactive protein to provide a basis for nutritional support for clinical patients. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art. In view of the problem that the single indicator of the prealbumin detection kit in the related art has limited guidance on malnutrition of clinical patients, a prealbumin and C-reactive protein combined detection kit and a preparation method are provided, which can effectively complete the evaluation of the nutritional and metabolic status of clinical patients, and help clinical physicians judge the changes in the condition and prognosis of critically ill patients.

[0006] The first aspect of the present invention is to provide a prealbumin and C-reactive protein combined detection kit, comprising: A test card (1), an IC card, a sample adding hole (2), a card reading window, and a sample diluent; wherein: The detection card (1) comprises a prealbumin detection reagent strip (11), a C-reactive protein detection reagent strip (12) and a card shell (13) for loading the prealbumin detection reagent strip and the C-reactive protein detection reagent strip; the prealbumin detection reagent strip (11) comprises a first reaction base plate, a first sample pad, a first conjugation pad, a first chromatographic membrane and a first absorbent paper; the C-reactive protein detection reagent strip (12) comprises a second reaction base plate, a second sample pad, a second conjugation pad, a second chromatographic membrane and a second absorbent paper; a quality control line and a detection line are arranged on the first chromatographic membrane and the second chromatographic membrane; The IC card is used for calibration; The sample loading holes (2) include a prealbumin sample loading hole (21) and a C-reactive protein sample loading hole (22); The card reading window is used to observe the detection process and collect the corresponding fluorescence signal; The sample diluent is used to dilute whole blood or serum samples; the prealbumin and C-reactive protein joint detection kit is loaded with two sample diluents, and the two sample diluents include a prealbumin sample diluent and a C-reactive protein sample diluent; The prealbumin detection reagent strip also includes: Prealbumin monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, microsphere preservation solution is added, and the solid phase is on the first conjugate pad; the first chromatographic membrane is coated with prealbumin monoclonal antibody as the first detection line (A1), and coated with rabbit anti-DNP antibody as the first quality control line (A2); The C-reactive protein detection reagent strip also includes: C-reactive protein monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, and microsphere preservation solution is added and solidified on the second conjugate pad; the second chromatographic membrane is coated with C-reactive protein monoclonal antibody as the second detection line (B1) and coated with rabbit anti-DNP antibody as the second quality control line (B2).

[0007] Preferably, the cartridge loaded with the prealbumin detection reagent strip and the C-reactive protein detection reagent strip is made of plastic; the prealbumin monoclonal antibody and the C-reactive protein monoclonal antibody are obtained by immunizing mice with human prealbumin; the first chromatographic membrane and the second chromatographic membrane are both nitrocellulose membranes; and the first absorbent paper and the second absorbent paper are both made of cotton and linen.

[0008] Preferably, the fluorescent microspheres labeled with prealbumin monoclonal antibody, the first fluorescent microspheres labeled with DNP-BSA, the fluorescent microspheres labeled with C-reactive protein monoclonal antibody and the second fluorescent microspheres labeled with DNP-BSA are polystyrene microspheres containing fluorescent substances, including one or more of Cy3 fluorescent microspheres, Cy5 fluorescent microspheres, quantum dot fluorescent microspheres and rare earth fluorescent microspheres.

[0009] The second aspect of the present invention provides a method for preparing the prealbumin and C-reactive protein combined detection kit according to the first aspect, comprising: S1, using fluorescent microspheres to label prealbumin monoclonal antibodies to form prealbumin fluorescent microsphere solution; S2, using fluorescent microspheres to label C-reactive protein monoclonal antibodies to form a C-reactive protein fluorescent microsphere solution; S3, using fluorescent microspheres to label DNP-BSA to form a DNP-BSA fluorescent microsphere solution; S4, solid phase treatment of the prealbumin fluorescent microsphere solution formed by the fluorescent microsphere labeled prealbumin monoclonal antibody; S5, solid phase treatment of the C-reactive protein fluorescent microsphere solution formed by the fluorescent microsphere-labeled C-reactive protein monoclonal antibody; S6, coating the fluorescent microspheres with prealbumin monoclonal antibodies; S7, coating the C-reactive protein monoclonal antibody; S8, preparing the prealbumin and C-reactive protein combined detection kit.

[0010] Preferably, the S1 comprises: S11, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, the concentration of the fluorescent microspheres being 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.005-0.02 mg / ml; then adding NHS to form a solution with a final concentration of 0.005-0.02 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S12, adding prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S11 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in 20-80 mmol / L boric acid buffer with a pH of 6.0-8.2; S13, add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S12, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S11, disperse the microspheres, and obtain a prealbumin fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S2 includes: S21, adding fluorescent microspheres to a 20-80 mmol / L MES buffer at a pH of 5.0-8.0, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.5-1.5 mg / ml; then adding NHS to form a solution with a final concentration of 2.5-7.5 mg / ml; activating for 20-60 minutes, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S22, adding C-reactive protein monoclonal antibody to the fluorescent microsphere solution activated in S21 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S23. Add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S22, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant, add the same volume of microsphere preservation solution as S21, disperse the microspheres, and obtain a C-reactive protein fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S3 includes: S31, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC and NHS to the same final concentration of 0.001-0.01 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2 to obtain an activated fluorescent microsphere solution; S32, adding DNP-BSA to the fluorescent microsphere solution activated in S31 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S33, add 5.0-10.0% BSA to the S32 microsphere solution; mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S1, disperse the microspheres, and obtain a DNP-BSA fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S4 includes: S41, diluting the prealbumin fluorescent microsphere solution formed by fluorescent microsphere-labeled prealbumin monoclonal antibody with microsphere storage solution 4 times; S42, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a first mixed solution; S43, spraying the first mixed solution onto the conjugate pad at 2-6 ul / cm and drying to obtain a finished fluorescent microsphere-labeled prealbumin conjugate pad; The S5 includes: S51, diluting the C-reactive protein fluorescent microsphere solution formed by labeling the C-reactive protein monoclonal antibody with the microsphere storage solution 4 times; S52, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a second mixed solution; S53, spraying the second mixed solution on the conjugate pad at 2-6 ul / cm and drying to obtain a finished fluorescent microsphere-labeled C-reactive protein conjugate pad; The S6 includes: S61, diluting the prealbumin detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a first spray solution; S62, spraying the first spraying liquid on the chromatographic membrane at a spraying volume of 0.5-2.0 ul / cm, and drying to obtain a finished prealbumin chromatographic membrane; The S7 includes: S71, diluting the C-reactive protein detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a second spray solution; S72, spraying the second spraying liquid on the chromatographic membrane at a spraying amount of 0.5-2.0 ul / cm, and drying to obtain a finished C-reactive protein chromatographic membrane; The S8 includes: The first sample pad, the first conjugate pad, the first chromatography membrane and the first absorbent paper are overlapped and fixed in sequence on the first reaction bottom plate, and the prealbumin detection reagent strip is obtained after being cut by a strip cutter; The second sample pad, the second conjugate pad, the second chromatography membrane and the second absorbent paper are overlapped and fixed in sequence on the second reaction bottom plate, and cut by a strip cutter to obtain a C-reactive protein detection reagent strip; The prealbumin detection reagent strip and the C-reactive protein detection reagent strip are loaded into the cartridge.

[0011] Preferably, the centrifugation conditions in S1, S2 and S3 are 10000-18000 rpm, and the centrifugation time is 10-20 minutes; the reaction is carried out on a shaker at room temperature, and the shaker speed is controlled at 100-200 rpm.

