MxA, CRP and SAA combined detection card and preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to an MxA, CRP and SAA combined detection card and a preparation method and application thereof, and the preparation method comprises the following steps: 1) preparing silicon-coated high-performance time-resolved fluorescent microspheres; (2) marking of an MxA antibody; (3) marking a CRP antibody; (4) marking an SAA antibody; (5) preparing a conjugate pad; (6) preparing a nitrocellulose membrane; and (7) assembling the reagent strip. The silicon-coated high-performance time-resolved fluorescent microspheres with the particle size range of 190-352 nm are prepared, the dye embedding rate is high, the sensitivity is high, the monodispersity is good, the stability is good, the hydrophilicity is high, agglomeration is not prone to occurring, and the dye embedding rate is increased. The MxA, CRP and SAA combined detection card prepared from the silicon-coated high-performance time-resolved fluorescent microspheres can improve the accuracy of identifying bacterial infection and virus infection, and is beneficial to early diagnosis and treatment of acute respiratory infectious diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and specifically relates to a combined detection card of MxA, CRP and SAA, a preparation method and use thereof. Background Art

[0002] Time-resolved fluorescence immunoassay is one of the three ultrasensitive immunoassay methods currently on par with chemiluminescence and electrochemiluminescence. Its principle is to use rare earth ions with a long fluorescence half-life as markers. It has the advantages of large Stokes shift and long life. During the test, as long as the measurement time is delayed and the signal of the marker is measured after the fluorescence of the background substance is fully decayed, the interference of various nonspecific fluorescence can be effectively eliminated, thereby improving the sensitivity of the detection.

[0003] Most of the traditional methods for preparing time-resolved fluorescent microspheres first prepare polystyrene microspheres by emulsion polymerization, and then embed the time-resolved fluorescent dye into the microspheres by swelling method. However, the dye embedding rate of the time-resolved fluorescent microspheres prepared by this method is low, the dye is easy to leak, and the polystyrene microspheres are highly hydrophobic and easy to agglomerate, affecting the performance. CN106221692A discloses a method for preparing monodisperse silica fluorescent microspheres, using silica as a seed core and silica-doped rare earth complexes as a surface coating, but this method has a low dye coverage rate.

[0004] Human myxovirus resistance protein A (MxA), full name Myxovirus resistance protein A, is an antiviral protein induced by type I interferon (IFN-α / β) with a molecular weight of about 78kD. MxA protein is connected to the smooth endoplasmic reticulum in the cytoplasm. By binding to the viral nucleocapsid, it changes its conformation, causing the virus to lose its nucleocapsid, thereby releasing viral nucleic acid, which is then degraded by nucleases in the cytoplasm to achieve an antiviral effect. The expression of MxA protein is closely related to a variety of viral infections and is very sensitive to viral reactions. Very small amounts of virus can induce cells to express MxA protein. Therefore, MxA protein can be used for the early diagnosis of viral infections and can also be used for the differential diagnosis of clinical viral infections and bacterial or other microbial infections.

[0005] C-reactive protein (CRP) is an acute phase protein with very low concentrations in healthy people. It increases sharply when the body is infected and tissues are damaged. C-reactive protein is a marker of inflammation and tissue damage. Its increase is related to the degree of infection. It can be used to detect routine inflammation and cardiovascular inflammation, and provide information for the diagnosis, treatment and monitoring of inflammatory diseases.

[0006] Serum amyloid A (SAA) begins to increase approximately 8 hours after the inflammatory response and increases significantly during viral infection, whereas CRP does not increase in viral infection without bacterial infection or may only increase slightly within a narrow range.

