H7 avian influenza virus antibody magnetic immunochemiluminescence detection kit and its application

Abstract

The invention discloses a H7 avian influenza virus antibody magnetic immunochemiluminescence detection kit and its application, belonging to the field of immunological detection. The kit contains enzyme-labeled antibodies, magnetic-labeled fluorescein isothiocyanate antibodies, fluorescein isothiocyanate modified antigens, standard solution, sample diluent, luminescent substrate liquid and concentrated washing solution; the antibody in the enzyme-labeled antibodies can specifically bind to the antigen in the fluorescein isothiocyanate modified antigen; the magnetic-labeled fluorescein isothiocyanate antibody is a core-shell structure; the core-shell structure is a fluorescein isothiocyanate antibody wrapped with magnetic nanoparticles; the luminescent substrate in the luminescent substrate liquid specifically binds to the enzyme in the enzyme-labeled antibody, so that the luminescent substrate emits fluorescence. The use of the kit to detect antibodies has the effects of high sensitivity, accuracy and strong specificity.

Description

Technical Field

[0001] The invention belongs to the field of immunological detection, and in particular, relates to a H7 avian influenza virus antibody chemiluminescence detection kit and a detection method thereof. Background Art

[0002] Avian influenza is a highly contagious disease of poultry caused by influenza A virus. The virus that causes this disease belongs to the family of Orthomyxoviridae, an RNA virus. It is approximately spherical in shape, with a diameter of 80 to 120 nm. The virus has an envelope on the outside, and a helical symmetrical nucleocapsid inside the envelope. The genome of influenza A virus consists of 8 segments. The glycoprotein antigens on the surface of the virus are divided into 16 hemagglutinins (HA subtypes) and 9 neuraminidase (NA subtypes). The genes between different subtypes of viruses can "hybridize" to form a large and complex family of viruses. In recent years, due to the antigenic drift of avian influenza virus (AIV), the virus has crossed the interspecies barrier and threatened human health. Among avian influenza viruses, H3, H5, H7, and H9 can be transmitted to humans, among which H5 is a highly pathogenic virus. There have been reports of different subtypes of avian influenza viruses, including H5N1, H7N7, H7N3, and H9N2, infecting humans and causing illness and death. The H7N9 subtype of avian influenza virus is one of them. As of January 10, 2015, 134 people have been confirmed to be infected with avian influenza in the country, 37 people have died, and the mortality rate is 27.6%.

[0003] H7 subtype highly pathogenic avian influenza is a compulsory immunization project. Its antibody is the main indicator for monitoring antibody levels after vaccination. The level of antibody is directly related to its immunity. The monitoring of antibody levels to formulate a reasonable and effective immunization program is the guarantee for improving the level of group immunity. The existing avian influenza virus antibody diagnostic techniques mainly include hemagglutination inhibition method, ELISA method, virus neutralization test, immunofluorescence method, colloidal gold test strips and other methods. Hemagglutination inhibition method is the most commonly used method for detecting avian influenza virus antibodies, but this technology has high requirements on the professional quality of operators, the process is cumbersome, the judgment of the results is also highly subjective, and high-throughput detection is basically impossible to achieve. The general scale and breeding density of poultry farming are large, and the sample size required for detection is also large. Therefore, it is urgent to develop a detection method that is simple to operate, highly sensitive, highly accurate, and high-throughput. Summary of the invention

[0004] The invention solves the technical problems in the prior art that antibody detection has low specificity, sensitivity and accuracy.

[0005] To achieve the above object, the present invention provides a method for preparing a magnetically labeled fluorescein isothiocyanate antibody, comprising the following steps:

[0006] (1) After stimulating an organism with fluorescein isothiocyanate as an immune antigen, serum is extracted to obtain crude fluorescein isothiocyanate antibody A, and casein, ovalbumin or bovine serum albumin is added to the serum to form crude fluorescein isothiocyanate antibody B; or after purifying the serum, fluorescein isothiocyanate antibody is extracted, and casein, ovalbumin or bovine serum albumin is added to the fluorescein isothiocyanate antibody to form crude fluorescein isothiocyanate antibody C;

