Protein antigen on-line fluorescence quantitative detection method based on immunoreaction

Abstract

The invention relates to a protein antigen on-line fluorescence quantitative detection method based on immunoreaction. The detection method comprises the following steps: preparing an immune complex to-be-detected sample containing to-be-detected protein antigens and immune complex standard samples containing different concentration gradients of protein antigens; constructing a real-time fluorescence imaging system which specifically comprises an electrophoresis tank, an excitation light source, an imaging device and a band-pass filter; loading the immune complex sample into a gel sample hole, and carrying out electrophoretic separation; in the electrophoresis process, a fluorescence image is collected through a real-time fluorescence imaging system, and the immunoelectrophoresis process is judged by observing migration and separation of a fluorescence band; and acquiring brightness information of a corresponding band of the free fluorescence-labeled antibody, and quantitatively analyzing the protein antigen concentration. Compared with the prior art, the protein antigen online fluorescent quantitative detection method has the advantages of high detection speed, simplicity and convenience in operation, low cost, high specificity, accurate quantification and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of protein detection, and in particular relates to an on-line fluorescence quantitative detection method of protein antigen based on immune response. Background Art

[0002] The specific quantitative detection of target proteins is of great significance in the fields of disease diagnosis, drug development and biomarker screening. At present, the main methods of protein immunological detection include Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofixation electrophoresis.

[0003] Among them, Western blot, as a classic protein-specific detection technology, can achieve the identification and semi-quantitative analysis of specific proteins by combining electrophoretic separation with immune recognition. This method first separates the protein mixture by polyacrylamide gel electrophoresis (PAGE), then transfers the separated proteins to the membrane, recognizes the target protein by specific antibodies and performs color detection. However, Western blot has many limitations, such as cumbersome operation steps, a long time span for the entire detection process, usually requiring 2-3 days; poor repeatability, unstable protein transfer efficiency after electrophoresis, and large differences between batches in the final semi-quantitative results; inability to detect online, traditional chemiluminescence or color detection methods rely on endpoint detection, making it difficult to achieve real-time monitoring; Western blot is mainly used for semi-quantitative analysis of proteins, and it is difficult to obtain accurate quantitative results, let alone absolute quantitative detection.

[0004] ELISA is another widely used immunological quantitative detection method for proteins, etc., with high sensitivity and specificity. This method achieves the capture and detection of target proteins by coating specific antibodies on the surface of a solid phase carrier through antigen-antibody specific binding. However, ELISA also has obvious shortcomings, such as high cost. Commercial ELISA kits are expensive, which increases the cost of detection; cumbersome operation, requiring multiple incubation and washing steps during the detection process; ELISA has high requirements for the quality of antibodies, and the specificity and affinity of antibodies directly affect the accuracy of the test results; poor anti-interference ability, due to the lack of separation steps, interferences in complex matrices may affect the accuracy of detection.

[0005] Immunofixation electrophoresis is also a protein immunoassay method widely used in clinics and laboratories, mainly used for typing analysis of myeloma serum immunoglobulin markers, etc. This method first separates immunoglobulin markers by electrophoresis, then applies specific antibodies directly to the surface of the electrophoresis gel, forms a precipitation band through antigen-antibody reaction, and then performs chemical staining for detection. However, this method also has some limitations, such as low sensitivity and poor anti-interference ability; it requires multiple steps of manual operation, which is labor-intensive and time-consuming; the result judgment is interfered by personnel experience, and the detection process requires professional personnel to interpret the results, which is subjective; the antibody consumption is large and the detection cost is high.

[0006] In recent years, with the deepening of biomedical research and the growth of clinical diagnosis needs, the requirements for protein immunoquantitative detection methods have continued to increase. Especially in the fields of disease marker detection, drug screening and biological product quality control, there is an urgent need for a fast, accurate, simple and low-cost protein-specific quantitative detection method. At present, although a variety of detection technologies have been developed, it is still difficult to simultaneously meet the requirements of fast detection speed, simple operation, low cost, strong specificity and accurate quantification. Therefore, the development of a new protein-specific quantitative detection method has important theoretical and practical application value. Summary of the invention

[0007] The purpose of the present invention is to provide an online fluorescence quantitative detection method for protein antigens based on immune response in order to overcome the defects of the prior art that it is difficult to simultaneously meet the requirements of fast detection speed, simple operation, low cost, strong specificity and accurate quantification.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention provides an on-line fluorescence quantitative detection method for protein antigens based on immune response, and the detection method comprises the following steps:

[0010] S1. Preparation of immune complex samples: preparing immune complex test samples containing the protein antigen to be tested and the fluorescently labeled antibody, and a series of immune complex standard samples containing the protein antigen and the fluorescently labeled antibody at different concentration gradients;

[0011] S2. Construct a real-time fluorescence imaging system, which specifically includes: an electrophoresis tank for loading a flat plate gel for electrophoresis and assisting fluorescence imaging; an excitation light source for exciting the fluorescent labeled antibody to generate a fluorescence signal; an imaging device for collecting the fluorescence image on the flat plate gel; and a bandpass filter provided at the front end of the imaging device lens;

[0012] S3, electrophoretic separation of immune complexes: loading the prepared immune complex samples into sample wells of polyacrylamide gel, followed by electrophoretic separation;

[0013] S4, collecting electrophoresis fluorescence images: During the electrophoresis process, the real-time fluorescence imaging system constructed in S2 is used to collect fluorescence images, and the progress of immunoelectrophoresis is judged by observing the migration and separation of the fluorescence bands;

[0014] S5. Quantitative analysis of protein antigen concentration based on fluorescence images: Obtain the brightness information of the corresponding bands of free fluorescent-labeled antibodies and quantitatively analyze the protein antigen concentration.