[0007] Acute respiratory infections are extremely common in clinical practice. They are usually mild and have a good prognosis, but the hospitalization rate and mortality rate of severe patients will increase significantly. Therefore, how to identify viral and bacterial infections early and diagnose and treat them in time has become an important clinical issue. The combined detection of MxA, SAA and CRP can make up for the lack of significant differences in CRP levels during viral infection, which is conducive to the early diagnosis of acute respiratory infectious diseases. However, there is currently no combined detection method for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A on the market. Summary of the invention

[0008] In view of the problems in the prior art such as easy aggregation of microspheres, low dye embedding rate, low detection sensitivity, and difficulty in early identification of viral and bacterial infections, the present invention aims to provide a combined detection card of MxA, CRP and SAA and its preparation method and application. The combined detection card is a detection card for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A based on silicon-coated high-performance time-resolved fluorescent microspheres.

[0009] Specifically, the present invention provides the following technical solutions: A method for preparing a combined detection card of MxA, CRP and SAA, characterized in that it comprises the following steps: (1) Preparation of high-performance time-resolved fluorescent microspheres coated with silicon SiO was first prepared by the sol-gel method. 2 Microspheres, and then the time-resolved fluorescent dye Eu (III) is embedded in the microspheres by ultrasonic swelling method; then the surface of the microspheres is double-bonded, and the time-resolved fluorescent dye Eu (III) is embedded in the microspheres by ultrasonic swelling method; finally, the surface of the microspheres is carboxy-modified by seed emulsion polymerization, and the time-resolved fluorescent dye Eu (III) is embedded in the microspheres by ultrasonic swelling method. This method uses silicon-coated time-resolved fluorescent microspheres. The large number of hydrophilic groups on the surface of the microspheres greatly improves the monodispersity of the microspheres. The multi-step embedding of the dye also greatly improves the embedding rate of the dye. The performance is stable. In the field of immunochromatography, the goal of quantitative detection of the object to be detected can be achieved with high sensitivity.

[0010] (2) Labeling of MxA antibodies ① Cleaning: Take the silica-coated high-performance time-resolved fluorescent microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; add the MxA-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 hours; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolve: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light; (3) CRP antibody markers ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; add the CRP-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 hours; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolve: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light; (4) Labeling of SAA antibodies ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; add the SAA-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 hours; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolve: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light; (5) Preparation of conjugate pad The MxA, CRP, and SAA antibody complexes labeled with silicon-coated high-performance time-resolved fluorescent microspheres were sprayed onto glass cellulose and dried at 45°C overnight to prepare a conjugate pad; (6) Preparation of nitrocellulose membrane Sheep anti-mouse IgG antibody, MxA coated antibody, CRP coated antibody and SAA coated antibody were sprayed onto nitrocellulose membrane using a streak meter, respectively, as quality control line C, MxA test line, CRP test line, SAA test line 3, and dried at 45°C overnight; (7) Reagent strip assembly The prepared nitrocellulose membrane is attached to the PVC base plate, the end coated with the silicon-coated high-performance time-resolved fluorescent microsphere antibody complex is attached to the binding pad, the sample pad is placed on the binding pad, and one end of the quality control line is attached to the absorbent pad.

[0011] Furthermore, the step (1) specifically includes: ① Preparation of SiO by sol-gel method 2 Microspheres: Add ammonia water to the ethanol aqueous solution, stir and disperse evenly, then add tetraethyl orthosilicate dropwise, and react overnight at 25-45°C; wherein the volume ratio of ethanol to water is 1:0-0.2, and the volume ratio of tetraethyl orthosilicate to ammonia water is 1:0.1-1.

[0012] ②Use ultrasonic swelling method to embed the time-resolved fluorescent dye Eu(III) complex: disperse the product in step ① in SDS aqueous solution to prepare emulsion 1, and dissolve the Eu(III) complex in DCM solution to prepare solution 1; then pour solution 1 into emulsion 1, and oscillate ultrasonically in the dark for 20-60 min; wherein the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1-1.

[0013] ③ Modify the double bonds on the surface of the microspheres by silane coupling reaction: disperse the product in step ② in ethanol, add ammonia water, stir and disperse evenly, add coupling agent vinyl triethoxy silane dropwise, and react at room temperature overnight; the volume ratio of vinyl triethoxy silane to ammonia water is 1:0.1-1.