[0007] (2) adding superparamagnetic nanoparticles to the fluorescein isothiocyanate crude antibody A, fluorescein isothiocyanate crude antibody B or fluorescein isothiocyanate crude antibody C described in step (1) to obtain a crude emulsion;

[0008] (3) adding a reducing agent to the crude emulsion of step (2) so that the disulfide bonds of the proteins in the crude emulsion are opened under the action of the reducing agent, thereby exposing the sulfhydryl groups and the hydrophobic regions; the proteins in the crude emulsion are connected to the superparamagnetic nanoparticles through sulfhydryl coordination and hydrophobic interaction to obtain magnetically labeled fluorescein isothiocyanate antibodies; the magnetically labeled fluorescein isothiocyanate antibodies have a core of superparamagnetic nanoparticles and an outer shell of fluorescein isothiocyanate antibodies and a closed protein outside the fluorescein isothiocyanate antibody site.

[0009] According to another aspect of the present invention, a magnetically labeled fluorescein isothiocyanate antibody prepared by the method is provided, wherein the magnetically labeled fluorescein isothiocyanate antibody is a core-shell structure; the core of the core-shell structure is a superparamagnetic nanoparticle, and the shell of the core-shell structure is a fluorescein isothiocyanate antibody and a blocking protein outside the fluorescein isothiocyanate antibody site; the superparamagnetic nanoparticle is connected to the fluorescein isothiocyanate antibody and the blocking protein through thiol coordination and hydrophobic interaction.

[0010] Preferably, the superparamagnetic nanoparticles are superparamagnetic ferroferric oxide nanoparticles or superparamagnetic ferrous oxide nanoparticles.

[0011] According to another aspect of the present invention, a magnetic immunochemiluminescent detection kit for H7 avian influenza virus antibodies is provided, the kit comprising the following reagents: enzyme-labeled H7 avian influenza virus monoclonal antibody, magnetic-labeled fluorescein isothiocyanate antibody, fluorescein isothiocyanate-modified H7 avian influenza virus antigen, standard solution, sample diluent, luminescent substrate solution and concentrated washing solution;

[0012] The magnetically labeled fluorescein isothiocyanate antibody specifically binds to the fluorescein isothiocyanate on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen;

[0013] The antibody on the enzyme-labeled H7 avian influenza virus monoclonal antibody specifically binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen;

[0014] The enzyme on the enzyme-labeled H7 avian influenza virus monoclonal antibody degrades the luminescent substrate in the luminescent substrate solution, causing the luminescent substrate to emit light.

[0015] Preferably, the enzyme-labeled H7 avian influenza virus monoclonal antibody is an alkaline phosphatase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinositol)-phenyl-1,2-dioxetane or 3-aminophenyl dicarboxylic acid hydrazide.

[0016] Preferably, the enzyme-labeled H7 avian influenza virus monoclonal antibody is a horseradish peroxidase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution is 3-aminophthalhydrazide.

[0017] Preferably, the enzyme-labeled H7 avian influenza virus monoclonal antibody is obtained by reacting alkaline phosphatase activated with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimidyl ester sodium salt with the H7 avian influenza virus monoclonal antibody thiolated with 2-iminothiolane hydrochloride.

[0018] According to another aspect of the present invention, there is provided a method for using the kit to detect antibodies, comprising the following steps:

[0019] (1) mixing a magnetically labeled fluorescein isothiocyanate antibody with a fluorescein isothiocyanate-modified H7 avian influenza virus antigen, wherein the magnetically labeled fluorescein isothiocyanate antibody specifically binds to the fluorescein isothiocyanate on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen;

[0020] (2) adding a standard solution or a sample to be tested to the product obtained in step (1), reacting at 20°C-37°C for 10min-15min, so that the antibody in the standard solution or the sample to be tested specifically binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, and then performing magnetic separation for 30s-2min, discarding the supernatant and washing the precipitate with a concentrated washing solution; adding an enzyme-labeled H7 avian influenza virus monoclonal antibody to the washed precipitate, reacting at 20°C-37°C for 10min-15min, so that the antibody on the enzyme-labeled H7 avian influenza virus monoclonal antibody binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, and then performing magnetic separation for 30s-2min, discarding the supernatant and washing the precipitate with a concentrated washing solution;

[0021] (3) Adding a luminescent substrate solution to the product obtained in step (2), allowing the luminescent substrate in the luminescent substrate solution to react with the enzyme in the enzyme-labeled H7 avian influenza virus monoclonal antibody bound to the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, detecting the luminescence value, drawing a standard curve with the luminescence value of the standard solution added, and calculating the antibody content in the sample to be tested from the standard curve with the luminescence value of the sample to be tested.