[0012] Preferably, the drying conditions of the finished fluorescent microsphere-labeled prealbumin binding pad, the finished fluorescent microsphere-labeled C-reactive protein binding pad, the finished prealbumin chromatography membrane and the finished C-reactive protein chromatography membrane are 30-45° C. and the drying time is 12-24 hours.

[0013] Preferably, the binding pad is pretreated by a pretreatment binding pad solution before the first mixed solution is sprayed on the binding pad and the second mixed solution is sprayed on the binding pad. The method for preparing the pretreatment binding pad solution is: 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, 0.1-5% Tween 20, pH 7.4; the binding pad is laid flat in the pretreatment binding pad solution and allowed to stand for 1-5 minutes, and then air-dried at 40-60°C for 1-2 hours.

[0014] Preferably, the first sample pad and the second sample pad are pretreated with a sample pad pretreatment solution, and the method for preparing the sample pad pretreatment solution is: 10-50mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA, 0.1%-2% S9, 0.1-2% Tween 20, pH 7.0-8.0.

[0015] The third aspect of the present invention is to provide a method for using the prealbumin and C-reactive protein combined detection kit of the second aspect, comprising: The prealbumin and C-reactive protein combined detection kit is loaded with a prealbumin sample diluent and a C-reactive protein sample diluent; Take 5ul of whole blood or serum sample, add it to the C-reactive protein sample diluent, mix well to form a first sample diluent, take 100ul of the first sample diluent and add it to the C-reactive protein sample well; 50ul of the first sample diluent after mixing is taken, and added to the prealbumin sample diluent, and mixed to form a second sample diluent, and 100ul of the second sample diluent is taken and added to the prealbumin sample well; After a period of time, the test card is inserted into the card slot of the adapter instrument to read the results; wherein, when the whole blood or serum sample is tested, the whole blood or serum sample is added to the corresponding sample addition hole, and under the capillary action, the whole blood or serum sample is chromatographed in the direction of the first absorbent paper, and the prealbumin antigen in the sample is combined with the prealbumin antibody on the first binding pad to form a prealbumin antigen-antibody complex, which is chromatographed onto the first chromatographic membrane and captured by the prealbumin antibody on the first detection line to form a prealbumin antibody-antigen-prealbumin antibody complex. The higher the prealbumin concentration in the whole blood or serum sample, the higher the fluorescent signal on the first detection line, and the fluorescent signal is collected by the detection instrument; the fluorescent signal on the first binding pad During the chromatography process, the DNP-BSA labeled with the optical microspheres is captured by the rabbit anti-DNP antibody on the first chromatographic membrane to form a DNP-BSA antigen-rabbit anti-DNP antibody complex, and the fluorescence signal of the first quality control line is collected by the detection instrument; the fluorescence intensity ratio of the test line and the quality control line is calculated, and it is substituted into the corresponding standard curve to obtain the concentration value of prealbumin in the whole blood or serum sample; similarly, the fluorescence intensity ratio of the second test line and the second quality control line of C-reactive protein is calculated and substituted into the corresponding standard curve to obtain the concentration value of C-reactive protein in the whole blood or serum sample; the normal reference range of the concentration value is: prealbumin 200-400 mg / L, C-reactive protein <10 mg / L.

[0016] Beneficial effects of the method and system of the present invention: It can effectively complete the evaluation of the nutritional and metabolic status of clinical patients, help clinical physicians judge the changes in critical illness conditions and prognosis, and solve the problem that the single indicator of the prealbumin detection kit in related technologies has limited guidance on malnutrition of clinical patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 A schematic diagram of the structure of a joint test kit provided according to an embodiment of the present invention; Figure 2A correlation diagram of the concentration of prealbumin in clinical serum samples detected by the combined test kit provided in Example 1 of the present invention and the concentration of prealbumin detected by the Biosino-Biotech test kit; Figure 3 This is a correlation diagram of the C-reactive protein concentration in clinical serum samples detected by the joint test kit provided in Example 1 of the present invention and the C-reactive protein concentration detected by the Sinopharm Beijing Control kit. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] 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. Embodiment 1

[0022] Provided is a prealbumin and C-reactive protein combined detection kit, comprising: A test card (1), an IC card, a sample adding hole (2), a card reading window, and a sample diluent; wherein: The detection card (1) comprises a prealbumin detection reagent strip (11), a C-reactive protein detection reagent strip (12) and a card shell (13) for loading the prealbumin detection reagent strip and the C-reactive protein detection reagent strip; the prealbumin detection reagent strip (11) comprises a first reaction base plate, a first sample pad, a first conjugation pad, a first chromatographic membrane and a first absorbent paper; the C-reactive protein detection reagent strip (12) comprises a second reaction base plate, a second sample pad, a second conjugation pad, a second chromatographic membrane and a second absorbent paper; a quality control line and a detection line are arranged on the first chromatographic membrane and the second chromatographic membrane; The IC card is used for calibration; The sample loading holes (2) include a prealbumin sample loading hole (21) and a C-reactive protein sample loading hole (22); The card reading window is used to observe the detection process and collect the corresponding fluorescence signal; The sample diluent is used to dilute whole blood or serum samples; the prealbumin and C-reactive protein joint detection kit is loaded with two sample diluents, and the two sample diluents include a prealbumin sample diluent and a C-reactive protein sample diluent.

[0023] As a preferred embodiment, the prealbumin detection reagent strip further comprises: Prealbumin monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, a microsphere preservation solution is added, and the solid phase is on the first conjugate pad; the first chromatographic membrane is coated with prealbumin monoclonal antibody as the first detection line (A1), and coated with rabbit anti-DNP antibody as the first quality control line (A2).

[0024] As a preferred embodiment, the C-reactive protein detection reagent strip further comprises: C-reactive protein monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, and microsphere preservation solution is added and solidified on the second conjugate pad; the second chromatographic membrane is coated with C-reactive protein monoclonal antibody as the second detection line (B1) and coated with rabbit anti-DNP antibody as the second quality control line (B2).

[0025] As a preferred embodiment, the card housing for loading the prealbumin detection reagent strip and the C-reactive protein detection reagent strip is made of plastic. Of course, those skilled in the art should know that the card housing can also be made of other lightweight materials, all within the scope of protection of the present invention.

[0026] As a preferred embodiment, the prealbumin monoclonal antibody and the C-reactive protein monoclonal antibody are obtained by immunizing mice with human prealbumin.

[0027] As a preferred embodiment, the first chromatographic membrane and the second chromatographic membrane are both nitrocellulose membranes, which have good chromatographic performance and strong protein adsorption capacity.

[0028] As a preferred embodiment, the first absorbent paper and the second absorbent paper are both made of cotton and linen, which can enable the sample to undergo antibody-antigen reaction from the sample pad to the chromatography membrane under capillary action.

[0029] As a preferred embodiment, the fluorescent microspheres labeled with prealbumin monoclonal antibody, the first fluorescent microspheres labeled with DNP-BSA, the fluorescent microspheres labeled with C-reactive protein monoclonal antibody and the second fluorescent microspheres labeled with DNP-BSA are polystyrene microspheres containing fluorescent substances, including one or more of Cy3 fluorescent microspheres, Cy5 fluorescent microspheres, quantum dot fluorescent microspheres and rare earth fluorescent microspheres.