[0014] ④ The time-resolved fluorescent dye Eu(Ⅲ) complex is embedded in the ultrasonic swelling method: the product in step ③ is dispersed in the SDS aqueous solution to prepare emulsion 2, and the Eu(Ⅲ) complex is dissolved in the DCM solution to prepare solution 2; then solution 2 is poured into emulsion 2, and oscillate ultrasonically for 20-60 min in the dark; wherein the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1-1.

[0015] ⑤Use seed emulsion polymerization to modify the surface carboxyl groups of microspheres: Disperse the product in step ④ in aqueous solution and pass N 2Deoxygenate, then add a proper amount of surfactant sodium dodecyl sulfate, after it dissolves, add a proper amount of carboxylic acid monomer methacrylic acid, add initiator potassium persulfate at 60°C, and react at 70-85°C overnight; wherein the mass ratio of carboxylic acid monomer methacrylic acid to initiator is 1:0.01-0.03.

[0016] ⑥Use ultrasonic swelling method to embed the time-resolved fluorescent dye Eu (III) complex: disperse the product in step ⑤ in SDS aqueous solution to prepare emulsion 3, and dissolve the Eu (III) complex in DCM solution to prepare solution 3; then pour solution 3 into emulsion 3, and oscillate ultrasonically in the dark for 20-60 min; wherein the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1-1.

[0017] The present invention uses the above-mentioned silicon-coated high-performance time-resolved fluorescent microspheres as tracer markers, which are coupled with labeled antibodies. When encountering antigens in the sample, they bind to them to form antigen-antibody complexes. The complexes flow on the reaction membrane and form double-antibody sandwich complexes with the detection antibodies fixed on the membrane. The method is fast, simple, and low-cost, and is widely used in content detection.

[0018] The three-in-one combined detection card of MxA, CRP and SAA includes a nitrocellulose membrane, a conjugation pad, a sample pad and a water absorbent pad arranged on the bottom plate. Among them, sheep anti-mouse IgG antibody, MxA coated antibody, CRP coated antibody and SAA coated antibody are arranged on the nitrocellulose membrane as quality control line C, detection line 1 (MxA), detection line 2 (CRP) and detection line 3 (SAA) respectively; the conjugation pad is placed on one end of the NC membrane, and the conjugation pad is coated with MxA labeled antibody, CRP labeled antibody and SAA labeled antibody labeled with silicon-coated high-performance time-resolved fluorescent microspheres; the sample pad is placed on the conjugation pad; the water absorbent pad is arranged at one end of the quality control line. By using silicon-coated high-performance time-resolved fluorescent microspheres to label MxA antibody, CRP antibody and SAA antibody, the three-in-one product can meet the performance requirements of the three detection indicators at the same time with the same sample diluent, the same sample dosage, the same reaction time and the same reagent strip, with high sensitivity, aiming to improve the accuracy of distinguishing bacterial infection and viral infection.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention prepares a silica-encapsulated high-performance time-resolved fluorescent microsphere with a particle size range of 190-352 nm, which has a high dye embedding rate, high sensitivity, good monodispersity, good stability, strong hydrophilicity, and is not easy to agglomerate.

[0020] (2) The silicon-encapsulated high-performance time-resolved fluorescent microspheres of the present invention use layer-by-layer encapsulation of the dye, which greatly improves the encapsulation rate of the dye.

[0021] (3) The product of the present invention can achieve the goal of high-sensitivity quantitative detection of the object to be detected in the field of immunochromatography.

[0022] (4) The present invention uses the silicon-coated high-performance time-resolved fluorescent microspheres to prepare a three-in-one detection kit for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid protein A. Combined detection can improve the accuracy of distinguishing bacterial infection from viral infection, which is beneficial for the early diagnosis and treatment of acute respiratory infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the MxA standard curve.

[0024] Figure 2 is the CRP standard curve.