[0022] Preferably, the standard curve in step (3) is established by the following method:

[0023] S1: The concentrations of the standards corresponding to the hemagglutination inhibition titers of 0, 2log2, 4log2, 6log2, 8log2 and 12log2 are defined as 0AU / mL, 0.1AU / mL, 0.3AU / mL, 0.5AU / mL, 1AU / mL and 2AU / mL, respectively;

[0024] S2: The concentration of the standard substance in step S1 is used as the horizontal coordinate, and the luminescence value corresponding to the concentration of the standard substance is used as the vertical coordinate, and a standard curve is obtained by fitting a four-parameter logistic curve.

[0025] Preferably, the enzyme-labeled H7 avian influenza virus monoclonal antibody in step (2) is an alkaline phosphatase-labeled H7 avian influenza virus monoclonal antibody; the luminescent substrate in the luminescent substrate solution in step (3) is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinosyl)-phenyl-1,2-dioxetane or 3-aminophthalhydrazide;

[0026] Preferably, the enzyme-labeled H7 avian influenza virus monoclonal antibody in step (2) is a horseradish peroxidase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution in step (3) is 3-aminophthalhydrazide.

[0027] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0028] (1) The magnetic immunochemiluminescence detection kit for H7 avian influenza virus antibodies of the present invention has high sensitivity and specificity, and the detection sensitivity of H7 avian influenza virus antibodies can reach 0.04AU / mL; and according to different sample titers of the hemagglutination inhibition test, such as all-negative samples, 2log2, 4log2, 6log2, 8log2, and 12log2, a linear relationship corresponding to the titer of the hemagglutination inhibition test samples is established, and a new measurement unit AU / mL is established, thereby realizing the quantitative detection of H7 avian influenza virus antibodies, and a large number of samples can be quantitatively detected at the same time, which is a significant technical improvement; by detecting the antibody content of the sample, the antibody level can be monitored, and a reasonable and effective immunization program can be formulated based on the monitoring results to improve the level of group immunity.

[0029] (2) The magnetic bead-labeled fluorescein isothiocyanate (FITC) antibody of the present invention adopts a preparation method of reducing self-assembled FITC antibody-coated superparamagnetic nanoparticles. The reducing agent opens the disulfide bonds in the FITC antibody and the foreign protein, exposing the hydrophobic region and the thiol group in the antibody. The magnetic microspheres are tightly combined with the antibody through coordination bonds and hydrophobic effects to form magnetic microspheres with strong stability. The preparation conditions of the magnetic labeled FITC antibody are mild. The FITC antibody can be directly fixed during the preparation process to obtain FITC antibody magnetic microspheres in one step. The magnetic microspheres are active, not easy to aggregate, and have low non-specific adsorption. They are used in chemiluminescence detection kits with strong specificity and high accuracy.

[0030] (3) The kit provided by the present invention has high sensitivity, specificity and accuracy when used to detect H7 avian influenza antibodies, and realizes fully automated detection, shortens detection time, improves detection throughput, and reduces human operation errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the standard curve of the magnetic immunochemiluminescence detection kit of the present invention.

[0032] Figure 2 Diagram of the preparation process of magnetically labeled fluorescein isothiocyanate (FITC) antibody.

[0033] Figure 3 Transmission electron micrograph of magnetically labeled fluorescein isothiocyanate (FITC) antibody. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Example 1: Preparation of specific components of the kit

[0036] 1. Preparation of enzyme-labeled antibodies

[0037] A. Alkaline phosphatase activation

[0038] Use DMSO as solvent to prepare 10mM SMCC solution; add 240uL 5mg / mL alkaline phosphatase to 29.5uL 10mM SMCC solution, mix well and let stand at room temperature for 15min, then add 2.7uL 1M pH 7.3 glycine buffer and let stand at room temperature for 5min; desalt by ultrafiltration of 0.01M pH 7.4 PBS solution, measure the absorbance at 280nm, enzyme concentration: ABS280*dilution factor / 1.0, enzyme mass: enzyme concentration*product volume (mg / mL), store at 2-8℃.

