A combined detection card for MxA, CRP and SAA, its preparation method and application

Abstract

The present invention relates to the field of biological detection technology, in particular to a combined detection card for MxA, CRP and SAA, and its preparation method and application. The preparation method includes the following steps: 1) Preparation of silica-coated high-performance time-resolved fluorescence microspheres; 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. The present invention prepares silica-coated high-performance time-resolved fluorescence microspheres with a particle size range of 190-352 nm, which have a high dye embedding rate, high sensitivity, good monodispersity, good stability, strong hydrophilicity, are not easy to agglomerate, and improve the dye embedding rate. The combined detection card for MxA, CRP and SAA prepared by using the silica-coated high-performance time-resolved fluorescence microspheres of the present invention can improve the accuracy of differentiating bacterial infection and viral infection, and is beneficial to the 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 for MxA, CRP and SAA, a preparation method and uses thereof. Background Art

[0002] As one of the three hypersensitive immunoassay methods currently alongside chemiluminescence and electrochemiluminescence, the principle of time-resolved fluorescence immunoassay is to use rare earth ions with a relatively long fluorescence half-life as labels, which has advantages such as a large Stokes shift and a long lifetime. During the testing process, as long as the measurement time is delayed and the fluorescence of the background substances is allowed to decay sufficiently before measuring the signal of the label, various non-specific fluorescence interferences can be effectively eliminated, and the detection sensitivity can be improved.

[0003] Most traditional methods for preparing time-resolved fluorescence microspheres first prepare polystyrene microspheres by emulsion polymerization and then embed time-resolved fluorescent dyes into the microspheres by a swelling method. However, the time-resolved fluorescence microspheres prepared by this method have a low dye embedding rate, easy dye leakage, and the polystyrene microspheres have high hydrophobicity and are prone to aggregation, affecting the performance. CN106221692A discloses a method for preparing monodisperse silica fluorescent microspheres, using silica as the seed core and silica-doped rare earth complexes as the surface coating, but this method has a low dye coating rate.

[0004] Human myxovirus resistance protein A (MxA), also known as Myxovirus resistance protein A, is an antiviral protein induced by type I interferon (IFN-α / β), with a molecular weight of about 78 kD. The MxA protein is connected to the smooth endoplasmic reticulum in the cytoplasm. By binding to the viral nucleocapsid and changing its configuration, the virus loses its nucleocapsid, thereby releasing viral nucleic acids, which are then degraded by endonucleases in the cytoplasm, achieving an antiviral effect. The expression of the MxA protein is closely related to various viral infections, is very sensitive to viruses, and a very small amount of virus can induce cells to express the MxA protein. Therefore, the MxA protein can be used for the early diagnosis of viral infections and also for the differential diagnosis of clinical viral infections and bacterial or other microbial infections.

[0005] C-reactive protein (CRP) is an acute-phase reaction protein, with a very low concentration in healthy people and a sharp increase when the body is infected and tissues are damaged. C-reactive protein is a marker of inflammation and tissue damage, and the degree of its increase is related to the degree of infection. It can be used for the detection of conventional inflammation and cardiovascular inflammation, providing information for the diagnosis, treatment and monitoring of inflammatory diseases.

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

[0007] Acute respiratory infections are extremely common in clinical practice. Usually, the condition is mild and the prognosis is good, but the hospitalization rate and mortality rate of severe patients will increase significantly. Therefore, how to early identify viral and bacterial infections and provide timely diagnosis and treatment has become an important clinical issue. The combined detection of MxA, SAA, and CRP can make up for the lack of significant difference in CRP levels during viral infections, which is beneficial to the early diagnosis of acute respiratory infectious diseases. However, there is currently no combined detection method for human mucovirus resistance protein A, C-reactive protein, and human serum amyloid A on the market. Summary of the Invention

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

[0009] Specifically, the present invention provides the following technical solutions:

[0010] A preparation method of a combined detection card for MxA, CRP, and SAA, characterized by comprising the following steps:

[0011] (1) Preparation of silica-coated high-performance time-resolved fluorescence microspheres

[0012] First, SiO2 microspheres are prepared by the sol-gel method, and then the time-resolved fluorescent dye Eu(Ⅲ) is embedded into the microspheres by the ultrasonic swelling method; then the surface of the microspheres is modified with double bonds, and then the time-resolved fluorescent dye Eu(Ⅲ) is embedded into the microspheres by the ultrasonic swelling method; finally, the surface of the microspheres is modified with carboxyl groups by the seed emulsion polymerization method, and then the time-resolved fluorescent dye Eu(Ⅲ) is embedded into the microspheres by the ultrasonic swelling method. This method uses silica-coated time-resolved fluorescence microspheres. A large number of hydrophilic groups on the surface of the microspheres greatly improve the monodispersity of the microspheres, and the multi-step embedding of dyes also greatly improves the embedding rate of the dyes. The performance is stable, and the goal of quantitative detection of the analyte can be achieved in the field of immunochromatography, with high sensitivity.