[0025] Figure 3 is the SAA standard curve.

[0026] Figure 4 The results are compared between the detection kit of the present invention and the Zhongyuan detection kit.

[0027] Figure 5 The results are compared between the detection kit of the present invention and the detection kit of Mindray.

[0028] Figure 6 The results are compared between the detection kit of the present invention and the detection kit of Novozymes. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0030] Example 1 (1) Preparation of time-resolved fluorescent dye Eu(III) 366 mg EuCl 3 6H 2 O and 672 mg DBM were dissolved in 20 mL anhydrous ethanol, and then 300 μL Et 3 N, and finally add 283 mg of phen. After precipitation occurs, stir for another 30 min, filter and dry, and store in dark place for later use.

[0031] (2) Preparation of high-performance time-resolved fluorescent microspheres coated with silicon ①SiO 2 Preparation of microspheres SiO was prepared by sol-gel method. 2 Microspheres, 10 mL of water and 90 mL of ethanol were measured in a three-necked flask, ultrasonically mixed, and the three-necked flask was transferred to a constant temperature water bath, the temperature was set to 40 ° C, and the stirring speed was 280 rpm. Then 5 mL of ammonia water was quickly added, stirred for 10 minutes, and finally 5 mL of tetraethyl orthosilicate was added dropwise, and the reaction was allowed to proceed overnight. After the reaction, the anhydrous ethanol was washed three times, the pure water was washed three times, and finally stored in pure water for use.

[0032] ②Eu(Ⅲ) embedded dye Take 1 g of the microspheres in step ① and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 1; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonic to prepare solution 1; pour solution 1 into emulsion 1 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with pure water and three times with anhydrous ethanol, and finally store it in anhydrous ethanol for use.

[0033] ③SiO 2 Double bond modification on microsphere surface Disperse the product in step ② in 200 mL of anhydrous ethanol, and place the reaction bottle in a 30°C water bath after ultrasonic dispersion. Then add 2 mL of ammonia water, stir at 280 rpm for 10 min, add 3 mL of vinyl triethoxysilane dropwise, and react overnight. After the reaction, wash three times with anhydrous ethanol and three times with pure water, and finally store in pure water for later use.

[0034] ④Eu(Ⅲ) embedded dye Take 1 g of the microspheres in step ③ and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 2; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonic to prepare solution 2; pour solution 2 into emulsion 2 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with anhydrous ethanol and three times with pure water, and finally store it in pure water for use.

[0035] ⑤ Carboxyl modification of microsphere surface The carboxyl group was modified by seed emulsion polymerization. Take 1 g of the product in step ④ and disperse it in 200 mL of pure water. Fill the system with nitrogen for 30 min to remove oxygen. Then add 60 mg SDS and stir to dissolve for 10 min. Add 5 mL of methacrylic acid and set the water bath temperature to 75 °C. When the temperature reaches 60 °C, add 50 mg KPS and react at 300 rpm overnight. After the reaction, wash with pure water three times, wash with anhydrous ethanol three times, and finally save the anhydrous ethanol for use.

[0036] ⑥Eu(Ⅲ) embedded dye Take 1 g of the microspheres in step ⑤ and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 3; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonication to prepare solution 3; pour solution 3 into emulsion 3 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with anhydrous ethanol and three times with pure water, and finally store it in pure water for use, and adjust the solid content to 1% for use.

[0037] Example 2 (1) Preparation of time-resolved fluorescent dye Eu(III) 366 mg EuCl 3 6H 2 O and 672 mg DBM were dissolved in 20 mL anhydrous ethanol, and then 300 μL Et 3 N, and finally add 283 mg of phen. After precipitation occurs, stir for another 30 min, filter and dry, and store in dark place for later use.

