A photochemiluminescence method for detecting myxovirus resistance protein a

Abstract

The application discloses a photochemical chemiluminescence method for detecting myxovirus resistance protein A, and belongs to the technical field of marker detection. The application provides a detection system of the myxovirus resistance protein A, which comprises MxA Ab1-receptor microsphere complex and MxA Ab2-donor microsphere complex. The application provides the detection system of the myxovirus resistance protein A, which is used for the detection of MxA, is homogeneous and free of washing, simple in operation, high in sensitivity, and wide in linear range. The application is verified through experiments, and the detection limit of the myxovirus resistance protein A antigen is 1.37 ng / mL, and the linear range is 5-1229 ng / mL. The application is good in specificity and repeatability, can be used for rapidly detecting a virus infection marker, and has application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of marker detection, in particular to a light initiated chemiluminescent assay for detecting myxovirus resistance protein A. BACKGROUND

[0002] Myxovirus resistance protein A (MxA) is a protein induced by interferon (IFN), which has activity against multiple viruses. MxA plays an important role in host defense against viral infection by directly acting on the viral genetic material in cells, so when the level of MxA in the blood increases, it reflects viral infection in the body and is a specific marker of viral infection. The detection of MxA can be used for the identification of respiratory viral infection in children and postoperative patients.

[0003] Light initiated chemiluminescent assay (LICA) is a chemiluminescent technology that modifies nanoscale microspheres to make the microspheres have corresponding functions for light emission. In this process, the photosensitive microspheres are connected to the secondary antibody as donor microspheres, and the light-emitting microspheres are connected to the primary antibody as acceptor microspheres. When the target antigen to be detected exists in the system, the three will approach each other to form an antibody-antigen-antibody immune sandwich complex. The distance is less than 200 nm. At this time, the donor microspheres are excited by 680 nm light, and the donor microspheres generate singlet oxygen to transfer energy to the acceptor microspheres, and the acceptor microspheres emit 610-620 nm light to detect the luminescence signal. LICA has high sensitivity, homogeneous reaction system, wide linear range, etc. At present, there is no report on using LICA technology to detect MxA. SUMMARY

[0004] The purpose of the present application is to provide a light initiated chemiluminescent assay for detecting myxovirus resistance protein A to solve the problems existing in the prior art.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] One of the technical solutions of the present application is a detection system for myxovirus resistance protein A, which comprises MxA Ab1-acceptor microsphere complex and MxA Ab2-donor microsphere complex.

[0007] The second technical solution of the present application is a detection method for myxovirus resistance protein A for non-disease diagnosis or treatment purposes, which utilizes the detection system to detect myxovirus resistance protein A in the sample to be detected.

[0008] Based on the above technical solutions, the present application has the following technical effects:

[0009] The application provides a detection system of myxovirus resistance protein A, which is used for the detection of MxA, is homogenous, wash-free, simple in operation, high in sensitivity and wide in linear range. The detection limit of the myxovirus resistance protein A antigen is 1.37 ng / mL, the linear range is 5-1229 ng / mL, the specificity is good, the repeatability is good, the detection method can be used for rapid detection of virus infection markers, and has application value. BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0011] Figure 1 Optimization chart for the ratio of donor microspheres to acceptor microspheres.

[0012] Figure 2 Optimization chart for the optimal amount of myxovirus resistance protein A antigen.

[0013] Figure 3 Optimization chart for the reaction temperature.

[0014] Figure 4 Optimization chart for the reaction time.

[0015] Figure 5 Hook effect chart and standard curve of the myxovirus resistance protein A antigen.

[0016] Figure 6 Repeatability of the detection method of the present application.

[0017] Figure 7 Specificity of the detection method of the present application.

[0018] Figure 8 Anti-interference property of the detection method of the present application.

[0019] Figure 9 Principle chart of the experiment of the present application. DETAILED DESCRIPTION

[0020] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0021] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value or subrange within that range is also specifically disclosed. Each of the smaller ranges is also individually and specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also endpoints of the range, subject to any specifically excluded endpoint.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0023] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples given are exemplary only.

[0024] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0025] Unless otherwise specified, the technical solutions described in the present application are conventional solutions in the art, and the reagents or raw materials used are purchased from commercial channels or are publicly known.

[0026] The embodiment of the present application provides a detection system of a myxovirus resistance protein A, which comprises an MxA Ab1-receptor microsphere complex and an MxA Ab2-donor microsphere complex.