[0013] (2) Labeling of MxA antibody

[0014] ① Washing: Take the silica-coated high-performance time-resolved fluorescence microspheres and add them to a centrifuge tube containing MES buffer. Mix well by sonication and centrifuge to discard the supernatant.

[0015] ② Activation: Add MES buffer to the washed silica-coated high-performance time-resolved fluorescence microspheres. After mixing well by sonication, add freshly prepared EDC and NHS, and activate at room temperature in the dark for 30 min. Centrifuge to discard the supernatant.

[0016] ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescence microspheres and mix well by sonication. Add the antibody to be conjugated with MxA, vortex to mix well, and couple at room temperature in the dark for 2 h.

[0017] ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant.

[0018] ⑤ Reconstitution: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescence microsphere antibody complex and store in the dark at 2 - 8 °C.

[0019] (3) Labeling of CRP antibody

[0020] ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer. Mix well by sonication and centrifuge to discard the supernatant.

[0021] ② Activation: Add MES buffer to the washed silica-coated high-performance time-resolved fluorescence microspheres. After mixing well by sonication, add freshly prepared EDC and NHS, and activate at room temperature in the dark for 30 min. Centrifuge to discard the supernatant.

[0022] ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescence microspheres and mix well by sonication. Add the antibody to be conjugated with CRP, vortex to mix well, and couple at room temperature in the dark for 2 h.

[0023] ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant.

[0024] ⑤ Reconstitution: Add the microsphere reconstitution solution to the silica-coated high-performance time-resolved fluorescence microsphere antibody complex and store in the dark at 2 - 8 °C.

[0025] (4) Labeling of SAA antibody

[0026] ① Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer. Mix well by sonication and centrifuge to discard the supernatant.

[0027] ② Activation: Add MES buffer to the washed silica-coated high-performance time-resolved fluorescence microspheres. After mixing well by sonication, add freshly prepared EDC and NHS, and activate at room temperature in the dark for 30 min. Centrifuge to discard the supernatant.

[0028] ③ Coupling: Add MES buffer to the activated high-performance time-resolved fluorescence microspheres encapsulated with silicon, and mix well by ultrasonic treatment; add the antibody labeled with SAA to be coupled, vortex to mix well, and couple at room temperature in the dark for 2 h;

[0029] ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant;

[0030] ⑤ Reconstitution: Add microsphere reconstitution solution to the antibody complex of high-performance time-resolved fluorescence microspheres encapsulated with silicon, and store in the dark at 2 - 8 °C;

[0031] (5) Preparation of conjugate pad

[0032] Spray the antibody complexes of MxA, CRP, and SAA labeled with high-performance time-resolved fluorescence microspheres encapsulated with silicon onto glass cellulose, and dry overnight at 45 °C to prepare a conjugate pad;

[0033] (6) Preparation of nitrocellulose membrane

[0034] Spray goat anti-mouse IgG antibody, MxA coating antibody, CRP coating antibody, and SAA coating antibody onto the nitrocellulose membrane respectively with a scribing instrument as the quality control line C, MxA test line, CRP test line, and SAA test line 3, and dry overnight at 45 °C;

[0035] (7) Assembly of test strip

[0036] Stick the prepared nitrocellulose membrane on the PVC bottom plate, stick the conjugate pad at the end coated with the antibody complex of high-performance time-resolved fluorescence microspheres encapsulated with silicon, place the sample pad on the conjugate pad, and stick the quality control line end on the absorbent pad.

[0037] Further, the step (1) specifically includes:

[0038] ① Prepare SiO2 microspheres by sol-gel method: Add ammonia water to an ethanol aqueous solution, stir and disperse evenly, and then dropwise add tetraethyl orthosilicate, 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.

[0039] ② Embed the time-resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ① in an SDS aqueous solution to prepare emulsion 1, dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 1; then pour solution 1 into emulsion 1, and oscillate and ultrasonicate 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.