[0038] (2) Preparation of high-performance time-resolved fluorescent microspheres coated with silicon ①SiO 2 Preparation of microspheres SiO was prepared by sol-gel method. 2 Microspheres, measure 10 mL of water and 90 mL of ethanol in a three-necked flask, mix with ultrasound, transfer the three-necked flask to a constant temperature water bath, set the temperature to 40°C, and stir at 280rpm. Then quickly add 10 mL of ammonia water, stir for 10 min, and finally add 10 mL of tetraethyl orthosilicate dropwise, and react overnight. After the reaction, wash three times with anhydrous ethanol, wash three times with pure water, and finally store in pure water for use.

[0039] ②Eu(Ⅲ) embedded dye Take 1 g of the microspheres in step ① and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 1; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonic to prepare solution 1; pour solution 1 into emulsion 1 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with pure water and three times with anhydrous ethanol, and finally store it in anhydrous ethanol for use.

[0040] ③SiO 2 Double bond modification on microsphere surface Disperse the product in step ② in 200 mL of anhydrous ethanol, and place the reaction bottle in a 30°C water bath after ultrasonic dispersion. Then add 2 mL of ammonia water, stir at 280 rpm for 10 min, add 3 mL of vinyl triethoxysilane dropwise, and react overnight. After the reaction, wash three times with anhydrous ethanol and three times with pure water, and finally store in pure water for later use.

[0041] ④Embedding dye Eu(Ⅲ) Take 1 g of the microspheres in step ③ and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 2; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonic to prepare solution 2; pour solution 2 into emulsion 2 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with anhydrous ethanol and three times with pure water, and finally store it in pure water for use.

[0042] ⑤ Carboxyl modification of microsphere surface The carboxyl group was modified by seed emulsion polymerization. Take 1 g of the product in step ④ and disperse it in 200 mL of pure water. Fill the system with nitrogen for 30 min to remove oxygen. Then add 60 mg SDS and stir to dissolve for 10 min. Add 5 mL of methacrylic acid and set the water bath temperature to 75 °C. When the temperature reaches 60 °C, add 50 mg KPS and react at 300 rpm overnight. After the reaction, wash with pure water three times, wash with anhydrous ethanol three times, and finally save the anhydrous ethanol for use.

[0043] ⑥Eu(Ⅲ) embedded dye Take 1 g of the microspheres in step ⑤ and disperse them in 20 mL of 0.25% SDS aqueous solution to prepare emulsion 3; weigh 200 mg of time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of swelling agent DCM and dissolve it by ultrasonication to prepare solution 3; pour solution 3 into emulsion 3 and oscillate and ultrasonicate for 30 min at room temperature in the dark. After the reaction, wash it three times with anhydrous ethanol and three times with pure water, and finally store it in pure water for use, and adjust the solid content to 1% for use.

[0044] It can be seen from Table 1 that the particle sizes of the silicon-coated time-resolved fluorescent microspheres in Example 1 and Example 2 are 190.9 nm and 352 nm, respectively, and the dispersion index is less than 0.05. The particle size is uniform and has good monodispersity.