[0027] In some specific embodiments, the volume ratio of the MxA Ab1-receptor microsphere complex and the MxA Ab2-donor microsphere complex is 5:4;

[0028] The concentration of the MxA Ab1-receptor microsphere complex is 0.007818 mg / mL;

[0029] The concentration of the MxA Ab2-donor microsphere complex is 0.01221 mg / mL.

[0030] In some specific embodiments, the method for preparing the MxA Ab1-receptor microsphere complex comprises the following steps: after activating the receptor microspheres, adding the MxA Ab1, an antibody against the resistance protein A of the myxovirus, to obtain the MxA Ab1-receptor microsphere complex.

[0031] In some specific embodiments, the mass ratio of the receptor microspheres to the MxA Ab1, an antibody against the resistance protein A of the myxovirus, is 127.66:1.

[0032] The method for activating the receptor microspheres comprises the following steps: adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and N-hydroxysuccinimide into the receptor microspheres, uniformly mixing, and then sealing and incubating; the volume ratio of the receptor microspheres, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, and the N-hydroxysuccinimide is 100:6:66; the incubation condition is 50 r / min, 25℃, and incubation for 20 min.

[0033] The method for activating the receptor microspheres further comprises two steps of washing before and after the activation.

[0034] In some specific embodiments, the method for preparing the MxA Ab2-donor microsphere complex comprises the following steps: after activating the donor microspheres, adding the MxA Ab2, a secondary antibody against the resistance protein A of the myxovirus, to obtain the MxA Ab2-donor microsphere complex.

[0035] In some specific embodiments, the mass ratio of the donor microspheres to the MxA Ab2, a secondary antibody against the resistance protein A of the myxovirus, is 81.63:1.

[0036] The method for activating the donor microspheres comprises the following steps: adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and N-hydroxysuccinimide into the donor microspheres, uniformly mixing, and then sealing and incubating; the volume ratio of the donor microspheres, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, and the N-hydroxysuccinimide is 100:6:66; the incubation condition is 50 r / min, 25℃, and incubation for 20 min.

[0037] The method for activating the donor microspheres further comprises two steps of washing before and after the activation.

[0038] The embodiments of the present application also provide a method for detecting the resistance protein A of the myxovirus for non-disease diagnosis or treatment purposes, which utilizes the detection system to detect the resistance protein A of the myxovirus in a sample to be detected.

[0039] In some specific embodiments, the reaction temperature of the detection is 25℃, and the reaction time is 8 min.

[0040] In some specific embodiments, the volume ratio of the sample to be tested to the MxA Ab1-receptor microsphere complex in the detection system is 3:5.

[0041] Example 1

[0042] 1. Coupling of microspheres and antibodies

[0043] 1.1. Washing of microspheres

[0044] Take 100 μL of 10 mg / mL donor microspheres (lisa donor microspheres, for Biolegend, Inc., Cat. No.: 67500001) and add 1 mL of activation buffer (pH 6.2, 0.05 mM MES) to a 2 mL transparent centrifuge tube. Mix well after ultrasonic treatment for 7-8 s, then centrifuge at 12000 rpm and 4°C for 40 min, and discard the supernatant.

[0045] 1.2. Activation of microspheres

[0046] Add 1 mL of activation buffer to the precipitate, mix well after ultrasonic treatment for 5 s, and then continue ultrasonic treatment for 7-8 s, take out and mix well by inverting, repeat 4-5 times. Then, immediately add 6 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution (10 μg / μL) to the dispersed microspheres, mix well after oscillation, then add 66 μL of N-hydroxysuccinimide (10 μg / μL), mix well after oscillation, seal with sealing film, and incubate in a constant temperature shaker instrument at 50 r / min and 25°C for 20 min.

[0047] 1.3. Washing of microspheres

[0048] ① After the activation is completed, centrifuge at 12000 rpm and 4°C for 40 min, and discard the supernatant.

[0049] ② Add 1 mL of activation buffer to the precipitate, mix well after ultrasonic treatment for 5 s, and then continue ultrasonic treatment for 7-8 s, take out and mix well by inverting, repeat 4-5 times. Then, centrifuge at 12000 rpm and 4°C for 40 min, and discard the supernatant.

[0050] ③ Add 1 mL of ultrapure water to the precipitate, mix well after ultrasonic treatment, then centrifuge at 12000 rpm and 4°C for 40 min, and discard the supernatant.