[0030] In this embodiment, rare earth fluorescent microspheres are preferably used. By adopting this technical solution, the fluorescent microspheres used in the present invention have an excitation light of 340nm, an emission light of 610nm, a relatively large Stokes shift, and a small signal interference between the absorption spectrum and the emission spectrum. The detection instrument can more accurately monitor the fluorescent signal, thereby improving the accuracy of the detection result.

[0031] like Figure 1 As shown, the albumin detection reagent strip and the C-reactive protein detection reagent strip card housing are made of plastic material, and the albumin detection reagent strip and the C-reactive protein detection reagent strip are placed in the card housing respectively, thereby ensuring that clinical samples do not interfere with each other and improving the accuracy of detection. Embodiment 2

[0032] The preparation method of the prealbumin and C-reactive protein combined detection kit of Example 1 adopts the following technical scheme: S1, using fluorescent microspheres to label prealbumin monoclonal antibodies to form prealbumin fluorescent microsphere solution; S2, using fluorescent microspheres to label C-reactive protein monoclonal antibodies to form a C-reactive protein fluorescent microsphere solution; S3, using fluorescent microspheres to label DNP-BSA to form a DNP-BSA fluorescent microsphere solution; S4, solid phase treatment of the prealbumin fluorescent microsphere solution formed by the fluorescent microsphere labeled prealbumin monoclonal antibody; S5, solid phase treatment of the C-reactive protein fluorescent microsphere solution formed by the fluorescent microsphere-labeled C-reactive protein monoclonal antibody; S6, coating the fluorescent microspheres with prealbumin monoclonal antibodies; S7, coating the C-reactive protein monoclonal antibody; S8, preparing the prealbumin and C-reactive protein combined detection kit.

[0033] As a preferred embodiment, the S1 includes: S11, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, the concentration of the fluorescent microspheres being 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.005-0.02 mg / ml; then adding NHS to form a solution with a final concentration of 0.005-0.02 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S12, adding prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S11 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in 20-80 mmol / L boric acid buffer with a pH of 6.0-8.2; S13, add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S12, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S11, disperse the microspheres, and obtain a prealbumin fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml.

[0034] As a preferred embodiment, the S2 includes: S21, adding fluorescent microspheres to a 20-80 mmol / L MES buffer at a pH of 5.0-8.0, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.5-1.5 mg / ml; then adding NHS to form a solution with a final concentration of 2.5-7.5 mg / ml; activating for 20-60 minutes, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S22, adding C-reactive protein monoclonal antibody to the fluorescent microsphere solution activated in S21 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S23. Add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S22, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant, add the same volume of microsphere preservation solution as S21, disperse the microspheres, and obtain a C-reactive protein fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml.

[0035] As a preferred implementation, S3 includes: S31, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC and NHS to the same final concentration of 0.001-0.01 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2 to obtain an activated fluorescent microsphere solution; S32, adding DNP-BSA to the fluorescent microsphere solution activated in S31 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S33, add 5.0-10.0% BSA to the S32 microsphere solution; mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S1, disperse the microspheres, and obtain a DNP-BSA fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml.

[0036] As a preferred embodiment, the centrifugation conditions in steps S1, S2 and S3 are 10000-18000 rpm, and the centrifugation time is 10-20 minutes. By adopting this technical scheme, the present invention strictly controls the centrifugation conditions in the process of fluorescent microsphere labeling antibodies. A lower centrifugation speed will cause insufficient centrifugation and excessive loss of raw materials. A higher centrifugation speed can settle all components to the bottom of the centrifuge tube, reducing the loss rate of raw materials.

[0037] As a preferred embodiment, the reactions in steps S1, S2 and S3 are carried out on a shaker at room temperature, and the shaker speed is controlled at 100-200 rpm. By adopting this technical scheme, the present invention fully mixes the prealbumin monoclonal antibody, the C-reactive protein monoclonal antibody and the DNP-BSA with the fluorescent microspheres under certain conditions, and fully couples them with the fluorescent microspheres, thereby improving the coupling efficiency of the antibody, DNP-BSA and the fluorescent microspheres.

[0038] As a preferred embodiment, step S3 requires adding BSA blocking agent and reacting for 1-2 hours at room temperature on a shaker at a speed of 100-200 rpm; using this technical solution, the experimental purpose is to reduce the non-specific binding of antibodies.

[0039] As a preferred implementation, S4 includes: S41, diluting the prealbumin fluorescent microsphere solution formed by fluorescent microsphere-labeled prealbumin monoclonal antibody with microsphere storage solution 4 times; S42, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a first mixed solution; S43, spraying the first mixed solution onto the conjugate pad at 2-6 ul / cm and drying the mixture to obtain a finished fluorescent microsphere-labeled prealbumin conjugate pad.

[0040] As a preferred implementation, S5 includes: S51, diluting the C-reactive protein fluorescent microsphere solution formed by labeling the C-reactive protein monoclonal antibody with the microsphere storage solution 4 times; S52, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a second mixed solution; S53, spraying the second mixed solution onto the conjugate pad at 2-6 ul / cm and drying the mixture to obtain a finished fluorescent microsphere-labeled C-reactive protein conjugate pad.

[0041] As a preferred implementation, S6 includes: S61, diluting the prealbumin detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a first spray solution; S62, spraying the first spraying liquid on the chromatographic membrane at a spraying volume of 0.5-2.0 ul / cm, and drying to obtain a finished prealbumin chromatographic membrane.

[0042] As a preferred implementation, the S7 includes: S71, diluting the C-reactive protein detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a second spray solution; S72, spraying the second spraying liquid on the chromatographic membrane at a spraying volume of 0.5-2.0 ul / cm, and drying to obtain a finished C-reactive protein chromatographic membrane.

[0043] As a preferred embodiment, the drying conditions of the finished fluorescent microsphere-labeled prealbumin binding pad, the finished fluorescent microsphere-labeled C-reactive protein binding pad, the finished prealbumin chromatography membrane and the finished C-reactive protein chromatography membrane are 30-45°C, and the drying time is 12-24 hours. By adopting this technical solution, the present invention strictly controls the drying temperature and time. If the drying time is too long, the antibody will be easily inactivated and the detection effect will be reduced. If the drying temperature is too low, it will be easily contaminated by harmful microorganisms, affecting the accuracy of the detection results.

[0044] As a preferred embodiment, the binding pad is pretreated by a pretreatment binding pad solution before the first mixed solution is sprayed on the binding pad and the second mixed solution is sprayed on the binding pad. The method for preparing the pretreatment binding pad solution is: 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, 0.1-5% Tween 20, pH 7.4; the binding pad is laid flat in the pretreatment binding pad solution and allowed to stand for 1-5 minutes, and then air-dried at 40-60°C for 1-2 hours.

[0045] As a preferred embodiment, the first sample pad and the second sample pad used in the present invention are pretreated with a sample pad pretreatment solution, and the method for preparing the sample pad pretreatment solution is: 10-50mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA, 0.1%-2% S9, 0.1-2% Tween 20, pH 7.0-8.0.

