CN112964870B - Influenza virus rapid detection method based on SPR biosensor - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The application belongs to the technical field of virus detection, and relates to a rapid influenza virus detection method based on an SPR biosensor. The rapid influenza virus detection method based on the SPR biosensor can obtain the detection result of the sample within 18 minutes, and has obvious advantages compared with the traditional nucleic acid PCR detection and ELISA detection methods.
Description
Technical Field
The application belongs to the technical field of virus detection, and relates to a rapid influenza virus detection method based on an SPR biosensor.
Background
Influenza virus is an important infectious disease pathogen for human and veterinary co-occurrence, has wide hosts and strong transmission capacity, causes a plurality of global pandemics, causes regional outbreaks every year and causes serious losses to human health and livestock breeding industries. Traditional influenza virus detection methods employ PCR detection or ELISA methods. However, these methods are time-consuming and labor-consuming, and cannot meet the requirements of in-situ detection on site.
SPR biosensors are widely used in environmental monitoring, pathogen diagnosis and food safety, and are the first choice for studying biomolecular interactions. Since SPR technology greatly shortens the time required for affinity detection, this method is also widely used in vaccine screening and design-related studies. The study of intermolecular interactions using SPR biosensors requires the coupling of a detection-specific antibody to the surface of a sensor chip, generally in the following steps: coupling pH screening, antibody coupling, affinity constant determination, binding force analysis and the like. Antibodies suitable for the test substance need to be screened before the relevant test can be performed. The quality of the antibody and the buffer environment of the detection object are two important factors for determining the detection result.
Disclosure of Invention
The invention provides a rapid influenza virus detection method based on an SPR biosensor, and the method is evaluated in terms of simplicity, sensitivity, specificity and the like of the detection method, so that a new means is provided for rapid influenza virus detection.
The technical scheme of the invention is as follows:
an influenza virus rapid detection method based on an SPR biosensor comprises the following steps:
step 1: equipment, materials and reagents were prepared, including SPR biosensor, CM5 chip, 10mmol/L sodium acetate solution with pH values of 4.0, 4.5, 5.0 and 5.5, 50mmol/L NaOH, HBS-EP buffer with pH 7.4, amino coupling kit, influenza virus specific antibody, influenza virus, etc.
Step 2: screening influenza virus antibody optimal coupling pH
Influenza virus antibodies were diluted to 10 μg/ml using 10mmol/L sodium acetate solutions at pH 4.0, 4.5, 5.0 and 5.5, respectively. And (3) inserting the CM5 chip into an SPR biosensor, running an SPR biosensor antibody screening program, placing the diluted antibody into a sample injection groove, setting the sample injection flow rate to be 10 mu L/min, running for 2min, cleaning the CM5 chip with 25 mu L of 50mmol/L NaOH after the analysis program is finished, and then analyzing the next antibody sample. After all four diluted antibodies are analyzed, the reaction value is selected to be not lower than 8000pg/mm 2 (RU, RU can be used to describe the amount of increase in biosensor surface mass for binding between biomolecules, 1ru=1 pg/mm 2 ) As the optimal coupling pH of the antibody.
Step 3: influenza virus antibody coupling chip
After the optimal coupling pH was determined, a 10mmol/L sodium acetate solution at this pH was used as coupling buffer, and the influenza antibody was diluted to 10. Mu.g/mL. The CM5 chip was inserted into the SPR biosensor and the antibody was coupled to the chip surface using an amino coupling kit and an SPR biosensor automated procedure. CM5 chip F2 channel coupled influenza specific antibody, F1 channel as blank reference, coupling target is reaction value 8000pg/mm 2 。
Step 4: analysis of affinity kinetic constants of samples to be tested and antibodies
The samples to be detected are diluted according to the proportion of 1:5,1:10,1:20,1:40 and 1:80 by utilizing HBS-EP buffer solution with pH of 7.4, and the samples are respectively injected for detection. Analysis was performed using SPR biosensor affinity kinetic analysis procedure, and after each detection run, the chip was regenerated (20. Mu.L/min, 30 s) using 10mmol/L Glycine-HCL (pH 2.5) at a total process temperature of 25 ℃. Binding constant (K) pairs using SPR biosensor software program a ) Dissociation constant (K) d ) And affinity equilibrium constant (K) A ) Analysis was performed.