[0051] 1.4. Coupling of antibodies

[0052] ① Add 1 mL of microsphere coupling solution (phosphate buffered saline (1X)) to the washed microspheres, mix well after ultrasonic treatment, then carefully add:

[0053] To 1 mL of acceptor microspheres (1 mg / mL), add 2.8 μL of 2.8 mg / mL of the first antibody of the myxovirus resistance protein A (MxA Ab1) (Hangzhou Huakuijinpei Biotechnology Co., Ltd., Catalog No.: M30133) to form a MxA Ab1-acceptor microsphere complex;

[0054] To 1 mL of donor microspheres (1 mg / mL), add 3.5 μL of 3.5 mg / mL of the second antibody of the myxovirus resistance protein A (MxA Ab2) (Hangzhou Huakuijinpei Biotechnology Co., Ltd., Catalog No.: M30132) to form a MxA Ab2-donor microsphere complex.

[0055] ②After careful shaking and mixing, seal with sealing film, incubate in a constant temperature shaking instrument, 50 r / min, 25℃, activate for 2 h.

[0056] ③After 2 h, add 100 μL of microsphere blocking solution (QuickBlock TM Western blocking solution) to the microspheres, shake and mix, seal with sealing film, incubate in a constant temperature shaking instrument, 50 r / min, 25℃, block overnight.

[0057] 1.5 Washing and storage

[0058] The next day, centrifuge the centrifuge tube in a centrifuge at 12000 rpm, 4℃ for 40 min, discard the supernatant. Add 1 mL of microsphere washing solution (0.1% Tween-20) to the precipitate, shake and mix, then place it in a centrifuge, centrifuge at 12000 rpm, 4℃ for 40 min, discard the supernatant. Add 1 mL of luminescence storage solution (homogeneous luminescence diluent, Hangzhou Huake Biotechnology Co., Ltd., Catalog No.: HI0101A-3) and store at 2-8℃ in the dark.

[0059] 2 Optimization of the volume ratio of donor microspheres to acceptor microspheres

[0060] Take 25 μL of the first antibody of the myxovirus resistance protein A (0.02 mg / mL) coated on the acceptor microspheres in a 96-well test plate, add 10 μL of 0.001 mg / mL of the myxovirus resistance protein A antigen to it, place it in an incubator and shake for 8 min, then immediately add different volumes of the second antibody of the myxovirus resistance protein A (0.02 mg / mL) coated on the donor microspheres, the volumes are: 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, shake in the incubator for 8 min, and immediately measure the luminescence signal value with a BPCL photochemical luminescence instrument after shaking.

[0061] After the donor microspheres and the acceptor microspheres are coupled with the paired antibodies of the adeno-associated virus resistance protein A respectively, the antibodies are diluted in different proportions to form an immune sandwich complex with the adeno-associated virus resistance protein A antigen, and then the complex is detected on the machine to investigate the most suitable dosage ratio of the donor microspheres and the acceptor microspheres. The results are shown in Figure 1 As the ratio of the donor microspheres and the acceptor microspheres increases, the trend is first increased and then decreased. When the volume ratio of the acceptor microspheres to the donor microspheres is 5:4, the luminescence intensity is the best, and therefore the ratio is the most suitable ratio.

[0062] 3. Optimal dosage of the standard

[0063] 25 μL of the acceptor microspheres coated with the adeno-associated virus resistance protein A primary antibody complex (0.02 mg / mL) is taken in a 96-well test plate, and different volumes of 0.001 mg / mL of the adeno-associated virus resistance protein A antigen are added, and the volumes are 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, and 35 μL. The mixture is placed in an incubator and shaken for 8 min, and then 20 μL of the donor microspheres coated with the adeno-associated virus resistance protein A secondary antibody complex (0.02 mg / mL) is immediately added, and the mixture is shaken in the incubator for 8 min. After the shaking is completed, the luminescence signal value is measured by using the BPCL photochemical luminescence instrument.

[0064] When the dosages of the donor microspheres (20 μL) and the acceptor microspheres (25 μL) are fixed, the optimal dosage of the adeno-associated virus resistance protein A antigen in the detection process is investigated. The results are shown in Figure 2 As the dosage of the adeno-associated virus resistance protein A antigen increases, the trend is first increased and then decreased. When the dosage of the adeno-associated virus resistance protein A antigen is 15 μL, the signal is the best, and therefore 15 μL is the optimal dosage of the adeno-associated virus resistance protein A antigen.