[0046] As a preferred embodiment, the S8 includes: The first sample pad, the first conjugate pad, the first chromatography membrane and the first absorbent paper are overlapped and fixed in sequence on the first reaction bottom plate, and the prealbumin detection reagent strip is obtained after being cut by a strip cutter; The second sample pad, the second conjugate pad, the second chromatography membrane and the second absorbent paper are overlapped and fixed in sequence on the second reaction bottom plate, and cut by a strip cutter to obtain a C-reactive protein detection reagent strip; The prealbumin detection reagent strip and the C-reactive protein detection reagent strip are loaded into the cartridge. Embodiment 3

[0047] A method for using the prealbumin and C-reactive protein combined detection kit of Example 2 is provided, comprising: The prealbumin and C-reactive protein combined detection kit is loaded with a prealbumin sample diluent and a C-reactive protein sample diluent; Take 5ul of whole blood or serum sample, add it to the C-reactive protein sample diluent, mix well to form a first sample diluent, take 100ul of the first sample diluent and add it to the C-reactive protein sample well; 50ul of the first sample diluent after mixing is taken, and added to the prealbumin sample diluent, and mixed to form a second sample diluent, and 100ul of the second sample diluent is taken and added to the prealbumin sample well; After 20 minutes, the test card is inserted into the card slot of the adapter instrument to interpret the results; wherein, when the whole blood or serum sample is tested, the whole blood or serum sample is added to the corresponding sample loading hole, and under the capillary action, the whole blood or serum sample is chromatographed in the direction of the first absorbent paper, and the prealbumin antigen in the sample is combined with the prealbumin antibody on the first binding pad to form a prealbumin antigen-antibody complex, which is chromatographed onto the first chromatographic membrane and captured by the prealbumin antibody on the first detection line to form a prealbumin antibody-antigen-prealbumin antibody complex. The higher the prealbumin concentration in the whole blood or serum sample, the higher the fluorescence signal on the first detection line, and the fluorescence signal is collected by the detection instrument; on the first binding pad During the chromatography process, DNP-BSA labeled with fluorescent microspheres is captured by the rabbit anti-DNP antibody on the first chromatographic membrane to form a DNP-BSA antigen-rabbit anti-DNP antibody complex, and the fluorescence signal of the first quality control line is collected by the detection instrument; the fluorescence intensity ratio of the test line and the quality control line is calculated, and it is substituted into the corresponding standard curve to obtain the concentration value of prealbumin in the whole blood or serum sample; similarly, the fluorescence intensity ratio of the second test line and the second quality control line of C-reactive protein is calculated and substituted into the corresponding standard curve to obtain the concentration value of C-reactive protein in the whole blood or serum sample; the normal reference range of the concentration value is: prealbumin 200-400 mg / L, C-reactive protein <10 mg / L.

[0048] like Figure 1 As shown, the prealbumin and C-reactive protein combined detection kit comprises: 1. A test card, comprising: ①. A prealbumin detection reagent strip, comprising: a. Use fluorescent microspheres to label prealbumin monoclonal antibodies and DNP-BSA, add microsphere storage solution, and solid phase onto the binding pad; b. The detection line is coated with prealbumin monoclonal antibody and the quality control line is coated with rabbit anti-DNP antibody; ②. C-reactive protein detection reagent strip, the C-reactive protein detection reagent strip comprises: a. Use fluorescent microspheres to label C-reactive protein monoclonal antibody and DNP-BSA, add microsphere storage solution, and solid phase onto the binding pad; b. The detection line is coated with C-reactive protein monoclonal antibody and the quality control line is coated with rabbit anti-DNP antibody; ③. The card housing for loading the prealbumin test strip and the C-reactive protein test strip can be made of plastic material; 2. IC card, used for calibration; 3. Sample diluent, used to dilute whole blood or serum samples.

[0049] The solid phase on the fluorescent microsphere-labeled prealbumin binding pad contains a mixed solution of fluorescent microspheres labeled with prealbumin monoclonal antibodies and fluorescent microspheres labeled with DNP-BSA.

[0050] The solid phase of the fluorescent microsphere-labeled C-reactive protein binding pad contains a mixed solution of fluorescent microspheres labeled with C-reactive protein monoclonal antibodies and fluorescent microspheres labeled with DNP-BSA.

[0051] The prealbumin chromatography membrane is coated with a prealbumin monoclonal antibody as a detection line, and is coated with a rabbit anti-DNP antibody as a quality control line.

[0052] The C-reactive protein chromatographic membrane is coated with a C-reactive protein monoclonal antibody as a detection line, and is coated with a rabbit anti-DNP antibody as a quality control line.

[0053] The present application embodiment discloses a prealbumin and C-reactive protein combined detection kit, such as Figure 1 As shown, the kit includes a prealbumin detection reagent strip and a C-reactive protein detection reagent strip, and a plastic card shell wrapped around the outside of the reagent strip. There are quality control lines and detection lines on the chromatographic membrane of the reagent strip to form a card reading window for convenient observation and fluorescence signal collection.

[0054] When testing the sample, the sample is added to the sample well. Under the capillary action, the sample is chromatographed in the direction of the absorbent paper. The prealbumin antigen in the sample is combined with the prealbumin antibody on the binding pad to form a prealbumin antigen-antibody complex. The complex is chromatographed on the chromatographic membrane and captured by the prealbumin antibody on the detection line to form a prealbumin antibody-antigen-prealbumin antibody complex. The higher the concentration of prealbumin in the sample, the higher the fluorescence signal on the detection line, and the fluorescence signal is collected by the detection instrument; the DNP-BSA labeled with fluorescent microspheres on the binding pad is captured by the rabbit anti-DNP antibody on the chromatographic membrane during the chromatography process to form a DNP-BSA antigen-rabbit anti-DNP antibody complex, and the fluorescence signal of the quality control line is collected by the detection instrument; the fluorescence intensity ratio of the prealbumin detection line and the quality control line is calculated, and it is brought into the corresponding standard curve to obtain the concentration value of the prealbumin in the sample; similarly, the fluorescence intensity ratio of the C-reactive protein detection line and the quality control line is calculated and brought into the corresponding standard curve to obtain the concentration value of the C-reactive protein in the sample.

[0055] A prealbumin and C-reactive protein combined detection kit in the present application embodiment is prepared by the following embodiment, and the antibody raw materials used are as follows: The prealbumin antibody of the present application is provided by Shanghai Medix Medical Technology Co., Ltd., with the catalog number Medix100830; The prealbumin detection antibody of the present application is provided by Shanghai Medix Medical Technology Co., Ltd., with the catalog number Medix 100828; The C-reactive protein antibody of the present application was produced by Wuhan Aokebot Biotechnology Co., Ltd., with clone number 15E3; The C-reactive protein detection antibody of this application is produced by Wuhan Aokebot Biotechnology Co., Ltd., with clone number 16D12; The DNP-BSA of the present application is produced by Beijing InnoWorld Biotechnology Co., Ltd., with the product number Inno681; The rabbit anti-DNP antibody of the present application is produced by Beijing InnoWorld Biotechnology Co., Ltd., with the product number Inno0532.

[0056] The present application discloses a solution preparation scheme for the kit, as shown below: Preparation Example 1 Preparation of pH 7.0, 0.05 mol / L Tris-HCl buffer: weigh 6.057 g Tris and add it to 950 mL purified water, adjust the pH to 7.0 ± 0.05, add purified water to make up to 1000 mL, and store at 2-8 °C for later use.

[0057] Preparation Example 2 Preparation of pH 8.0, 0.05 mol / L Tris-HCl buffer: weigh 6.057 g Tris and add it to 950 mL purified water, adjust the pH to 8.0 ± 0.05, add purified water to make up to 1000 mL, and store at 2-8°C for later use.

[0058] Preparation Example 3 Preparation of pH 7,0 0.02 mol / L phosphate buffer: Preparation of 0.02 mol / L Na2HPO4: Weigh 7.163 g of Na2HPO4·12H2O solid and dissolve it in purified water to make up to 1000 mL.