Step 5: binding force analysis of test sample and antibody
The samples to be detected were diluted in a ratio of 1:5,1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560,1:5120,1:10240 with HBS-EP buffer at pH 7.4, and were individually subjected to sample injection detection. Analysis was performed using the SPR biosensor binding force analysis program. After each detection run, the chip was regenerated (20. Mu.L/min, 30 s) using 10mmol/L Glycine-HCL (pH 2.5) at 25℃for the whole process.
Step 6: and judging positive results. If the response curve is in a parabolic continuous rising form after the sample is injected, and the response curve is in a parabolic continuous falling form after 10mmol/L glycine-HCL (pH 2.5) is injected, and the response value and the sample concentration show obvious gradient effect, judging that the sample to be detected is positive to influenza virus, otherwise, judging that the sample to be detected is negative to influenza virus.
Further characterized in that the target value of antibody coupling is 8000pg/mm 2 。
Further characterized in that the sample to be measured is centrifuged at 12000rpm and 4 ℃ for 5min before sample introduction to remove impurities.
Further features, the detection can be carried out after the gradient dilution of influenza samples with known concentration, and the detection limit of the method is obtained.
The rapid influenza virus detection method based on the SPR biosensor can obtain the detection result of the sample within 18 minutes, and has obvious advantages compared with the traditional nucleic acid PCR detection and ELISA detection methods.
Drawings
FIG. 1 is a screen diagram of optimal pH conditions for coupling H5N1 influenza virus specific antibodies on the surface of CM5 chips.
FIG. 2 (a) is a graph of affinity kinetics analysis of a gradient diluted H5N1 (a) influenza virus with H5N 1-specific antibodies.
FIG. 2 (b) is a graph of affinity kinetics analysis of a gradient diluted H1N1 (b) influenza virus with H5N 1-specific antibodies.
FIG. 3 (a) is a graph showing the analysis of the binding force of H5N1 influenza virus diluted in a gradient to H5N1 specific antibodies on the chip surface.
FIG. 3 (b) is a graph showing the analysis of the binding force between the H5N1 influenza virus diluted in gradient and the H5N1 specific antibody on the chip surface.
Detailed Description
The invention is further described in detail below with reference to the drawings and the technical scheme.
Example 1:
a rapid H5N1 influenza virus detection method based on an SPR biosensor comprises the following steps:
step 1: preparation of equipment, materials and reagents: the SPR biosensor is Biacore 3000 biosensor of Biacore company, sweden; CM5 chip, 10mmol/L sodium acetate solution, 50mmol/L NaOH, HBS-EP buffer solution with pH value of 4.0, 4.5, 5.0 and 5.5 respectively, amino coupling kit is purchased from Biacore company, sweden; H5N 1-specific antibodies were purchased from Santa Cruz Biotechnology, inc., USA; goat anti-mouse IgG-HRP antibody was purchased from Abmart biomedical limited, shanghai; TMB substrate was purchased from Beijing Soy Bao technology Co., ltd; H5N1 and H1N1 influenza viruses are saved by military and veterinary research institute, and the virus titers are respectively 10 7.25 TCID 50 /mL and 10 7.75 TCID 50 /mL。
Step 2: screening influenza virus antibody optimal coupling pH
H5N 1-specific antibodies were diluted to 10. Mu.g/ml using 10mmol/L sodium acetate solutions at pH 4.0, 4.5, 5.0 and 5.5, respectively. CM5 chip is inserted into Biacore 3000 biosensor, and Biacore 3000 biosensor antibody screening program is run to diluteThe obtained antibody is placed in a sample injection groove, the sample injection flow rate is set to be 10 mu L/min, the operation time is set to be 2min, after the analysis program is finished, the CM5 chip is washed by 25 mu L of 50mmol/L NaOH, and then the analysis of the next antibody sample is carried out. After all four diluted antibodies are analyzed, the reaction value is selected to be not lower than 8000pg/mm 2 (RU, RU can be used to describe the amount of increase in biosensor surface mass for binding between biomolecules, 1ru=1 pg/mm 2 ) As the optimal coupling pH of the antibody. As shown in FIG. 1, the H5N 1-specific antibody had an optimal coupling pH of 5.0.