[0040] 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 to disperse evenly, and dropwise add the coupling agent vinyltriethoxysilane, and react overnight at room temperature; the volume ratio of vinyltriethoxysilane to ammonia water is 1:0.1 - 1.

[0041] ④ Entrap the time - resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ③ in an SDS aqueous solution to prepare emulsion 2, and dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 2; then pour solution 2 into emulsion 2, and ultrasonically oscillate in the dark for 20 - 60 min; among them, the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1 - 1.

[0042] ⑤ Modify the carboxyl groups on the surface of the microspheres by seed emulsion polymerization: Disperse the product in step ④ in an aqueous solution, purge with N2 to remove oxygen, then add an appropriate amount of the surfactant sodium dodecyl sulfate, and after dissolution, add an appropriate amount of the carboxylic acid monomer methacrylic acid, and add the initiator potassium persulfate at 60 °C, and react overnight at 70 - 85 °C; among them, the mass ratio of the carboxylic acid monomer methacrylic acid to the initiator is 1:0.01 - 0.03.

[0043] ⑥ Entrap the time - resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ⑤ in an SDS aqueous solution to prepare emulsion 3, and dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 3; then pour solution 3 into emulsion 3, and ultrasonically oscillate in the dark for 20 - 60 min; among them, the volume ratio of the SDS aqueous solution to the DCM solution is 1:0.1 - 1.

[0044] The present invention uses the above - mentioned silica - coated high - performance time - resolved fluorescence microspheres as a tracer marker, couples with a labeled antibody, and binds to the antigen in the sample when encountering it to form an antigen - antibody complex. This complex flows on the reaction membrane and forms a double - antibody sandwich complex with the detection antibody immobilized on the membrane. This method is fast, simple, and has a low cost, and is widely used in the detection of content.

[0045] The triple-combined detection card for MxA, CRP and SAA comprises a nitrocellulose membrane, a conjugate pad, a sample pad and an absorbent pad disposed on a bottom plate. Among them, goat anti-mouse IgG antibody, MxA coated antibody, CRP coated antibody and SAA coated antibody are disposed on the nitrocellulose membrane as the quality control line C, the detection line 1 (MxA), the detection line 2 (CRP) and the detection line 3 (SAA) respectively; one end of the conjugate pad is overlapped on the NC membrane, and the conjugate pad is coated with MxA labeled antibody, CRP labeled antibody and SAA labeled antibody labeled by silica-coated high-performance time-resolved fluorescence microspheres; the sample pad is disposed on the conjugate pad; the absorbent pad is disposed at one end of the quality control line. By using silica-coated high-performance time-resolved fluorescence microspheres to label MxA antibody, CRP antibody and SAA antibody, the triple-combined product can meet the performance requirements of three detection indexes simultaneously on the same sample diluent, the same sample dosage, the same reaction time and the same test strip, with high sensitivity, aiming to improve the accuracy of differentiating bacterial infection and viral infection.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] (1) The present invention prepares a silica-coated high-performance time-resolved fluorescence 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.

[0048] (2) The silica-coated high-performance time-resolved fluorescence microspheres of the present invention adopt layer-by-layer embedding of dyes, greatly improving the embedding rate of the dyes.

[0049] (3) The product of the present invention can achieve the goal of high-sensitivity quantitative detection of the analyte in the field of immunochromatography.

[0050] (4) The triple detection kit for human myxovirus resistance protein A, C-reactive protein and human serum amyloid A prepared by using the silica-coated high-performance time-resolved fluorescence microspheres of the present invention can improve the accuracy of differentiating bacterial infection and viral infection through combined detection, which is beneficial to the early diagnosis and treatment of acute respiratory infectious diseases. Description of the Drawings

[0051] Figure 1 It is the standard curve of MxA.

[0052] Figure 2 It is the standard curve of CRP.

[0053] Figure 3 It is the standard curve of SAA.

[0054] Figure 4 It is the detection comparison result between the detection kit of the present invention and the Zhongyuan detection kit.

[0055] Figure 5Detection comparison results between the detection kit of the present invention and Mindray detection kit.

[0056] Figure 6 Detection comparison results between the detection kit of the present invention and Novizan detection kit. Specific embodiments

[0057] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] Example 1

[0059] (1) Preparation of time-resolved fluorescent dye Eu(Ⅲ)

[0060] 366 mg of EuCl3·6H2O and 672 mg of DBM are dissolved in 20 mL of absolute ethanol, then 300 μL of Et3N is added, and finally 283 mg of phen is added. After precipitation occurs, stir for another 30 min, filter and dry, and store in the dark for later use.