[0045] Table 1 Particle size and dispersion index of silicon-encapsulated time-resolved fluorescent microspheres prepared in Example 1 and Example 2 Example 3 A method for preparing a detection card for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid protein A based on silicon-coated high-performance time-resolved fluorescent microspheres, the specific steps of which are: (1) Labeling of MxA antibody ① Cleaning: Take 50 μL of the silica-coated time-resolved fluorescent microspheres of Example 1 and add them to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer, mix by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add 1 mL of 50 mM MES (pH 6.0) buffer to the cleaned silica-coated time-resolved fluorescent microspheres of Example 1, add 10 μL each of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) after ultrasonic mixing, vortex mix, activate at room temperature in the dark for 30 min, centrifuge and discard the supernatant; ③ Coupling: add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescent microspheres of Example 1, and mix by ultrasonication; add the MxA labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add 50 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add 500 μL of microsphere reconstitution solution to the silicon-coated time-resolved fluorescent microsphere-antibody complex of Example 1 and store at 2-8°C in the dark. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2±0.05), 150 mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0046] (2) Labeling of CRP antibodies (For labeling of CRP antibodies, using large-diameter microspheres similar to those in Example 2 can improve the detection sensitivity. Of course, in other variations of the beneficial embodiments, using small-diameter microspheres similar to those in Example 1 is also possible) ① Cleaning: Take 50 μL of the silica-coated time-resolved fluorescent microspheres of Example 2 and add them to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer, mix by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add 1 mL of 50 mM MES (pH 6.0) buffer to the cleaned silica-coated time-resolved fluorescent microspheres of Example 2, add 10 μL each of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) after ultrasonic mixing, vortex mix, activate at room temperature in the dark for 30 min, and centrifuge to discard the supernatant; ③ Coupling: add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescent microspheres of Example 2, and mix by ultrasonication; add the CRP-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add 50 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add 500 μL of microsphere reconstitution solution to the silicon-coated time-resolved fluorescent microsphere-antibody complex of Example 2 and store at 2-8°C in the dark. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2±0.05), 150 mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0047] (3) Labeling of SAA antibodies ① Cleaning: Take 50 μL of the silica-coated time-resolved fluorescent microspheres of Example 1 and add them to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer, mix by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add 1 mL of 50 mM MES (pH 6.0) buffer to the cleaned silica-coated time-resolved fluorescent microspheres of Example 1, add 10 μL each of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) after ultrasonic mixing, vortex mix, activate at room temperature in the dark for 30 min, centrifuge and discard the supernatant; ③ Coupling: add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescent microspheres of Example 1, and mix by ultrasonication; add the SAA-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add 50 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add 500 μL of microsphere reconstitution solution to the silicon-coated time-resolved fluorescent microsphere-antibody complex of Example 1 and store at 2-8°C in the dark. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2±0.05), 150 mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0048] (4) Preparation of nitrocellulose membrane The MxA-coated antibody with a concentration of 2 mg / mL was drawn on the nitrocellulose membrane with a coating amount of 1 μL / cm as test line 1, the CRP-coated antibody with a concentration of 2 mg / mL was drawn on the nitrocellulose membrane with a coating amount of 1 μL / cm as test line 2, the SAA-coated antibody with a concentration of 2 mg / mL was drawn on the nitrocellulose membrane with a coating amount of 1 μL / cm as test line 3, and the goat anti-mouse IgG antibody with a concentration of 1 mg / mL was drawn on the nitrocellulose membrane with a coating amount of 1 μL / cm as quality control line, and dried at 45°C overnight.

[0049] (5) Preparation of conjugate pad The MxA antibody complex, CRP antibody complex, and SAA antibody complex labeled with silicon-coated time-resolved fluorescent microspheres were sprayed onto the glass cellulose membrane at a coating volume of 7 μL / cm and dried at 45° C. overnight to prepare a conjugate pad.

[0050] (6) Reagent strip assembly Stick the coated nitrocellulose membrane on the PVC base plate, stick one end of the marking line on the binding pad, put the sample pad on the binding pad, and stick one end of the quality control line on the absorbent pad.

[0051] Application Examples (1) MxA sensitivity and linear range detection Different concentrations of MxA calibrators were used for calibration test, the reaction time was 8 min, 80 μL of diluent was added to the sample or calibrator, and 80 μL of diluent was taken and then the sample was added to the test card of Example 3. The experimental results are shown in Table 2.

[0052] Table 2 The concentration of MxA antigen calibrator was used as the horizontal axis and the measured fluorescence signal value was used as the vertical axis to establish a fitting standard curve. The results are shown in Figure 1 As shown, the linearity of the detection results is good, and the concentration of MxA protein in the sample can be quantitatively tested by this standard curve.

[0053] (2) CRP sensitivity and linear range detection Different concentrations of CRP calibrators were used for calibration test. The reaction time was 8 minutes. 80 μL of diluent was added to the sample or calibrator. 80 μL of diluent was taken and then the sample was added to the test card. The experimental results are shown in Table 3.