[0059] Preparation of 0.02 mol / L NaH2PO4: Weigh 3.12 g of NaH2PO4·2H2O solid and dissolve it in purified water to make up to 1000 mL.

[0060] pH 7.0, 0.02 mol / L PB buffer: Swap 0.02 mol / L NaH2PO4 and 0.02 mol / L Na2HPO4 and mix well. Adjust the pH to 7.0±0.05 at room temperature in the workshop and store at 2-8℃ for later use.

[0061] Preparation Example 4 Preparation of pH 8.2, 0.05 mol / L boric acid buffer: Preparation of 0.05 mol / L borax: Weigh 19.07 g of solid borax and dissolve it in purified water to make up to 1000 mL.

[0062] Preparation of 0.2 mol / L boric acid: Weigh 12.37 g of solid boric acid and dissolve it in purified water to make up to 1000 mL.

[0063] pH 8.2, 0.05 mol / L boric acid buffer: mix 0.05 mol / L borax and 0.2 mol / L boric acid evenly, adjust the pH to 8.2±0.05 at room temperature in the workshop, and store at 2-8℃ for later use.

[0064] Preparation Example 5 Preparation of pH 6.0, 0.05 mol / L MES buffer: Weigh 21.325 g of MES solid and dissolve it in 900 ml of purified water. Adjust the pH value to 6.0±0.05 with NaOH solution, make up to 1000 mL and store at 2-8°C for later use.

[0065] Preparation Example 6 Blocking solution preparation: Weigh 10 g of bovine serum albumin and dissolve it in 100 mL of pure water and store it at -20°C.

[0066] Preparation Example 7 Preparation of binding pad treatment solution: Add 5g sodium caseinate, 10g bovine serum albumin, 60g sucrose, and 1g S9 to 900mL pH 7.0, 0.05 mol / L Tris hydrochloric acid buffer, make up to 1000mL, and store at -20℃.

[0067] Preparation Example 8 Sample diluent preparation: weigh Na 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, 0.2g KCl, 8.0g NaCl, add 900mL purified water to dissolve, make up to 1000mL, adjust the pH value to 7.4±0.05 at room temperature in the workshop, and store at 2-8℃ for later use. Example 1

[0068] A prealbumin and C-reactive protein combined detection kit and a preparation method thereof, comprising the following steps: Method for labeling prealbumin monoclonal antibody with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 10ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 90ul of the boric acid buffer of Preparation Example 4, mix well, add 10ug EDC and 10ug NHS, activate for 40min, 15000rpm, 15 minutes, discard the supernatant, add 100ul of the boric acid buffer of Preparation Example 4, and mix well.

[0069] S2. Fluorescent microspheres labeled with monoclonal antibody against prealbumin Add 10ug of prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S1, mix well, label on a shaker at room temperature for 2 hours, and disperse the microspheres by ultrasonic wave to avoid microsphere aggregation; S3. Microsphere blocking Add 10ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 400ul of microsphere preservation solution, disperse the microspheres, and obtain a prealbumin fluorescent microsphere solution; Method for labeling C-reactive protein monoclonal antibody with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 10ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 90ul of MES buffer prepared in Example 5, mix well, add 100ug EDC and 500ug NHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 100ul of phosphate buffer prepared in Example 3, and mix well.

[0070] S2. Fluorescent microspheres labeled with monoclonal antibody against prealbumin Add 10ug of prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S1, mix well, and label for 2 hours on a shaker at room temperature in the dark. Disperse the microspheres with ultrasound to avoid aggregation of the microspheres. S3. Microsphere blocking Add 10ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 400ul of microsphere preservation solution, disperse the microspheres, and obtain a C-reactive protein fluorescent microsphere solution; Method for labeling DNP-BSA with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 10ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 90ul of the boric acid buffer of Preparation Example 4, mix well, add 5ug EDC and 5ug NHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 100ul of the MES buffer of Preparation Example 5, and mix well.

[0071] S2. Fluorescent microspheres labeled with rabbit anti-DNP antibody Add 10ug DNP-BSA to the fluorescent microsphere solution activated in S1, mix well, and label for 1.5 hours on a shaker at room temperature in the dark. Disperse the microspheres with ultrasound to prevent aggregation of the microspheres. S3. Microsphere blocking Add 10ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 100ul of microsphere preservation solution, disperse the microspheres, and obtain a DNP-BSA fluorescent microsphere solution; The solid phase method of the finished prealbumin binding pad is as follows: add 4ul of DNP-BSA fluorescent microsphere solution to 400ul of prealbumin fluorescent microsphere solution, mix well, spray on the pretreated binding pad at 4ul / cm, and dry at 37°C for 24 hours to obtain the finished prealbumin binding pad; The solid phase method of the finished C-reactive protein binding pad is as follows: add 4ulDNP-BSA fluorescent microsphere solution to 400ulC-reactive protein fluorescent microsphere solution, mix well, spray on the pretreated binding pad at 4ul / cm, dry at 37°C for 24 hours, and obtain the finished C-reactive protein binding pad; Coating method of prealbumin monoclonal antibody: dilute prealbumin monoclonal antibody to 1.5 mg / ml, and dilute rabbit anti-DNP antibody to 1.0 mg / ml, and coat them on the chromatographic membrane with a spray volume of 1 ul / cm to form a prealbumin detection line and a rabbit anti-DNP quality control line, and place them at 37°C to dry for 24 hours to obtain a prealbumin finished chromatographic pad.

[0072] Coating method of C-reactive protein monoclonal antibody: dilute C-reactive protein monoclonal antibody to 1.0 mg / ml, dilute rabbit anti-DNP antibody to 1.0 mg / ml, use 1ul / cm spray volume to coat on the chromatographic membrane to form C-reactive protein detection line and rabbit anti-DNP quality control line, place at 37°C to dry for 24 hours, and obtain the finished C-reactive protein chromatographic pad.

[0073] Kit assembly method: overlap and paste the sample pad, the above-mentioned finished binding pad, the finished chromatography pad and the absorbent paper on the reaction base plate in sequence, and keep an overlap of 1-2 mm, use a strip cutter to cut into reagent strips with a width of 4 mm, and assemble the prealbumin detection reagent strip and the C-reactive protein detection reagent strip at the corresponding positions of the card shell, that is, assemble a prealbumin and C-reactive protein joint detection kit.

[0074] Standard curve detection Take 5ul of the calibration solution sample of the corresponding concentration, add it to 2ml of the C-reactive protein sample diluent of Preparation Example 8, mix well, take 100ul of the diluted solution and add it to the C-reactive protein spotting hole, then draw 50ul from the mixed C-reactive protein sample diluent, add it to the prealbumin diluent, mix well, and then take 100ul of the diluted solution and add it to the prealbumin spotting hole. After 20 minutes, insert the reagent card into the card slot of the fluorescent immunoassay analyzer to test the fluorescence intensity. There are 7 calibration solutions in total, and each is tested 5 times repeatedly. The average value is taken. The ratio of the fluorescence intensity of the test line and the quality control line and the concentration of the calibration solution are used to draw the prealbumin standard curve and the C-reactive protein standard curve. The prealbumin standard curve is a cubic equation, and the C-reactive protein standard curve is a 4-parameter equation. The signal value is the independent variable and the concentration value is the dependent variable. The data are shown in Tables 1 and 2; Correlation coefficient R of the standard curve equation of prealbumin 2 is 0.9969; C-reactive protein standard curve equation correlation coefficient R 2 is 0.999; The two calibration curves have a good fit and can be used as calibration formulas.