[0061] (2) Preparation of silica-coated high-performance time-resolved fluorescent microspheres

[0062] ① Preparation of SiO2 microspheres

[0063] SiO2 microspheres are prepared by the sol-gel method. Measure 10 mL of water and 90 mL of ethanol into a three-necked flask, mix well by ultrasonic, transfer the three-necked flask to a constant temperature water bath, set the temperature to 40 °C, and the stirring speed to 280 rpm. Then quickly add 5 mL of ammonia water, stir for 10 min, and finally dropwise add 5 mL of tetraethyl orthosilicate, and react overnight. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for later use.

[0064] ② Embedding dye Eu(Ⅲ)

[0065] Take 1 g of the microspheres in step ① and disperse them in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 1; weigh 200 mg of the time-resolved fluorescent dye Eu(Ⅲ) in 5 mL of the swelling agent DCM, and dissolve it by ultrasonic to prepare solution 1; pour solution 1 into emulsion 1, and oscillate and ultrasonic for 30 min at room temperature in the dark. After the reaction, wash three times with pure water and three times with absolute ethanol, and finally store in absolute ethanol for later use.

[0066] ③ Double bond modification on the surface of SiO2 microspheres

[0067] Disperse the product in step ② in 200 mL of absolute ethanol. After ultrasonic dispersion, place the reaction flask in a water bath at 30 °C. Then add 2 mL of ammonia water, stir at 280 rpm for 10 min, and add 3 mL of vinyltriethoxysilane dropwise. React overnight. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for later use.

[0068] ④ Embed the dye Eu(Ⅲ)

[0069] Take 1 g of the microspheres in step ③ and disperse them in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 2; weigh 200 mg of the time-resolved fluorescent dye Eu(Ⅲ) and dissolve it in 5 mL of the swelling agent DCM by ultrasonic treatment to prepare solution 2; pour solution 2 into emulsion 2, and ultrasonically oscillate at room temperature in the dark for 30 min. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for later use.

[0070] ⑤ Modify the carboxyl groups on the surface of the microspheres

[0071] Carboxyl modification is carried out by the seed emulsion polymerization method. Take 1 g of the product in step ④ and disperse it in 200 mL of pure water. Purge with nitrogen for 30 min to remove the oxygen in the system. Then add 60 mg of SDS and stir to dissolve for 10 min. Then add 5 mL of methacrylic acid, and set the temperature of the water bath to 75 °C. When the temperature reaches 60 °C, add 50 mg of KPS and react at 300 rpm overnight. After the reaction, wash three times with pure water and three times with absolute ethanol, and finally store in absolute ethanol for later use.

[0072] ⑥ Embed the dye Eu(Ⅲ)

[0073] Take 1 g of the microspheres in step ⑤ and disperse them in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 3; weigh 200 mg of the time-resolved fluorescent dye Eu(Ⅲ) and dissolve it in 5 mL of the swelling agent DCM by ultrasonic treatment to prepare solution 3; pour solution 3 into emulsion 3, and ultrasonically oscillate at room temperature in the dark for 30 min. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for later use. Adjust the solid content to 1% for later use.

[0074] Example 2

[0075] (1) Preparation of the time-resolved fluorescent dye Eu(Ⅲ)

[0076] Dissolve 366 mg of EuCl3·6H2O and 672 mg of DBM in 20 mL of absolute ethanol, then add 300 μL of Et3N, and finally add 283 mg of phen. After precipitation occurs, stir for another 30 min, filter and dry, and store in the dark for later use.

[0077] (2)Preparation of silica-coated high-performance time-resolved fluorescence microspheres

[0078] ① Preparation of SiO2 microspheres

[0079] SiO2 microspheres were prepared by the sol-gel method. Measure 10 mL of water and 90 mL of ethanol into a three-necked flask, mix well by ultrasonic, transfer the three-necked flask to a constant temperature water bath, set the temperature at 40 °C, and the stirring speed at 280 rpm. 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 absolute ethanol and three times with pure water, and finally store in pure water for use.