[0039] B. Activation of monoclonal antibodies against H7 avian influenza virus

[0040] Prepare 5 mM EDTA at pH 8.0 using 0.1 M borate as solvent. 2 O solution; take 400uL of 3mg / mL monoclonal antibody and use the above solution as mobile phase, pass through GE Healthcare LifeScience protein separation and purification system PD10 desalting column, collect the product, the product is 2mg / mL; prepare 0.02M 2-IT solution with 0.1M borate as solvent, take 4uL of 0.02M 2-IT solution and add it to 500uL of 2mg / mL desalted product, mix well and let stand at room temperature for 30min, then add 5uL of 1M pH7.3 glycine buffer and let stand at room temperature for 5min; ultrafiltration desalt with 0.01M pH 7.4 PBS solution, add 2 times volume of 1M MgCl 2 , measure the absorbance at 280nm, protein concentration: ABS280*dilution factor / 1.36, protein amount: enzyme concentration*product volume (mg / mL), store at 2-8℃.

[0041] C. Coupling

[0042] The activated alkaline phosphatase and the activated antibody were coupled at a mass ratio of 0.91:1, and the reaction was allowed to stand at 2-8°C for 16-18h; 10uL of 10mM maleimide solution was added, mixed and allowed to stand for 10min, and the preparation process of 10mM maleimide solution was as follows: 1.03mL DMF dissolved 10mg of maleimide solid, and then diluted 10 times with 0.01M pH7.4 PBS solution to prepare 10mM maleimide solution; 10uL of 100mM ethanolamine solution was added, mixed and allowed to stand for 10min, and the preparation process of 100mM ethanolamine solution was as follows: 20uL ethanolamine was dissolved in 1.98mL 0.01M pH7.4 PBS solution, and then 600uL of the above ethanolamine solution was taken and added to 400uL 0.01M pH7.4 PBS solution to obtain 100mM ethanolamine solution; Superdex was used. The above products were separated and purified on a GE Healthcare Life Science protein separation and purification system using a 200 column, and the elution buffer was 0.1 M pH 7.4 containing 5 mM MgCl 2 , 0.1 mM ZnCl 2 and 0.3% sodium azide in Tris-buffered saline, 0.1 M pH 7.4, containing 5 mM MgCl 2 , 0.1 mM ZnCl 2 and 0.3% sodium azide in Tris buffered saline. Store at 2-8°C.

[0043] 2. Preparation of magnetically labeled fluorescein isothiocyanate (FITC) antibody

[0044] The magnetically labeled fluorescein isothiocyanate (FITC) antibody is realized by reducing the self-assembled antibody to encapsulate the magnetic microspheres. First, the fluorescein isothiocyanate (FITC) immune antigen is used as a stimulus to produce fluorescein isothiocyanate (FITC) antibodies in organisms such as mice or rabbits. Serum is extracted to obtain crude antibodies containing foreign proteins, or crude antibodies are formed by adding appropriate amounts of foreign proteins (such as bovine serum albumin BSA) to the extracted serum, or crude antibodies are formed by artificially adding appropriate amounts of foreign proteins (such as bovine serum albumin BSA) to the extracted serum after purifying the antibodies; then superparamagnetic iron oxide SPIO nanoparticles are mixed with fluorescein isothiocyanate (FITC) antibodies by physical mixing to form a crude emulsion. Under the action of reducing agent, fluorescein isothiocyanate (FITC) antibody and foreign proteins open the disulfide bonds of the antibody, exposing the sulfhydryl and hydrophobic regions of the antibody. The antibody self-assembles with supermagnetic iron oxide nanoparticles (SPIO) through sulfhydryl coordination and hydrophobic interaction to form a core-shell structure of fluorescein isothiocyanate (FITC) antibody-coated magnetic microsphere reaction solution. Figure 2 This is a diagram of the preparation process of magnetically labeled fluorescein isothiocyanate (FITC) antibody. Figure 3This is a transmission electron micrograph of a magnetically labeled fluorescein isothiocyanate (FITC) antibody. Figure 3 It can be seen that the fluorescein isothiocyanate (FITC) antibody encapsulates the nanoparticles. The specific operation is as follows:

[0045] A. Physical mixing

[0046] Take 5mg superparamagnetic iron oxide nanoparticles (SPIO), add 3mL ethanol and vortex for 30s, collect SPIO with a magnet, remove the supernatant, add 100ul chloroform to dissolve SPIO again, then add 100ul ethanol and 1mL pH 7.3 Tris buffer solution. Ultrasonicate for 3min in an ultrasonic cleaner, transfer SPIO to a 25ml glass bottle and continue ultrasonicating for 1min, add 4mL pH 7.3 Tris buffer solution, ultrasonicate for 2min, add 50mg FITC crude antibody to SPIO, and mix on a plasma mixer for 12h to obtain a crude emulsion.

[0047] B. Reduction self-assembly encapsulation: The crude emulsion was transferred to a 25 ml round-bottom flask, and 2 mol / L mercaptoethanol was slowly added in a 37°C water bath and reacted for 6 min.

[0048] C. Separation and purification: The reaction was terminated with a 1:1 (V:V) ratio of the reaction solution to 0°C Tris buffered saline. Magnetic separation was performed for 2 minutes, the supernatant was discarded, and the supernatant was washed twice with pH 7.4 0.05M Tris buffered saline, and stored in pH 8.0, containing 0.02% WPS (V:V), 1% TritionX-100 (M:V), 0.5% BSA (M:V), 0.05M Tris buffered saline, with a final concentration of 2 mg / ml.

[0049] 3. Preparation of fluorescein isothiocyanate (FITC) labeled H7 avian influenza virus antigen

[0050] Take an appropriate amount of FITC, dissolve it with sodium bicarbonate at a concentration of 1.68% (M:V), and prepare a 1mg / ml solution. Prepare it and use it immediately, and store it in the dark. Slowly add 100ul FITC solution to 64ul H7 avian influenza virus antigen, and react in the dark at 2-8℃ overnight. Use sodium bicarbonate buffer as the mobile phase and pass it through the PD10 desalting column of the protein separation and purification system of GE Healthcare Life Science. Add glycerol at a ratio of 1:1 and store it in a pH 7.4, 0.05M Tris buffered saline solution, and store it at -20℃.

[0051] Example 2: Kit Assembly

[0052] A magnetic immunochemiluminescence detection kit for detecting antibodies to H7 avian influenza virus was constructed to contain the following components:

[0053] (1) Alkaline phosphatase-labeled H7 avian influenza virus monoclonal antibody marker

[0054] (2) Magnetic labeled fluorescein isothiocyanate (FITC) antibody

[0055] (3) Antigen labeled with fluorescein isothiocyanate (FITC)

[0056] (4) H7 avian influenza virus antibody standard solution

[0057] (5) Sample diluent

[0058] (6) Chemiluminescent substrate solution

[0059] (7) Concentrated washing solution

[0060] The enzyme-labeled antibody diluent used in the present invention is a 0.05M Tris buffered saline solution with a pH of 7.2 to pH 7.4.

[0061] The magnetic label fluorescein isothiocyanate (FITC) antibody diluent is pH 8.0, containing 0.02% WPS (V:V), 1% TritionX-100 (M:V), 0.5% BSA (M:V), and 0.05M Tris buffered saline.

[0062] The fluorescein isothiocyanate (FITC) modified antigen diluent of the present invention is a pH 7.4, 0.05 M Tris buffered saline solution.

[0063] The concentrations of H7 avian influenza virus antibody standard solutions are: 0AU / mL, 0.1AU / mL, 0.3AU / mL, 0.5AU / mL, 1AU / mL, 2AU / mL, and the standard diluent is pH 7.4, 0.05M Tris buffered saline.

[0064] The sample diluent is a 0.05 M Tris buffered saline solution at pH 7.2 to pH 7.4.

[0065] The buffer of the chemiluminescent substrate solution is pH 9.0, 0.1 M Tris buffered saline.

[0066] The concentrated washing solution is a 0.05M Tris buffered saline solution with a pH of 7.4 and containing 1% Tween-20 by volume.