[0075] Table 1 Example 1 Prealbumin standard curve data

[0076] Table 2 Example 1 C-reactive protein standard curve data Example 2

[0077] A prealbumin and C-reactive protein combined detection kit and a preparation method thereof, comprising the following steps: Method for labeling prealbumin monoclonal antibody with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 100ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 900ul of the boric acid buffer of Preparation Example 4, mix well, add 100ug EDC and 100ug NHS, activate for 40min, 15000rpm, 15 minutes, discard the supernatant, add 1000ul of the boric acid buffer of Preparation Example 4, and mix well.

[0078] S2. Fluorescent microspheres labeled with monoclonal antibody against prealbumin Add 100ug of prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S1, mix well, label on a shaker at room temperature for 2 hours, and disperse the microspheres with ultrasound to avoid microsphere aggregation; S3. Microsphere blocking Add 100ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 4000ul of microsphere preservation solution, disperse the microspheres, and obtain a prealbumin fluorescent microsphere solution; Method for labeling C-reactive protein monoclonal antibody with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 100ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 900ul of MES buffer prepared in Example 5, mix well, add 1000ug EDC and 5000ug NHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 1000ul of phosphate buffer prepared in Example 3, and mix well.

[0079] S2. Fluorescent microspheres labeled with C-reactive protein monoclonal antibody Add 100ug of C-reactive protein monoclonal antibody to the fluorescent microsphere solution activated in S1, mix well, and label for 2 hours on a shaker at room temperature in the dark. Disperse the microspheres with ultrasound to prevent aggregation of the microspheres. S3. Microsphere blocking Add 100ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 4000ul of microsphere preservation solution, disperse the microspheres, and obtain a C-reactive protein fluorescent microsphere solution; Method for labeling DNP-BSA with fluorescent microspheres: S1. Activation of fluorescent microspheres Take 50ul of fluorescent microspheres with a solid content of 1% (original concentration 10mg / ml) and add them to 450ul of the boric acid buffer of Preparation Example 4, mix well, add 25ug EDC and 25ug NHS, activate for 30min, 15000rpm, 15 minutes, discard the supernatant, add 500ul of the MES buffer of Preparation Example 5, and mix well.

[0080] S2. Fluorescent microspheres labeled with rabbit anti-DNP antibody Add 50ug DNP-BSA to the fluorescent microsphere solution activated in S1, mix well, and label for 1.5 hours on a shaker at room temperature in the dark. Disperse the microspheres with ultrasound to prevent aggregation of the microspheres. S3. Microsphere blocking Add 50ul of 5% BSA to the S2 microsphere solution, mix well, block on a shaker at room temperature for 1 hour, 10000rpm for 10 minutes, discard the supernatant, add 500ul of microsphere preservation solution, disperse the microspheres, and obtain DNP-BSA fluorescent microsphere solution; The solid phase method of the finished prealbumin binding pad is as follows: 40ul of DNP-BSA fluorescent microsphere solution is added to 4000ul of prealbumin fluorescent microsphere solution, mixed, sprayed on the pretreated binding pad at 4ul / cm, and dried at 37°C for 24 hours to obtain the finished prealbumin binding pad; The solid phase method of the finished C-reactive protein binding pad is as follows: 40ul of DNP-BSA fluorescent microsphere solution is added to 4000ul of C-reactive protein fluorescent microsphere solution, mixed, sprayed on the pretreated binding pad at 4ul / cm, and dried at 37°C for 24 hours to obtain the finished C-reactive protein binding pad; The coating method of the prealbumin monoclonal antibody and the C-reactive protein monoclonal antibody and the kit assembly method are the same as those in Example 1; Standard curve detection Take 5ul of the calibration solution sample of the corresponding concentration, add it to 2ml of the C-reactive protein sample diluent of Preparation Example 8, mix well, take 100ul of the diluted solution and add it to the C-reactive protein spotting well, then draw 50ul from the mixed C-reactive protein sample diluent, add it to the prealbumin diluent, mix well, and then take 100ul of the diluted solution and add it to the prealbumin spotting well. After 20 minutes, insert the reagent card into the card slot of the fluorescent immunoassay analyzer to test the fluorescence intensity. There are 7 calibration solutions in total, and each is tested 5 times. The average value is taken, and then the ratio of the fluorescence intensity of the test line and the quality control line and the concentration of the calibration solution are used to draw the prealbumin standard curve and the C-reactive protein standard curve. The prealbumin and C-reactive protein standard curves are both cubic equations, the signal value is the independent variable, and the concentration value is the dependent variable. The data are shown in Tables 3 and 4; Correlation coefficient R of the standard curve equation of prealbumin 2 is 0.999; C-reactive protein standard curve equation correlation coefficient R 2 is 0.999; The two calibration curves have a good fit and can be used as calibration formulas.

[0081] Table 3 Example 2 Prealbumin standard curve data

[0082] Table 4 Example 2 C-reactive protein standard curve data

[0083] Performance testing experiment 1. Perform linearity evaluation experiments on prealbumin and C-reactive protein detection kits a. Mix the high-concentration prealbumin sample (400 mg / L) close to the upper limit of the linear range and the low-concentration prealbumin sample (50 mg / L) close to the lower limit of the linear range in a certain proportion to obtain 5 concentration samples, namely 400, 312.5, 225, 127.5 and 50 mg / L. Take 5ul of the above concentration samples respectively, add them to the C-reactive protein diluent, mix them well, then take 50ul of each and add them to the prealbumin diluent, mix them well, then take 100ul of the diluted samples respectively, add them to the prealbumin spotting hole, insert the reagent card into the card slot of the fluorescent immunoassay analyzer for 20 minutes, read the reading, input the standard curve into the instrument in advance, and directly read the detection concentration value. Repeat the test 3 times for each concentration sample, and calculate the average concentration. The test data are shown in Tables 5 and 6.

[0084] Table 5 Prealbumin linearity test data of Example 1 (mg / L)

[0085] Table 6 Prealbumin linearity test data of Example 2 (mg / L)

[0086] b. Mix the high-concentration C-reactive protein sample (100 mg / L) close to the upper limit of the linear range and the low-concentration C-reactive protein sample (5 mg / L) close to the lower limit of the linear range in a certain proportion to obtain 5 concentration samples, namely 100, 76.25, 52.5, 28.75 and 5 mg / L. Take 5ul of the above concentration samples respectively, add them to the C-reactive protein diluent, mix well, and then take 100ul of the diluted samples respectively, add them to the C-reactive protein spotting hole, insert the reagent card into the card slot of the fluorescent immunoassay analyzer for 20 minutes, read the reading, and input the standard curve into the instrument in advance, so that the detection concentration value can be read directly. Repeat the test for each concentration sample 3 times and calculate the average concentration. The test data are shown in Tables 7 and 8.

[0087] Table 7 Example 1 C-reactive protein linear detection data (mg / L)

[0088] Table 8 Example 2 C-reactive protein linear detection data (mg / L)

[0089] 2. Precision test experiment For the precision test of the prealbumin and C-reactive protein joint test kit, 200 mg / L (prealbumin control) and 8 mg / L (C-reactive protein control) were selected respectively. The quality control products were diluted according to the dilution method of the samples in the above linear detection experiment. 100 ul of the diluted samples were added to the prealbumin spotting well and the C-reactive protein spotting well respectively. After 20 minutes, the reagent card was inserted into the card slot of the fluorescent immunoassay analyzer and the reading was read; the test was repeated 20 times respectively, and the CV value was calculated. The test data are shown in Tables 9, 10, 11 and 12.