[0080] ② Embedding dye Eu(Ⅲ)

[0081] Take 1 g of the microspheres in step ① and disperse them in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 1; weigh 200 mg of the time-resolved fluorescence dye Eu(Ⅲ) in 5 mL of the swelling agent DCM, dissolve it by ultrasonic to prepare solution 1; pour solution 1 into emulsion 1, and oscillate and ultrasonicate at room temperature in the dark for 30 min. After the reaction, wash three times with pure water and three times with absolute ethanol, and finally store in absolute ethanol for use.

[0082] ③ Double bond modification on the surface of SiO2 microspheres

[0083] Disperse the product in step ② in 200 mL of absolute ethanol, after ultrasonic dispersion, place the reaction flask in a 30 °C water bath. Then add 2 mL of ammonia water, stir at 280 rpm for 10 min, add 3 mL of vinyltriethoxysilane dropwise, and react overnight. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for use.

[0084] ④ Embedding dye Eu(Ⅲ)

[0085] Take 1 g of the microspheres in step ③ and disperse them in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 2; weigh 200 mg of the time-resolved fluorescence dye Eu(Ⅲ) in 5 mL of the swelling agent DCM, dissolve it by ultrasonic to prepare solution 2; pour solution 2 into emulsion 2, and oscillate and ultrasonicate at room temperature in the dark for 30 min. After the reaction, wash three times with absolute ethanol and three times with pure water, and finally store in pure water for use.

[0086] ⑤ Carboxyl modification on the surface of microspheres

[0087] Carboxyl modification was carried out by seed emulsion polymerization. 1 g of the product in step ④ was dispersed in 200 mL of pure water, and nitrogen was purged for 30 min to remove oxygen in the system. Then 60 mg of SDS was added and stirred to dissolve for 10 min. Then 5 mL of methacrylic acid was added, and the temperature of the water bath was set at 75 °C. When the temperature reached 60 °C, 50 mg of KPS was added, and the reaction was carried out overnight at 300 rpm. After the reaction, it was washed three times with pure water and three times with absolute ethanol, and finally stored with absolute ethanol for use.

[0088] ⑥Embedding the dye Eu(Ⅲ)

[0089] 1 g of the microspheres in step ⑤ was dispersed in 20 mL of an SDS aqueous solution with a mass fraction of 0.25% to prepare emulsion 3; 200 mg of the time-resolved fluorescent dye Eu(Ⅲ) was weighed and dissolved in 5 mL of the swelling agent DCM by ultrasonic treatment to prepare solution 3; solution 3 was poured into emulsion 3, and ultrasonic oscillation was carried out at room temperature in the dark for 30 min. After the reaction, it was washed three times with absolute ethanol and three times with pure water, and finally stored in pure water for use, and the solid content was adjusted to 1% for use.

[0090] As can be seen from Table 1, the particle sizes of the silica-coated time-resolved fluorescent microspheres in Example 1 and Example 2 were 190.9 nm and 352 nm respectively, the dispersion indices were both less than 0.05, the particle sizes were uniform, and they had good monodispersity.

[0091] Table 1 Particle sizes and dispersion indices of the silica-coated time-resolved fluorescent microspheres prepared in Example 1 and Example 2

[0092]

[0093] Example 3

[0094] A preparation method of a detection card for human mucin virus resistance protein A, C-reactive protein, and human serum amyloid A based on silica-coated high-performance time-resolved fluorescent microspheres, the specific steps are as follows:

[0095] (1) Labeling of MxA antibody

[0096] ① Cleaning: 50 μL of the silica-coated time-resolved fluorescent microspheres in Example 1 was added to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer solution, ultrasonically mixed evenly, and the supernatant was discarded by centrifugation;

[0097] ② Activation: 1 mL of 50 mM MES (pH 6.0) buffer solution was added to the cleaned silica-coated time-resolved fluorescent microspheres in Example 1, ultrasonically mixed evenly, then 10 μL of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) were added respectively, vortexed and mixed evenly, activated at room temperature in the dark for 30 min, and the supernatant was discarded by centrifugation;

[0098] ③ Coupling: Add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescence microspheres of Example 1, and mix well by ultrasonic; add the antibody labeled with MxA to be coupled, vortex to mix well, and couple at room temperature in the dark for 2 h;

[0099] ④ Blocking: Add 50 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant;

[0100] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the silica-coated time-resolved fluorescence microsphere-antibody complex of Example 1, and store in the dark at 2 - 8 °C. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0101] (2) Labeling of CRP antibody (For the labeling of CRP antibody, using large-sized microspheres similar to those in Example 2 can improve its detection sensitivity. Of course, in the variations of other beneficial embodiments, using small-sized microspheres similar to those in Example 1 is also possible)