[0065] 4. Optimization of the reaction temperature

[0066] 25 μL of the acceptor microspheres coated with the adeno-associated virus resistance protein A primary antibody complex (0.0002 mg / mL) is taken in a 96-well test plate, and 15 μL of 0.001 mg / mL of the adeno-associated virus resistance protein A antigen is added. The mixture is placed in an incubator and shaken at different temperatures (20°C, 25°C, 37°C, and 40°C) for 8 min, and then 20 μL of the donor microspheres coated with the adeno-associated virus resistance protein A secondary antibody complex (0.0002 mg / mL) is immediately added. The mixture is shaken in the incubator at different temperatures (20°C, 25°C, 37°C, and 40°C) for 8 min. After the shaking is completed, the luminescence signal value is measured by using the BPCL photochemical luminescence instrument.

[0067] The temperature of the whole reaction was optimized when the amount of donor microspheres (20 μL) and acceptor microspheres (25 μL) and the amount of antigen added (15 μL) were determined. The results are shown in Table 1. Figure 3 As shown in Table 1, when the reaction temperature was 25°C, the curve showed an increasing trend with the increase of the concentration of the antigen of the resistance protein A of myxovirus, and the signal was good. Therefore, it was considered that the reaction temperature of 25°C was the optimal reaction temperature.

[0068] 5 Optimization of reaction time

[0069] 25 μL of acceptor microspheres coated with the first antibody complex of the resistance protein A of myxovirus (0.002 mg / mL) was taken in a 96-well test plate, 15 μL of 0.001 mg / mL of the antigen of the resistance protein A of myxovirus was added, and the mixture was incubated in an incubator for different times (30 s, 2 min, 4 min, 8 min) with shaking, then 20 μL of the second antibody complex of the resistance protein A of myxovirus coated with donor microspheres (0.002 mg / mL) was immediately added, and the mixture was incubated in an incubator for different times (30 s, 2 min, 4 min, 8 min) with shaking. After the shaking was completed, the luminescence signal value was measured by a BPCL photochemical luminescence instrument.

[0070] After the reaction temperature was determined, the incubation time was optimized, and the results are shown in Table 2. Figure 4 As shown in Table 2, with the increase of the reaction time, the sandwich complex formed increased, and the signal value also increased. As shown in Table 2, when the reaction time was 8 min, the reaction effect was good, so 8 min was used as the incubation time in this experiment. Figure 4

[0071] ​Optimized detection process: Take the coupled receptor microspheres coated with adhesion virus resistance protein A primary antibody complex and donor microspheres coated with adhesion virus resistance protein A secondary antibody complex from the refrigerator, stand at room temperature, then dilute the receptor microspheres coated with adhesion virus resistance protein A primary antibody complex (0.007818 mg / mL) according to the volume ratio: receptor microspheres coated with adhesion virus resistance protein A primary antibody complex:diluent = 2:5; dilute the donor microspheres coated with adhesion virus resistance protein A secondary antibody complex (0.01221 mg / mL) according to the volume ratio: donor microspheres coated with adhesion virus resistance protein A secondary antibody complex:diluent = 1:3. Then dilute the antigen to be tested with 1xPBS, take 5 μL of the antigen to be tested (0.3 mg / mL) and add it to 175 μL of 1xPBS to prepare a stock solution, then take 80 μL of the stock solution and add it to 30 μL of 1xPBS for dilution. Add 15 μL of the known concentration of the antigen to be tested of different concentrations to the 96-well plate, immediately add 25 μL of the receptor microspheres coated with adhesion virus resistance protein A primary antibody complex, place it in an incubator at 25°C for 8 min, then add 20 μL of the donor microspheres coated with adhesion virus resistance protein A secondary antibody complex, place it in an incubator at 25°C for 8 min, and immediately detect the luminescence signal value.

[0072] Note: The dilution ratio of the receptor microspheres coated with adhesion virus resistance protein A primary antibody complex and the donor microspheres coated with adhesion virus resistance protein A complex to the diluent is not fixed, only affects the height of the luminescence signal value, and different dilution ratios have no effect on the optimal conditions of the optimization process.

[0073] 6 Linearity

[0074] Take 50 μL of adhesion virus resistance protein A antigen (0.002 mg / mL) in a test tube, add antigen diluent (phosphate buffer (1X) 15 μL, sequentially gradient dilution, then test the BPCL light-activated chemiluminescence value of the antigen of different concentrations according to the above steps.

[0075] Under the optimal detection conditions, different concentrations of adhesion virus resistance protein A antigen were determined, as shown in Figure 5 , the linear equation is y = 73.87x + 10475, R 2 = 0.9957, the linear range is 5-1229 ng / mL, and the detection limit is 1.37 ng / mL.