[0067] Example 3: Detection of H7 avian influenza virus antibodies in samples

[0068] Testing and result analysis:

[0069] The diluted enzyme-labeled antibody is loaded into the enzyme-labeled antibody working solution container of the chemiluminescence detector, wherein the volume ratio of the diluted enzyme-labeled antibody diluent is 1:1000 to 1:10000; the diluted magnetically labeled fluorescein isothiocyanate (FITC) antibody and the diluted fluorescein isothiocyanate (FITC) labeled antigen are loaded into the magnetic bead antigen working solution container of the chemiluminescence detector, wherein the volume ratio of the diluted fluorescein isothiocyanate (FITC) antibody and the corresponding diluent is 1:20 to 1:40 and 1:1000 to 10000 respectively; the sample diluent is loaded into the sample dilution working solution container of the chemiluminescence detector; the position on the sample rack is set for each sample / standard, and the sample information and the name of the test item to be detected are input; the sample tube / standard tube is placed in the set sample rack, and the chemiluminescence detector draws 10μL of sample, 100μL of sample diluent and 100μL of magnetic label respectively. Antigens are added to the reaction cup in sequence, reacted at 37°C for 10 minutes, and then magnetically separated for 30 seconds by a cleaning device. After discarding the supernatant, the precipitate is washed 3 to 5 times with 250μL to 500μL of washing solution; 50μL of enzyme-labeled antibody is taken and added to the reaction cup, reacted at 37°C for 10 minutes, and then magnetically separated for 30 seconds by a cleaning device, after discarding the supernatant, the precipitate is washed 3 to 5 times with 250μL to 500μL of washing solution; 100μL of chemiluminescent substrate solution is added to detect the emitted light intensity (RLU), the standard RLU is the ordinate, the corresponding standard concentration is the abscissa, and the standard curve is fitted with a four-parameter Logistic curve; the luminescence value of the sample is substituted into the standard curve, and the concentration of each sample can be read from the standard curve. If the content value is ≥0.3AU / mL, it is judged as positive; if the content value is <0.3AU / mL, it is judged as negative.

[0070] The present invention adopts 6 H7 avian influenza virus antibody standard products (0AU / mL, 0.1AU / mL, 0.3AU / mL, 0.5AU / mL, 1AU / mL, 2AU / mL) to carry out curve mapping. The method for establishing the measurement unit AU / mL is that, according to the hemagglutination inhibition test results of the samples, samples with titers of all negative samples, 2log2, 4log2, 6log2, 8log2, and 12log2 are selected, a linear relationship corresponding to the titers of the hemagglutination inhibition test samples is established, and standard products corresponding to the hemagglutination inhibition test titers of all negative samples, 2log2, 4log2, 6log2, 8log2, and 12log2 are established, the standard products are 0, 0.1, 0.3, 0.5, 1, and 2, and the measurement unit AU / mL is established. The standard product RLU is the ordinate, the content of the H7 avian influenza virus antibody is the abscissa, and a four-parameter Logistic curve is used to fit the standard curve, and the concentration of each sample can be read from the standard curve. If the content value is ≥0.3AU / mL, it is judged as positive; if the content value is <0.3AU / mL, it is judged as negative. Figure 1 shown.

[0071] Example 4: Determination of kit quality

[0072] 1. Kit specificity

[0073] According to the method of Example 3, H5 avian influenza virus, H9 avian influenza virus, Newcastle disease (NDV), Marek's virus (MDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), duck plague virus (DPV), goose plague virus (GPV), goose paramyxovirus (GPMV) positive samples were determined, and the test results showed that the contents of H5 avian influenza virus, H9 avian influenza virus, Newcastle disease (NDV), Marek's virus (MDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), duck plague virus (DPV), goose plague virus (GPV), goose paramyxovirus (GPMV) positive samples were all less than 0.1AU / mL, and there was no specific reaction. The data are as follows:

[0074]

[0075] 2. Detection limit of the kit

[0076] The detection limit (analytical sensitivity) of the kit is defined as: the average value of 20 negative samples plus 3 times the standard deviation. The detection limit of the kit is: 0.04AU / mL.