[0102] ① Washing: Take 50 μL of the silica-coated time-resolved fluorescence microspheres of Example 2 and add them to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer, mix well by ultrasonic, and centrifuge to discard the supernatant;

[0103] ② Activation: Add 1 mL of 50 mM MES (pH 6.0) buffer to the washed silica-coated time-resolved fluorescence microspheres of Example 2, after mixing well by ultrasonic, add 10 μL each of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL), vortex to mix well, activate at room temperature in the dark for 30 min, and centrifuge to discard the supernatant;

[0104] ③ Coupling: Add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescence microspheres of Example 2, mix well by ultrasonic; add the antibody labeled with CRP to be coupled, vortex to mix well, and couple at room temperature in the dark for 2 h;

[0105] ④ Blocking: Add 50 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant;

[0106] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the silica-coated time-resolved fluorescence microsphere-antibody complex of Example 2, and store it in the dark at 2 - 8°C. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0107] (3) Labeling of SAA antibody

[0108] ① Washing: Take 50 μL of the silica-coated time-resolved fluorescence microspheres of Example 1 and add them to a centrifuge tube containing 1 mL of 50 mM MES (pH 6.0) buffer. Mix well by ultrasound and centrifuge to discard the supernatant.

[0109] ② Activation: Add 1 mL of 50 mM MES (pH 6.0) buffer to the washed silica-coated time-resolved fluorescence microspheres of Example 1. After mixing well by ultrasound, add 10 μL each of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL), vortex mix, and activate at room temperature in the dark for 30 min. Centrifuge to discard the supernatant.

[0110] ③ Coupling: Add 1 mL of 50 mM MES (pH 6.0) buffer to the activated silica-coated time-resolved fluorescence microspheres of Example 1. Mix well by ultrasound; add the SAA-labeled antibody to be coupled, vortex mix, and couple at room temperature in the dark for 2 h.

[0111] ④ Blocking: Add 50 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant.

[0112] ⑤ Reconstitution: Add 500 μL of the microsphere reconstitution solution to the silica-coated time-resolved fluorescence microsphere-antibody complex of Example 1, and store it in the dark at 2 - 8°C. The formula of the microsphere reconstitution solution is: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0113] (4) Preparation of nitrocellulose membrane

[0114] The anti-MxA antibody coated at a concentration of 2 mg / mL was drawn onto the nitrocellulose membrane at a coating amount of 1 μL / cm as the test line 1, the anti-CRP antibody coated at a concentration of 2 mg / mL was drawn onto the nitrocellulose membrane at a coating amount of 1 μL / cm as the test line 2, the anti-SAA antibody coated at a concentration of 2 mg / mL was drawn onto the nitrocellulose membrane at a coating amount of 1 μL / cm as the test line 3, and the goat anti-mouse IgG antibody coated at a concentration of 1 mg / mL was drawn onto the nitrocellulose membrane at a coating amount of 1 μL / cm as the quality control line, and then dried overnight at 45°C.

[0115] (5)Preparation of the conjugate pad

[0116] The complexes of silicon-coated time-resolved fluorescence microspheres labeled with anti-MxA antibody, anti-CRP antibody, and anti-SAA antibody were sprayed onto the glass cellulose membrane at a coating amount of 7 μL / cm and dried overnight at 45°C to prepare the conjugate pad.

[0117] (6)Assembly of the test strip

[0118] The above-mentioned coated nitrocellulose membrane was pasted on the PVC bottom plate, with the marked line end pasted to the conjugate pad, the sample pad placed on the conjugate pad, and the quality control line end pasted to the absorbent pad.

[0119] Application examples

[0120] (1)Detection of the sensitivity and linear range of MxA

[0121] Calibration samples with different concentrations of MxA were used for the standard curve test. The reaction time was 8 min. The sample or calibration sample was added to 80 μL of diluent, and after taking 80 μL of the diluent, the sample was added to the test card of Example 3. The experimental results are shown in Table 2.

[0122] Table 2

[0123]

[0124] Taking the concentration of the MxA antigen calibration sample as the abscissa and the measured fluorescence signal value as the ordinate, a fitted standard curve was established. The results are as Figure 1 shown. The linearity of the detection results is good, and the concentration of MxA protein in the sample can be quantitatively tested through this standard curve.