Step 3: H5N1 specific antibody coupling chip
A10 mmol/L sodium acetate solution at pH5.0 was used as coupling buffer and influenza antibody was diluted to 10. Mu.g/mL. The CM5 chip was inserted into the Biacore 3000 biosensor, and the antibody was coupled to the chip surface using an amino coupling kit and Biacore 3000 biosensor automation program. CM5 chip F2 channel coupled influenza specific antibody, F1 channel as blank reference, coupling target is reaction value 8000pg/mm 2 。
Step 4: analysis of affinity kinetic constants of H5N1 and H1N1 influenza viruses with H5N 1-specific antibodies
H5N1 and H1N1 influenza viruses were diluted in the ratios 1:5,1:10,1:20,1:40,1:80 respectively using HBS-EP buffer at pH 7.4 and were assayed by sample injection. Analysis was performed using the Biacore 3000 biosensor affinity kinetic analysis program, and after each detection run, the chip was regenerated (20. Mu.L/min, 30 s) using 10mmol/L Glycine-HCL (pH 2.5) at a total process temperature of 25 ℃. Binding constants (K) using Biacore 3000 biosensor software program BIAevalution Software 4.1.1 a ) Dissociation constant (K) d ) And affinity equilibrium constant (K) A ) Analysis was performed. Fig. 2 (a) and fig. 2 (b) show dynamic change curves of reaction values after the samples were injected into H5N1 and H1N1, respectively. The affinity kinetic parameters of H5N1 and H1N1 influenza viruses with H5N1 specific antibodies are shown in table 1.
Table 1 shows the affinity kinetic analysis of H5N1, H1N1 influenza viruses with H5N1 monoclonal antibodies
Note that: 1: a1 Langmuir-binding model was used to determine the kinetic equilibrium constant K a Binding constant, K d Separation constant, k A Affinity equilibrium constant.
Step 5: binding force analysis of H5N1 influenza virus and H5N1 specific antibody
H5N1 influenza virus was diluted in a ratio of 1:5,1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560,1:5120,1:10240 using HBS-EP buffer pH 7.4 and separately assayed by injection. Analysis was performed using the Biacore 3000 biosensor binding force analysis program. After each detection run, the chip was regenerated (20. Mu.L/min, 30 s) using 10mmol/L Glycine-HCL (pH 2.5) at 25℃for the whole process. The binding force analysis of H5N1 influenza virus and H5N1 specific antibody is shown in FIG. 3 (a) and FIG. 3 (b).
Step 6: and judging positive results. As shown in fig. 2 (a) and 2 (b), after the H5N1 influenza virus is injected, the response curve is in a parabolic continuous rising form, after 10mmol/L glycine-HCL (pH 2.5) is injected, the response curve is in a parabolic continuous falling form, and the response value and the sample concentration show obvious gradient effect, so that the detection result is positive. After the H1N1 influenza virus is injected, the response curve is not parabolic and continuously rises, so that the sample is negative.
Step 7: detection sensitivity analysis (compared with traditional ELISA detection methods)
SPR biosensor detection and ELISA detection were performed using the same H5N1 influenza virus specific antibodies, respectively. As shown in Table 2, the detection limit of the SPR biosensor detection method is slightly higher than that of the ELISA detection method, but the antibody usage amount and the detection time are obviously better than those of the ELISA detection. Therefore, the SPR detection method has obvious advantages when facing a large amount of samples to be detected, and is suitable for rapid preliminary screening.
Table 2 shows a comparison of SPR biosensor detection methods with conventional ELISA detection methods.