[0076] 7 Reproducibility

[0077] Take 25 μL of the receptor microspheres coated with the virus resistance protein A primary antibody complex (0.002 mg / mL) in a 96-well test plate, add 15 μL of 0.00001 mg / mL of the virus resistance protein A antigen to the test plate, and place the test plate in an incubator for vibration incubation for 8 min, then immediately add 20 μL of the donor microspheres coated with the virus resistance protein A secondary antibody complex (0.002 mg / mL) to the test plate, and place the test plate in an incubator for vibration incubation for 8 min, and immediately measure the luminescence signal value of the test plate by using a BPCL photochemical luminescence instrument.

[0078] Under the optimal conditions, the repeatability of the virus resistance protein A is tested by repeating the determination for 16 times, and the relative standard deviation RSD is calculated, as shown in the following table. Figure 6

[0079] 8 Selectivity

[0080] The selectivity of the method is determined by selecting several possible interfering substances, and the photochemical luminescence values of the selected substances are measured according to the above detection steps.

[0081] The selectivity of the method is determined by selecting several possible interfering substances, and the photochemical luminescence values of the selected substances are measured according to the above detection steps. Figure 7 It can be seen that the detection method only has a signal for the virus resistance protein A antigen, and therefore the specificity is good.

[0082] 9. Anti-interference

[0083] The anti-interference of the method is determined by selecting several possible interfering substances in serum samples, and the photochemical luminescence values of the selected substances are measured according to the above detection steps.

[0084] The anti-interference of the method is determined by selecting several possible interfering substances, and the photochemical luminescence values of the selected substances are measured according to the above detection steps. Figure 8 It can be seen that the detection method has good anti-interference, and the interference rate is -1.31% to 1.70%, which indicates that the method has good anti-interference.

[0085] In summary, the detection method of MxA provided by the application is all-phase and wash-free, simple to operate, has high sensitivity, a wide linear range, good specificity, and good repeatability, can be used for rapid detection of virus infection markers, and has application value.

[0086] ​Obviously, the above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A detection system for a resistance protein A of a virus of the family of the Orthomyxoviridae, characterized in that, The MxA Ab1-receptor microsphere complex and the MxA Ab2-donor microsphere complex are included; The volume ratio of the MxA Ab1-receptor microsphere complex and the MxA Ab2-donor microsphere complex is 5:4; The concentration of the MxA Ab1-receptor microsphere complex is 0.007818 mg / mL; The concentration of the MxA Ab2-donor microsphere complex is 0.01221 mg / mL; The preparation method of the MxA Ab1-receptor microsphere complex comprises the following steps: after the receptor microspheres are activated, the MxA Ab1, the virus resistance protein A primary antibody, is added, so that the MxA Ab1-receptor microsphere complex is obtained; the mass ratio of the receptor microspheres to the MxA Ab1, the virus resistance protein A primary antibody, is 127.66:1; The method for activating the receptor microspheres comprises the following steps: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride solution and N-hydroxysuccinimide are added into the receptor microspheres, and then the mixture is uniformly mixed and sealed for incubation; the volume ratio of the receptor microspheres, the 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride solution and the N-hydroxysuccinimide is 100:6:66; the incubation is carried out at 50 r / min and 25°C for 20 min; The receptor microspheres further comprise two washing steps before and after the activation; The preparation method of the MxA Ab2-donor microsphere complex comprises the following steps: after the donor microspheres are activated, the MxA Ab2, the virus resistance protein A secondary antibody, is added, so that the MxA Ab2-donor microsphere complex is obtained; the mass ratio of the donor microspheres to the MxA Ab2, the virus resistance protein A secondary antibody, is 81.63:1; The method for activating the donor microspheres comprises the following steps: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride solution and N-hydroxysuccinimide are added into the donor microspheres, and then the mixture is uniformly mixed and sealed for incubation; the volume ratio of the donor microspheres, the 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride solution and the N-hydroxysuccinimide is 100:6:66; the incubation is carried out at 50 r / min and 25°C for 20 min; The donor microspheres further comprise two washing steps before and after the activation.

2. A method for detecting a resistance protein A of a myxovirus for a purpose other than disease diagnosis or treatment, characterized by, The detection system of claim 1 is used for detecting the virus resistance protein A in a sample to be detected.

3. The detection method according to claim 2, characterized in that, The reaction temperature of the detection is 25°C, and the reaction time is 8 min.

4. The method of claim 2, wherein The volume ratio of the sample to be detected to the MxA Ab1-receptor microsphere complex in the detection system is 3:5.

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