[0125] (2)Detection of the sensitivity and linear range of CRP

[0126] Calibration samples with different concentrations of CRP were used for the standard curve test. The reaction time was 8 min. The sample or calibration sample was added to 80 μL of diluent, and after taking 80 μL of the diluent, the sample was added to the test card. The experimental results are shown in Table 3.

[0127] Table 3

[0128]

[0129] Taking the concentration of CRP antigen calibrator as the abscissa and the measured fluorescence signal value as the ordinate, a fitting standard curve was established. The results are as Figure 2 shown. The linearity of the detection results is good, and the concentration of CRP protein in the sample can be quantitatively tested through this standard curve.

[0130] (3) Detection of SAA sensitivity and linear range

[0131] SAA calibrators with different concentrations were used for standard curve testing. The reaction time was 8 min. 80 μL of diluent was added to the sample or calibrator, and after taking 80 μL of the diluent, the sample was added to the test card. The experimental results are shown in Table 4.

[0132] Table 4

[0133]

[0134] Taking the concentration of SAA antigen calibrator as the abscissa and the measured fluorescence signal value as the ordinate, a fitting standard curve was established. The results are as Figure 3 shown. The linearity of the detection results is good, and the concentration of SAA protein in the sample can be quantitatively tested through this standard curve.

[0135] (4) Detection of clinical samples

[0136] Whole blood samples of 70 suspected respiratory infection patients in the fever clinic were collected. The concentration of MxA in the whole blood samples was detected and assigned values using the Zhongyuan detection kit. At the same time, a triple kit prepared with the detection cards for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A of the above-mentioned silica-coated high-performance time-resolved fluorescence microspheres was used for detection, and the corresponding concentrations were obtained by fitting. The results are as Figure 4 , R 2 = 0.9574, indicating that the detection results have a good correlation with the Zhongyuan detection.

[0137] Whole blood samples of 55 cases with CRP values assigned by Mindray 7500 were collected. At the same time, a triple kit prepared with the detection cards for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A of the silica-coated high-performance time-resolved fluorescence microspheres was used for detection, and the corresponding concentrations were calculated by fitting. The results are as Figure 5 , R 2 = 0.9785, indicating that the detection results have a good correlation with the Mindray target value.

[0138] Whole blood samples of 45 cases with SAA values tested by the Novizan serum amyloid A detection kit (quantum dot fluorescence immunoassay) were collected. At the same time, a triple kit prepared with the detection cards for human myxovirus resistance protein A, C-reactive protein, and human serum amyloid A of the silica-coated high-performance time-resolved fluorescence microspheres was used for detection, and the corresponding concentrations were obtained by fitting. The results are asFigure 6 , R 2 = 0.9588, indicating that the test results have a good correlation with those of Novoprotein.