Note that: the H5N1 detection sample was 140. Mu.L. The coupling amount of the H5N1 monoclonal antibody on the chip is 8000RU, and 1RU corresponds to 1pg/mm 2 . CM5 chip area was 0.75mm 2 Therefore, the amount of the antibody consumed was 0.0015. Mu.g (8000 pg/mm) 2 ×0.75mm 2 ÷4)。
Claims (5)
1. The rapid influenza virus detection method based on the SPR biosensor is characterized by comprising the following steps of:
step 1: preparing equipment, materials and reagents, including SPR biosensor, CM5 chip, 10mmol/L sodium acetate solution with pH value of 4.0, 4.5, 5.0 and 5.5, 50mmol/L NaOH, HBS-EP buffer with pH value of 7.4, amino coupling kit, influenza virus specific antibody and influenza virus;
step 2: screening influenza virus antibody optimal coupling pH
Diluting influenza virus antibodies to 10 μg/ml with 10mmol/L sodium acetate solution at pH 4.0, 4.5, 5.0 and 5.5, respectively; inserting a CM5 chip into an SPR biosensor, running an SPR biosensor antibody screening program, placing the diluted antibody into a sample injection groove, setting the sample injection flow rate to be 10 mu L/min, running for 2min, cleaning the CM5 chip with 25 mu L of 50mmol/LNaOH after the analysis program is finished, and then analyzing the next antibody sample; after all four diluted antibodies are analyzed, the reaction value is selected to be not lower than 8000pg/mm 2 As the optimal coupling pH of the antibody;
step 3: influenza virus antibody coupling chip
After the optimal coupling pH is determined, 10mmol/L sodium acetate solution with the pH value is used as a coupling buffer solution, and the influenza antibody is diluted to 10 mug/mL; the CM5 chip is inserted into an SPR biosensor, and an amino coupling kit and an SPR biosensor automation program are adopted to couple the antibody on the surface of the chip; CM5 chip F2 channel coupled influenza specific antibody, F1 channel as blank reference, coupling target is reaction value 8000pg/mm 2 ；
Step 4: analysis of affinity kinetic constants of samples to be tested and antibodies
Diluting a sample to be detected by using an HBS-EP buffer solution with the pH of 7.4 according to the proportion of 1:5,1:10,1:20,1:40 and 1:80, and respectively carrying out sample injection detection; analyzing by using an SPR biosensor affinity kinetics analysis program, and regenerating a chip by using 10mmol/L glycine-HCL with the pH value of 2.5, wherein the temperature of the whole process is 25 ℃ after each detection flow, and the temperature is 20 mu L/min for 30 s; binding constant K using SPR biosensor software program a Dissociation constant K d And affinity equilibrium constant K A Analyzing;
step 5: binding force analysis of test sample and antibody
Diluting a sample to be detected by using an HBS-EP buffer solution with the pH of 7.4 according to the proportion of 1:5,1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560,1:5120 and 1:10240, and respectively carrying out sample injection detection; analyzing by utilizing an SPR biosensor binding force analysis program; after each detection flow, regenerating the chip by using 10mmol/L glycine-HCL and pH 2.5, wherein the temperature of the whole process is 25 ℃ at 20 mu L/min for 30 s;
step 6: judging a positive result; if the response curve is in a parabolic continuous rising form after sample introduction of the sample to be detected, the pH is 2.5, the response curve is in a parabolic continuous falling form after sample introduction, and the response value and the concentration of the sample show obvious gradient effects, the sample to be detected is judged to be positive for influenza virus, otherwise, the sample to be detected is judged to be negative.
2. The rapid influenza virus detection method based on SPR biosensor according to claim 1, wherein the target value of antibody coupling is 8000pg/mm 2 。
3. A rapid influenza virus detection method based on SPR biosensor according to claim 1 or 2, wherein,
before sample injection, the sample to be detected should be centrifuged at 12000rpm and 4 ℃ for 5min to remove impurities.
4. A rapid influenza virus detection method based on SPR biosensor according to claim 1 or 2, wherein,
and carrying out gradient dilution by adopting an influenza sample with a known concentration, and then detecting to obtain the detection limit of the method.
5. The rapid detection method of influenza virus based on SPR biosensor as claimed in claim 3, wherein the detection limit of the method is obtained by performing the detection after the gradient dilution of the influenza sample with known concentration.
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