[0054] Table 3 The concentration of CRP antigen calibrator is the horizontal axis, and the measured fluorescence signal value is the vertical axis. The fitting standard curve is established. The results are as follows: Figure 2 As shown, the linearity of the test results is good, and the CRP protein concentration in the sample can be quantitatively tested through this standard curve.

[0055] (3) SAA sensitivity and linear range detection SAA calibrators of different concentrations were used for calibration curve test. The reaction time was 8 minutes. 80 μL of diluent was added to the sample or calibrator. 80 μL of diluent was taken and then the sample was added to the test card. The experimental results are shown in Table 4.

[0056] Table 4 The concentration of SAA antigen calibrator is the horizontal axis, and the measured fluorescence signal value is the vertical axis. The fitting standard curve is established. The results are as follows: Figure 3 As shown, the linearity of the detection results is good, and the SAA protein concentration in the sample can be quantitatively tested through this standard curve.

[0057] (4) Clinical sample testing Whole blood samples were collected from 70 patients with suspected respiratory tract infection in the fever clinic, and the MxA concentration of the whole blood samples was detected and assigned using the Zhongyuan detection kit. The three-in-one kit prepared by the above-mentioned silicon-coated high-performance time-resolved fluorescent microspheres for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid protein A detection card was used for simultaneous detection, and the corresponding concentrations were fitted. The results are shown in Figure 4 , R 2 =0.9574, indicating that the test result has a good correlation with the Zhongyuan test.

[0058] 55 whole blood samples with CRP values ​​assigned by Mindray 7500 were collected and tested simultaneously using a three-in-one test kit prepared by silicon-coated high-performance time-resolved fluorescent microspheres for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A test cards, and the corresponding concentrations were obtained by fitting and calculation. Figure 5 , R 2 =0.9785, indicating that the test result has a good correlation with Mindray's target value.

[0059] 45 whole blood samples were collected and tested for SAA value by Novozymes serum amyloid A test kit (quantum dot fluorescence immunoassay), and tested simultaneously using a three-in-one test kit prepared by silicon-coated high-performance time-resolved fluorescent microspheres for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A test card, and the corresponding concentrations were obtained by fitting. Figure 6 , R 2 =0.9588, indicating that the test results have a good correlation with the Norvegicus test.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a combined detection card of MxA, CRP and SAA, characterized in that: The following steps are involved: (1) Preparation of high-performance time-resolved fluorescent microspheres coated with silicon First, SiO2 microspheres were prepared by the sol-gel method, and then the time-resolved fluorescent dye Eu (III) was embedded in the microspheres by the ultrasonic swelling method; then the microsphere surface was double-bonded, and then the time-resolved fluorescent dye Eu (III) was embedded in the microspheres by the ultrasonic swelling method; finally, the microsphere surface was carboxyl-modified by the seed emulsion polymerization method, and then the time-resolved fluorescent dye Eu (III) was embedded in the microspheres by the ultrasonic swelling method; (2) Labeling of MxA antibody; (3) Labeling of CRP antibody; (4) Labeling of SAA antibody; (5) Preparation of conjugate pad; (6) Preparation of nitrocellulose membrane; (7) Assembly of reagent strip.

2. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 1, characterized in that: The step (1) specifically includes: ① The SiO2 microspheres were prepared by the sol-gel method: ammonia water was added to the ethanol aqueous solution, and after being stirred and dispersed evenly, tetraethyl orthosilicate was added dropwise, and the reaction was carried out at 25-45°C overnight; the volume ratio of ethanol to water was 1:0-0.2, and the volume ratio of tetraethyl orthosilicate to ammonia water was 1:0.1-1; ②Using ultrasonic swelling method to embed the time-resolved fluorescent dye Eu(III) complex: disperse the product in step ① in SDS aqueous solution to prepare emulsion 1, dissolve the Eu(III) complex in DCM solution to prepare solution 1; then pour solution 1 into emulsion 1, and oscillate ultrasonically in the dark; ③ Modification of double bonds on the surface of microspheres by silane coupling reaction: Disperse the product in step ② in ethanol, add ammonia water, stir and disperse evenly, add coupling agent vinyl triethoxysilane dropwise, and react at room temperature overnight; ④Using ultrasonic swelling method to embed the time-resolved fluorescent dye Eu(Ⅲ) complex: disperse the product in step ③ in SDS aqueous solution to prepare emulsion 2, dissolve the Eu(Ⅲ) complex in DCM solution to prepare solution 2; then pour solution 2 into emulsion 2, and oscillate ultrasonically in the dark; ⑤ Use seed emulsion polymerization to modify the surface of microspheres with carboxyl groups: disperse the product in step ④ in an aqueous solution, pass N2 to deoxygenate, then add an appropriate amount of surfactant sodium dodecyl sulfate, after it is dissolved, add an appropriate amount of carboxylic acid monomer methacrylic acid, add initiator potassium persulfate at 60°C, and react at 70-85°C overnight; ⑥Use ultrasonic swelling method to embed the time-resolved fluorescent dye Eu(III) complex: disperse the product in step ⑤ in SDS aqueous solution to prepare emulsion 3, dissolve the Eu(III) complex in DCM solution to prepare solution 3; then pour solution 3 into emulsion 3 and oscillate ultrasonically in the dark.

3. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 2, characterized in that: The step (2) is specifically as follows: ① Cleaning: Take the silica-coated high-performance time-resolved fluorescent microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; Add the MxA-labeled antibody to be coupled, vortex to mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light.

4. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 3, characterized in that: The step (3) is specifically as follows: ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; Add the CRP-labeled antibody to be coupled, vortex to mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light.

5. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 4, characterized in that: The step (4) specifically comprises: ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer, mix them by ultrasonication, centrifuge and discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescent microspheres, mix by ultrasonication, add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 minutes, centrifuge and discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescent microspheres and mix them by ultrasonication; Add the SAA-labeled antibody to be coupled, vortex to mix, and couple at room temperature in the dark for 2 h; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block for 1 hour at room temperature in the dark, and centrifuge and discard the supernatant; ⑤ Redissolution: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescent microsphere-antibody complex and store at 2-8℃ away from light.

6. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 5, characterized in that: When the time-resolved fluorescent dye Eu(III) complex is embedded by ultrasonic swelling method, the ultrasonic wave is oscillated for 20-60 min in a dark environment; wherein the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1-1; When the double bonds on the microsphere surfaces are modified by silane coupling reaction, the volume ratio of vinyltriethoxysilane to ammonia water is 1:0.1-1; When the surface of the microspheres is modified with carboxyl groups by seed emulsion polymerization, the mass ratio of the carboxylic acid monomer methacrylic acid to the initiator is 1:0.01-0.

03.

7. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 6, characterized in that: The step (5) specifically comprises spraying the MxA, CRP, and SAA antibody complex labeled with silicon-coated high-performance time-resolved fluorescent microspheres onto glass cellulose, and drying at 45° C. overnight to prepare a conjugate pad.

8. The method for preparing the combined detection card of MxA, CRP and SAA according to claim 7, characterized in that: The step (6) specifically comprises spraying goat anti-mouse IgG antibody, MxA coated antibody, CRP coated antibody and SAA coated antibody onto nitrocellulose membrane respectively using a streaking instrument as quality control line C, MxA detection line, CRP detection line and SAA detection line 3, and drying at 45° C. overnight; The step (7) specifically comprises attaching the prepared nitrocellulose membrane to a PVC base plate, attaching one end coated with the silicon-coated high-performance time-resolved fluorescent microsphere antibody complex to a binding pad, placing the sample pad on the binding pad, and attaching one end of the quality control line to a water-absorbing pad.

9. A combined detection card of MxA, CRP and SAA prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the combined detection card according to claim 9 in preparing a detection kit for MxA, CRP and SAA.

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