[0139] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a combined detection card for MxA, CRP and SAA, characterized in that, It includes the following steps: (1) Preparation of silica-coated high-performance time-resolved fluorescence microspheres ① Preparation of SiO2 microspheres by sol-gel method: Add ammonia water to an ethanol aqueous solution, stir and disperse evenly, then dropwise add tetraethyl orthosilicate, and react overnight at 25-45 °C; 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; ② Embedding the time-resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ① in an SDS aqueous solution to prepare emulsion 1, and dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 1; then pour solution 1 into emulsion 1, and oscillate and ultrasonicate in the dark; ③ Modifying the double bond 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, then dropwise add the coupling agent vinyltriethoxysilane, and react overnight at room temperature; ④ Embedding the time-resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ③ in an SDS aqueous solution to prepare emulsion 2, and dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 2; then pour solution 2 into emulsion 2, and oscillate and ultrasonicate in the dark; ⑤ Modifying the carboxyl group on the surface of the microspheres by seed emulsion polymerization method: Disperse the product in step ④ in an aqueous solution, deoxygenate by passing N2, then add an appropriate amount of surfactant sodium dodecyl sulfate, and after dissolution, add an appropriate amount of carboxylic acid monomer methacrylic acid, and add the initiator potassium persulfate at 60 °C, and react overnight at 70-85 °C; ⑥ Embedding the time-resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method: Disperse the product in step ⑤ in an SDS aqueous solution to prepare emulsion 3, and dissolve the Eu(Ⅲ) complex in a DCM solution to prepare solution 3; then pour solution 3 into emulsion 3, and oscillate and ultrasonicate in the dark; (2) Labeling of MxA antibody ① Cleaning: Take the silica-coated high-performance time-resolved fluorescence microspheres and add them to a centrifuge tube containing MES buffer, ultrasonicate and mix evenly, and centrifuge to discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescence microspheres, ultrasonicate and mix evenly, then add freshly prepared EDC and NHS, activate at room temperature and in the dark for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescence microspheres, ultrasonicate and mix evenly; Add the MxA-labeled antibody to be coupled, vortex and mix evenly, and couple at room temperature and in the dark for 2 h; ④ Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature and in the dark for 1 h, and centrifuge to discard the supernatant; ⑤ Redissolution: Add the microsphere redissolution solution to the silica-coated high-performance time-resolved fluorescence microsphere antibody complex, and store in the dark at 2-8 °C; (3) Labeling of CRP antibody ① Cleaning: Take the microspheres and add them to a centrifuge tube containing MES buffer, ultrasonicate and mix evenly, and centrifuge to discard the supernatant; ② Activation: Add MES buffer to the cleaned silica-coated high-performance time-resolved fluorescence microspheres, ultrasonicate and mix evenly, then add freshly prepared EDC and NHS, activate at room temperature and in the dark for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add MES buffer to the activated silica-coated high-performance time-resolved fluorescence microspheres, ultrasonicate and mix evenly; Add the antibody labeled with CRP to be conjugated, vortex to mix well, and conjugate at room temperature in the dark for 2 h; ④Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant; ⑤Redissolution: Add the microsphere redissolution solution to the silicon-coated high-performance time-resolved fluorescence microsphere antibody complex, and store it at 2-8 °C in the dark; (4)Labeling of SAA antibody ①Washing: Take the microspheres and add them to a centrifuge tube containing MES buffer, mix well by ultrasound, and centrifuge to discard the supernatant; ②Activation: Add MES buffer to the washed silicon-coated high-performance time-resolved fluorescence microspheres, mix well by ultrasound, then add freshly prepared EDC and NHS, activate at room temperature in the dark for 30 min, and centrifuge to discard the supernatant; ③Coupling: Add MES buffer to the activated silicon-coated high-performance time-resolved fluorescence microspheres, and mix well by ultrasound; Add the antibody labeled with SAA to be conjugated, vortex to mix well, and conjugate at room temperature in the dark for 2 h; ④Blocking: Add BSA and ethanolamine to the above coupling buffer, block at room temperature in the dark for 1 h, and centrifuge to discard the supernatant; ⑤Redissolution: Add the microsphere redissolution solution to the silicon-coated high-performance time-resolved fluorescence microsphere antibody complex, and store it at 2-8 °C in the dark; (5)Preparation of conjugate pad; (6)Preparation of nitrocellulose membrane; (7)Assembly of test strip.

2. The preparation method of the combined detection card for MxA, CRP and SAA according to claim 1, wherein: When embedding the time-resolved fluorescence dye Eu(Ⅲ) complex by ultrasonic swelling method, oscillate and ultrasonicate 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; When modifying the double bond on the surface of the microsphere by silane coupling reaction, the volume ratio of vinyltriethoxysilane to ammonia water is 1:0.1-1; When modifying the carboxyl group on the surface of the microsphere by seed emulsion polymerization method, the mass ratio of the carboxylic acid monomer methacrylic acid to the initiator is 1:0.01-0.

03.

3. The preparation method of the combined detection card for MxA, CRP and SAA according to claim 2, characterized in that: The specific step (5) is to spray the complexes of MxA, CRP, and SAA antibodies labeled with silicon-coated high-performance time-resolved fluorescence microspheres onto glass cellulose, and dry them overnight at 45 °C to prepare a conjugate pad.

4. The preparation method of the combined detection card for MxA, CRP and SAA according to claim 3, wherein: The specific step (6) is to spray goat anti-mouse IgG antibody, MxA coating antibody, CRP coating antibody, and SAA coating antibody onto the nitrocellulose membrane respectively with a scribing instrument as the quality control line C, MxA test line, CRP test line, and SAA test line 3, and dry them overnight at 45 °C; The specific step (7) is to attach the prepared nitrocellulose membrane to the PVC bottom plate, attach the end coated with the silicon-coated high-performance time-resolved fluorescence microsphere antibody complex to the conjugate pad, place the sample pad on the conjugate pad, and attach the quality control line end to the absorbent pad.

5. A combined detection card for MxA, CRP, and SAA prepared by the preparation method according to any one of claims 1-4.

6. Use of the combined detection card according to claim 5 in the preparation of a detection kit for MxA, CRP, and SAA.

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