[0090] Table 9 Prealbumin linearity test data of Example 1 (200 mg / L)

[0091] Table 10 Example 2 Prealbumin linear detection data (200 mg / L)

[0092] Table 11 Example 1 C-reactive protein linear detection data (8 mg / L)

[0093] Table 12 Example 2 C-reactive protein linear detection data (8 mg / L)

[0094] 3. Clinical sample testing experiment The serum samples were tested using the kit of Example 1 of the present application, the Sinopharm BH system and the matching prealbumin and C-reactive protein reagents. The Sinopharm BH reagents were tested according to the reagent instructions and the biochemical analyzer operating instructions. The kit of Example 1 of the present application, 5ul of serum sample was added to the C-reactive protein diluent, mixed, 100ul of diluent was added to the C-reactive protein spotting well in the test card, and then 50ul of C-reactive protein diluent with serum sample was added to the prealbumin diluent, mixed, and then 100ul was added to the prealbumin spotting well, left at room temperature for 20 minutes, and inserted into the fluorescent immunoassay analyzer to detect the concentration of prealbumin and C-reactive protein in the serum. The experimental results are shown in Table 13, Figure 2 and Table 14, Figure 3 .

[0095] Table 13 Example 1 Prealbumin clinical sample comparison data

[0096] Table 14 Example 1 C-reactive protein clinical sample comparison data

[0097] From Table 13 and Figure 2It can be seen that the results of the test kit of the present application for detecting the concentration of prealbumin in serum samples are compared with the test results using the Sinopharm Biotechnology Co., Ltd. biochemical instrument and the supporting kit, and the correlation coefficient is R2=0.9809, and the linear equation is y=1.0099x-1.6578. The clinical experimental results show that the test kit of the present application has a good correlation with the Sinopharm Biotechnology Co., Ltd. detection system and can be used for clinical diagnosis.

[0098] From Table 14 and Figure 3 It can be seen that the results of the test kit of the present application for detecting the concentration of C-reactive protein in serum samples are compared with the test results using the Sinopharm Biotechnology Co., Ltd. biochemical instrument and the supporting kit. The correlation coefficient is R2=0.9826, and the linear equation is y=0.9693x+0.584. The clinical experimental results show that the test kit of the present application has a good correlation with the Sinopharm Biotechnology Co., Ltd. detection system and can be used for clinical diagnosis.

[0099] 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.

[0100] 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 and C-reactive protein combined detection kit, characterized in that: include: A test card (1), an IC card, a sample adding hole (2), a card reading window, and a sample diluent; wherein: The detection card (1) comprises a prealbumin detection reagent strip (11), a C-reactive protein detection reagent strip (12) and a card shell (13) for loading the prealbumin detection reagent strip and the C-reactive protein detection reagent strip; the prealbumin detection reagent strip (11) comprises a first reaction base plate, a first sample pad, a first conjugation pad, a first chromatographic membrane and a first absorbent paper; the C-reactive protein detection reagent strip (12) comprises a second reaction base plate, a second sample pad, a second conjugation pad, a second chromatographic membrane and a second absorbent paper; a quality control line and a detection line are arranged on the first chromatographic membrane and the second chromatographic membrane; The IC card is used for calibration; The sample loading holes (2) include a prealbumin sample loading hole (21) and a C-reactive protein sample loading hole (22); The card reading window is used to observe the detection process and collect the corresponding fluorescence signal; The sample diluent is used to dilute whole blood or serum samples; the prealbumin and C-reactive protein joint detection kit is loaded with two sample diluents, and the two sample diluents include a prealbumin sample diluent and a C-reactive protein sample diluent; The prealbumin detection reagent strip also includes: Prealbumin monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, microsphere preservation solution is added, and the solid phase is on the first conjugate pad; the first chromatographic membrane is coated with prealbumin monoclonal antibody as the first detection line (A1), and coated with rabbit anti-DNP antibody as the first quality control line (A2); The C-reactive protein detection reagent strip also includes: C-reactive protein monoclonal antibody and DNP-BSA are labeled with fluorescent microspheres, and microsphere preservation solution is added and solidified on the second conjugate pad; the second chromatographic membrane is coated with C-reactive protein monoclonal antibody as the second detection line (B1) and coated with rabbit anti-DNP antibody as the second quality control line (B2).

2. A prealbumin and C-reactive protein combined detection kit according to claim 1, characterized in that: The card shell loaded with the prealbumin detection reagent strip and the C-reactive protein detection reagent strip is made of plastic material; the prealbumin monoclonal antibody and the C-reactive protein monoclonal antibody are obtained by immunizing mice with human prealbumin; the first chromatographic membrane and the second chromatographic membrane are both nitrocellulose membranes; the first absorbent paper and the second absorbent paper are both made of cotton and linen.

3. A prealbumin and C-reactive protein combined detection kit according to claim 2, characterized in that: The fluorescent microspheres labeled with prealbumin monoclonal antibody, the first fluorescent microspheres labeled with DNP-BSA, the fluorescent microspheres labeled with C-reactive protein monoclonal antibody and the second fluorescent microspheres labeled with DNP-BSA are polystyrene microspheres containing fluorescent substances, including one or more of Cy3 fluorescent microspheres, Cy5 fluorescent microspheres, quantum dot fluorescent microspheres and rare earth fluorescent microspheres.

4. A method for preparing a prealbumin and C-reactive protein combined detection kit according to any one of claims 1 to 3, characterized in that: include: S1, using fluorescent microspheres to label prealbumin monoclonal antibodies to form prealbumin fluorescent microsphere solution; S2, using fluorescent microspheres to label C-reactive protein monoclonal antibodies to form a C-reactive protein fluorescent microsphere solution; S3, using fluorescent microspheres to label DNP-BSA to form a DNP-BSA fluorescent microsphere solution; S4, solid phase treatment of the prealbumin fluorescent microsphere solution formed by the fluorescent microsphere labeled prealbumin monoclonal antibody; S5, solid phase treatment of the C-reactive protein fluorescent microsphere solution formed by the fluorescent microsphere-labeled C-reactive protein monoclonal antibody; S6, coating the fluorescent microspheres with prealbumin monoclonal antibodies; S7, coating the C-reactive protein monoclonal antibody; S8, preparing the prealbumin and C-reactive protein combined detection kit.