[0077] 3. Accuracy of the kit

[0078] Accuracy refers to the degree of conformity between the measured value and the true value. 20 samples with different hemagglutination inhibition titers were selected, and the hemagglutination inhibition test was used as the standard. The hemagglutination inhibition titer ≥4log2 was positive, and the titer <4log2 was negative. The content value of this kit ≥0.3AU / mL was judged as positive; the content value <0.3AU / mL was judged as negative. The data show that the positive and negative conformity rate of this kit and the hemagglutination inhibition test is 100%, and the data are as follows:

[0079]

[0080]

[0081] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a magnetically labeled fluorescein isothiocyanate antibody, characterized in that: Contains the following steps: (1) After stimulating an organism with fluorescein isothiocyanate as an immune antigen, serum is extracted to obtain crude fluorescein isothiocyanate antibody A, and casein, ovalbumin or bovine serum albumin is added to the serum to form crude fluorescein isothiocyanate antibody B; or after purifying the serum, fluorescein isothiocyanate antibody is extracted, and casein, ovalbumin or bovine serum albumin is added to the fluorescein isothiocyanate antibody to form crude fluorescein isothiocyanate antibody C; (2) adding superparamagnetic nanoparticles to the fluorescein isothiocyanate crude antibody A, fluorescein isothiocyanate crude antibody B or fluorescein isothiocyanate crude antibody C described in step (1) to obtain a crude emulsion; (3) adding a reducing agent to the crude emulsion of step (2), so that the disulfide bonds of the proteins in the crude emulsion are opened under the action of the reducing agent, thereby exposing the sulfhydryl groups and the hydrophobic regions; the proteins in the crude emulsion are connected to the superparamagnetic nanoparticles through sulfhydryl coordination and hydrophobic interaction to obtain a magnetically labeled fluorescein isothiocyanate antibody; the magnetically labeled fluorescein isothiocyanate antibody has a core of superparamagnetic nanoparticles and an outer shell of fluorescein isothiocyanate antibody and a closed protein outside the fluorescein isothiocyanate antibody site; The specific steps include: A. Physical mixing Take 5 mg of superparamagnetic iron oxide nanoparticles SPIO, add 3 mL of ethanol and vortex for 30 seconds, collect SPIO with a magnet, remove the supernatant, add 100 ul of chloroform to dissolve SPIO again, then add 100 ul of ethanol and 1 mL of pH 7.3 Tris buffer solution; sonicate in an ultrasonic cleaner for 3 minutes, transfer SPIO to a 25 ml glass bottle and continue sonicating for 1 minute, add 4 mL of pH 7.3 Tris buffer solution, sonicate for 2 minutes, add 50 mg of FITC crude antibody to SPIO, and mix on a plasma mixer for 12 hours to obtain a crude emulsion; B. Reduction self-assembly encapsulation: The crude emulsion was transferred to a 25 ml round-bottom flask, and 2 mol / L mercaptoethanol was slowly added in a 37°C water bath for 6 min; C. Separation and purification: terminate the reaction with the reaction solution obtained in step B and 0°C Tris buffered saline in a ratio of 1:1 (V:V); perform magnetic separation for 2 minutes and discard the supernatant.

2. The magnetically labeled fluorescein isothiocyanate antibody prepared by the method of claim 1, characterized in that: The magnetic label fluorescein isothiocyanate antibody is a core-shell structure; the core of the core-shell structure is a superparamagnetic nanoparticle, and the shell of the core-shell structure is the fluorescein isothiocyanate antibody and the blocking protein outside the fluorescein isothiocyanate antibody site; the superparamagnetic nanoparticle is connected to the fluorescein isothiocyanate antibody and the blocking protein through thiol coordination and hydrophobic interaction.

3. The magnetically labeled fluorescein isothiocyanate antibody according to claim 2, characterized in that: The superparamagnetic nanoparticles are superparamagnetic ferroferric oxide nanoparticles or superparamagnetic ferric oxide nanoparticles.

4. A magnetic immunochemiluminescence detection kit for H7 avian influenza virus antibodies, characterized in that: The kit contains the following reagents: enzyme-labeled H7 avian influenza virus monoclonal antibody, magnetic-labeled fluorescein isothiocyanate antibody according to claim 2 or claim 3, fluorescein isothiocyanate-modified H7 avian influenza virus antigen, standard solution, sample diluent, luminescent substrate solution and concentrated washing solution; The magnetically labeled fluorescein isothiocyanate antibody of claim 2 or claim 3 specifically binds to the fluorescein isothiocyanate on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen; The antibody on the enzyme-labeled H7 avian influenza virus monoclonal antibody specifically binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen; The enzyme on the enzyme-labeled H7 avian influenza virus monoclonal antibody degrades the luminescent substrate in the luminescent substrate solution, causing the luminescent substrate to emit light.