5. The method for preparing the prealbumin and C-reactive protein combined detection kit according to claim 4, characterized in that: The S1 includes: S11, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, the concentration of the fluorescent microspheres being 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.005-0.02 mg / ml; then adding NHS to form a solution with a final concentration of 0.005-0.02 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer solution with a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S12, adding prealbumin monoclonal antibody to the fluorescent microsphere solution activated in S11 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in 20-80 mmol / L boric acid buffer with a pH of 6.0-8.2; S13, add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S12, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S11, disperse the microspheres, and obtain a prealbumin fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S2 includes: S21, adding fluorescent microspheres to a 20-80 mmol / L MES buffer at a pH of 5.0-8.0, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC to form a solution with a final concentration of 0.5-1.5 mg / ml; then adding NHS to form a solution with a final concentration of 2.5-7.5 mg / ml; activating for 20-60 minutes, centrifuging and discarding the supernatant, and resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, thereby obtaining an activated fluorescent microsphere solution; S22, adding C-reactive protein monoclonal antibody to the fluorescent microsphere solution activated in S21 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant, and resuspending the precipitated microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S23. Add 5.0-10.0% BSA to the fluorescent microsphere solution after resuspending in S22, mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant, add the same volume of microsphere preservation solution as S21, disperse the microspheres, and obtain a C-reactive protein fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S3 includes: S31, adding fluorescent microspheres to a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2, wherein the concentration of the fluorescent microspheres is 0.5-2 mg / ml; then adding EDC and NHS to the same final concentration of 0.001-0.01 mg / ml; activating for 20-60 min, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres with a 20-80 mmol / L boric acid buffer at a pH of 6.0-8.2 to obtain an activated fluorescent microsphere solution; S32, adding DNP-BSA to the fluorescent microsphere solution activated in S31 to form a solution with a final concentration of 0.05-0.2 mg / ml; mixing, labeling on a shaker at room temperature in the dark for 1-3 hours, centrifuging and discarding the supernatant; resuspending the precipitated fluorescent microspheres in a 20-80 mmol / L phosphate buffer with a pH of 6.0-8.0; S33, add 5.0-10.0% BSA to the S32 microsphere solution; mix well, block on a shaker at room temperature for 1-2 hours, centrifuge and discard the supernatant; add the same volume of microsphere preservation solution as S1, disperse the microspheres, and obtain a DNP-BSA fluorescent microsphere solution with a final concentration of 0.05-0.2 mg / ml; The S4 includes: S41, diluting the prealbumin fluorescent microsphere solution formed by fluorescent microsphere-labeled prealbumin monoclonal antibody with microsphere storage solution 4 times; S42, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a first mixed solution; S43, spraying the first mixed solution onto the conjugate pad at 2-6 ul / cm and drying to obtain a finished fluorescent microsphere-labeled prealbumin conjugate pad; The S5 includes: S51, diluting the C-reactive protein fluorescent microsphere solution formed by labeling the C-reactive protein monoclonal antibody with the microsphere storage solution 4 times; S52, mixing with a DNP-BSA fluorescent microsphere solution formed by labeling DNP-BSA with fluorescent microspheres at a ratio of 100:(0.5-2) to form a second mixed solution; S53, spraying the second mixed solution on the conjugate pad at 2-6 ul / cm and drying to obtain a finished fluorescent microsphere-labeled C-reactive protein conjugate pad; The S6 includes: S61, diluting the prealbumin detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a first spray solution; S62, spraying the first spraying liquid on the chromatographic membrane at a spraying volume of 0.5-2.0 ul / cm, and drying to obtain a finished prealbumin chromatographic membrane; The S7 includes: S71, diluting the C-reactive protein detection antibody and the rabbit anti-DNP quality control antibody to 0.5-2.0 mg / ml respectively to form a second spray solution; S72, spraying the second spraying liquid on the chromatographic membrane at a spraying amount of 0.5-2.0 ul / cm, and drying to obtain a finished C-reactive protein chromatographic membrane; The S8 includes: The first sample pad, the first conjugate pad, the first chromatography membrane and the first absorbent paper are overlapped and fixed on the reaction bottom plate in sequence, and the prealbumin detection reagent strip is obtained after being cut by a strip cutter; The second sample pad, the second conjugate pad, the second chromatography membrane and the second absorbent paper are overlapped and fixed on the reaction bottom plate in sequence, and cut by a strip cutter to obtain a C-reactive protein detection reagent strip; The prealbumin detection reagent strip and the C-reactive protein detection reagent strip are loaded into the cartridge.

6. The method for preparing the prealbumin and C-reactive protein combined detection kit according to claim 5, characterized in that: The centrifugal conditions in S1, S2 and S3 are 10000-18000 rpm, and the centrifugal time is 10-20 minutes; the reaction is carried out on a shaker at room temperature, and the shaker speed is controlled at 100-200 rpm.

7. The method for preparing the prealbumin and C-reactive protein combined detection kit according to claim 6, characterized in that: The drying conditions of the finished fluorescent microsphere-labeled prealbumin binding pad, the finished fluorescent microsphere-labeled C-reactive protein binding pad, the finished prealbumin chromatography membrane and the finished C-reactive protein chromatography membrane are 30-45° C. and the drying time is 12-24 hours.

8. The method for preparing the prealbumin and C-reactive protein combined detection kit according to claim 7, characterized in that: The conjugate pad is pretreated by a pretreatment conjugate pad solution before the first mixed solution is sprayed on the conjugate pad and the second mixed solution is sprayed on the conjugate pad. The method for preparing the pretreatment conjugate pad solution is: 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, 0.1-5% Tween 20, pH 7.4; the conjugate pad is laid flat in the pretreatment conjugate pad solution and allowed to stand for 1-5 minutes, and then air-dried at 40-60°C for 1-2 hours.

9. The method for preparing the prealbumin and C-reactive protein combined detection kit according to claim 8, characterized in that: The first sample pad and the second sample pad are pretreated with a sample pad pretreatment solution, and the sample pad pretreatment solution is prepared by: 10-50mmol / L PB, 1%-5% sucrose, 0.1%-2% PVP, 0.5-5% BSA, 0.1%-2% S9, 0.1-2% Tween 20, pH 7.0-8.

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10. A method for using the prealbumin and C-reactive protein combined detection kit according to any one of claims 1 to 3, characterized in that: include: The prealbumin and C-reactive protein combined detection kit is loaded with a prealbumin sample diluent and a C-reactive protein sample diluent; Take 5ul of whole blood or serum sample, add it to the C-reactive protein sample diluent, mix well to form a first sample diluent, take 100ul of the first sample diluent and add it to the C-reactive protein sample well; 50ul of the first sample diluent after mixing is taken, and added to the prealbumin sample diluent, and mixed to form a second sample diluent, and 100ul of the second sample diluent is taken and added to the prealbumin sample well; After a period of time, the test card is inserted into the card slot of the adapter instrument to read the results; wherein, when the whole blood or serum sample is tested, the whole blood or serum sample is added to the corresponding sample addition hole, and under the capillary action, the whole blood or serum sample is chromatographed in the direction of the first absorbent paper, and the prealbumin antigen in the sample is combined with the prealbumin antibody on the first binding pad to form a prealbumin antigen-antibody complex, which is chromatographed onto the first chromatographic membrane and captured by the prealbumin antibody on the first detection line to form a prealbumin antibody-antigen-prealbumin antibody complex. The higher the prealbumin concentration in the whole blood or serum sample, the higher the fluorescent signal on the first detection line, and the fluorescent signal is collected by the detection instrument; the fluorescent signal on the first binding pad During the chromatography process, the DNP-BSA labeled with the optical microspheres is captured by the rabbit anti-DNP antibody on the first chromatographic membrane to form a DNP-BSA antigen-rabbit anti-DNP antibody complex, and the fluorescence signal of the first quality control line is collected by the detection instrument; the fluorescence intensity ratio of the test line and the quality control line is calculated, and it is substituted into the corresponding standard curve to obtain the concentration value of prealbumin in the whole blood or serum sample; similarly, the fluorescence intensity ratio of the second test line and the second quality control line of C-reactive protein is calculated and substituted into the corresponding standard curve to obtain the concentration value of C-reactive protein in the whole blood or serum sample; the normal reference range of the concentration value is: prealbumin 200-400 mg / L, C-reactive protein <10 mg / L.

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