5. The H7 avian influenza virus antibody magnetic immunochemiluminescence detection kit as claimed in claim 4, characterized in that: The enzyme-labeled H7 avian influenza virus monoclonal antibody is an alkaline phosphatase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane or 3-aminophenyl dicarboxylic acid hydrazide.

6. The H7 avian influenza virus antibody magnetic immunochemiluminescence detection kit according to claim 4, characterized in that: The enzyme-labeled H7 avian influenza virus monoclonal antibody is a horseradish peroxidase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution is 3-aminophthalhydrazide.

7. The H7 avian influenza virus antibody magnetic immunochemiluminescence detection kit according to claim 4, characterized in that: The enzyme-labeled H7 avian influenza virus monoclonal antibody is obtained by reacting alkaline phosphatase activated by 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt with the H7 avian influenza virus monoclonal antibody thiolated by 2-iminothiolane hydrochloride.

8. A method for detecting antibodies using the kit according to any one of claims 4 to 7, characterized in that: The method is a non-disease diagnosis method, comprising the following steps: (1) mixing a magnetically labeled fluorescein isothiocyanate antibody with a fluorescein isothiocyanate-modified H7 avian influenza virus antigen, wherein the magnetically labeled fluorescein isothiocyanate antibody specifically binds to the fluorescein isothiocyanate on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen; (2) adding a standard solution or a sample to be tested to the product obtained in step (1), reacting at 20°C-37°C for 10min-15min, so that the antibody in the standard solution or the sample to be tested specifically binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, and then performing magnetic separation for 30s-2min, discarding the supernatant and washing the precipitate with a concentrated washing solution; adding an enzyme-labeled H7 avian influenza virus monoclonal antibody to the washed precipitate, reacting at 20°C-37°C for 10min-15min, so that the antibody on the enzyme-labeled H7 avian influenza virus monoclonal antibody binds to the H7 avian influenza virus antigen on the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, and then performing magnetic separation for 30s-2min, discarding the supernatant and washing the precipitate with a concentrated washing solution; (3) Adding a luminescent substrate solution to the product obtained in step (2), allowing the luminescent substrate in the luminescent substrate solution to react with the enzyme in the enzyme-labeled H7 avian influenza virus monoclonal antibody bound to the fluorescein isothiocyanate-modified H7 avian influenza virus antigen, detecting the luminescence value, drawing a standard curve with the luminescence value of the standard solution added, and calculating the antibody content in the sample to be tested from the standard curve with the luminescence value of the sample to be tested.

9. The method for detecting antibodies according to claim 8, characterized in that: The standard curve in step (3) is established by the following method: S1: The concentrations of the standards corresponding to the hemagglutination inhibition titers of 0, 2log2, 4log2, 6log2, 8log2 and 12log2 are defined as 0AU / mL, 0.1AU / mL, 0.3AU / mL, 0.5AU / mL, 1AU / mL and 2AU / mL, respectively; S2: The concentration of the standard substance in step S1 is used as the horizontal coordinate, and the luminescence value corresponding to the concentration of the standard substance is used as the vertical coordinate, and a standard curve is obtained by fitting a four-parameter logistic curve.

10. The method for detecting antibodies according to claim 8, characterized in that: The enzyme-labeled H7 avian influenza virus monoclonal antibody in step (2) is an H7 avian influenza virus monoclonal antibody labeled with alkaline phosphatase; the luminescent substrate in the luminescent substrate solution in step (3) is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinositol)-phenyl-1,2-dioxetane or 3-aminophenyl dicarboxylic acid hydrazide.

11. The method for detecting antibodies according to claim 8, characterized in that: The enzyme-labeled H7 avian influenza virus monoclonal antibody in step (2) is a horseradish peroxidase-labeled H7 avian influenza virus monoclonal antibody, and the luminescent substrate in the luminescent substrate solution in step (3) is 3-aminophthalhydrazide.

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