[0015] Furthermore, in step S1, the specific preparation method of the immune complex sample is: the protein antigen and the fluorescently labeled antibody are mixed and then incubated to allow the protein antigen and the fluorescently labeled antibody to combine to form an immune complex; after cross-linking and fixing with formaldehyde, excess formaldehyde is quenched with a Tris buffer solution, and then the cross-linked immune complex is mixed with a protein loading buffer and heated to perform SDS treatment on the immune complex and protein for use.

[0016] Furthermore, in step S2, the flat plate gel is fastened to the front end of the electrophoresis tank by transparent glass and wedges.

[0017] Furthermore, in step S2, the excitation light source is an LED light board, whose central wavelength is adapted to the excitation wavelength of the fluorescent-labeled antibody.

[0018] The excitation light source can use an LED light board with a central wavelength of 470nm, which can be adapted to the excitation wavelength of FTIC; or other central wavelengths of LED light boards can be used, which can be adapted to the excitation wavelength range of other fluorescent groups. Specifically, the LED light board with a central wavelength of 480-495nm can be adapted to the excitation wavelength of Cy3; the LED light board with a central wavelength of 630-640nm can be adapted to the excitation wavelength of Cy5; the LED light board with a central wavelength of 480-495nm can be adapted to the excitation wavelength of PE. The corresponding selection relationship between other fluorescent-labeled antibodies and LED light boards is similar, and can be selected according to specific scenarios and needs.

[0019] Preferably, the excitation light sources are symmetrically arranged on both sides of the flat gel.

[0020] Furthermore, the horizontal angle between the excitation light source and the flat gel is 10-70°, preferably 30°.

[0021] Furthermore, in step S2, the bandpass filter is a filter that can transmit fluorescence of 520-560nm.

[0022] Furthermore, in step S2, the imaging device includes a camera lens with adjustable focal length and aperture and a CMOS image sensor.

[0023] Furthermore, the distance between the camera lens and the flat gel imaging surface is 200-300 mm.

[0024] Furthermore, in step S3, the voltage of the electrophoretic separation is 80-200V.

[0025] Furthermore, in step S3, the total time of the electrophoretic separation is 50-100 min.

[0026] Furthermore, in step S4, the exposure time used for the fluorescence image acquisition is 1000-9000 ms, and the gain parameter is 1-20.

[0027] Furthermore, in step S5, the specific steps of quantitatively analyzing the protein concentration are: using image analysis software to measure the fluorescence intensity value of the band corresponding to the free fluorescent-labeled antibody in each lane, and drawing a standard curve of fluorescence intensity and protein antigen concentration; quantitatively estimating the protein antigen concentration in the immune complex sample to be tested based on the standard curve.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The protein antigen online fluorescence quantitative detection method of the present invention does not require complicated gel disassembly and time-consuming fixation and staining steps, and can achieve specific quantitative detection of the target protein through the immune binding principle and online fluorescence imaging.

[0030] (2) The protein antigen online fluorescence quantitative detection method of the present invention can quickly complete the detection within 1.5 hours, which greatly shortens the detection time compared with the traditional Western blot method.

[0031] (3) The protein antigen online fluorescence quantitative detection method of the present invention can be used for real-time online monitoring during protein immunoelectrophoresis, without the need for complicated and time-consuming steps such as gel disassembly, membrane transfer and washing, fixation and staining, and scanning and imaging, thereby effectively avoiding the in-gel broadening of protein bands and improving the separation resolution.

[0032] (4) Compared with the ELISA kit, the protein antigen online fluorescence quantitative detection method of the present invention only requires a small amount of inexpensive antibodies to complete the detection, and the equipment can be reused, which significantly reduces the detection cost.

[0033] (5) The present invention constructs a standard curve of band fluorescence intensity and protein amount through fluorescence spectrum analysis. The quantitative standard curve has a wide dynamic range and a good linear fitting coefficient, which can achieve accurate quantification of the target protein.

[0034] (6) The present invention combines the specificity of immune recognition with the resolution of electrophoretic separation. This method can effectively avoid interference from impurities in complex samples and achieve specific and highly sensitive detection of low-abundance proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the immune binding and formaldehyde cross-linking molecular reaction principle of the present invention.

[0036] Figure 2 It is a schematic diagram of the electrophoretic separation and online fluorescence imaging principle of the present invention.

[0037] Figure 3 The figure is a schematic diagram of the optical system composition and imaging light path of the present invention.

[0038] Figure 4 This is a graph showing the free antibody fluorescent bands and protein antigen quantitative standard curve of Example 2.

[0039] Figure 5 It is the free antibody fluorescent band and protein antigen quantitative standard curve diagram of Example 3.

[0040] Description of the markings in the figure:

[0041] 1-electrophoresis tank, 2-slab gel, 3-excitation light source, 4-imaging device, 41-camera lens, 42-CMOS image sensor, 5-bandpass filter. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0043] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] The anti-HER2 used in the subsequent tests of the present invention is only for testing purposes and is not intended to limit the scope of protein antigens. Anti-HER2 can be obtained commercially or prepared and purified. The anti-HER2 mAb of the present invention is expressed using expi293F cells and purified by a protein A affinity chromatography column (MabSelect-SuRe). For details, refer to Y.Min, Y.Chen, L.Wang, Y.Ke, F.Rong, Q.He, P.Paerhati, H.Zong, J.Zhu, Y.Wang, B.Zhang, Supramolecularantibody-drug conjugates for combined antibody therapy and photothermaltherapy targeting HER2-positive cancers, International Journal of BiologicalMacromolecules 278 (2024) 134622. https: / / doi.org / https: / / doi.org / 10.1016 / j.ijbiomac.2024.134622, which will not be repeated here.

[0045] The present invention provides an on-line fluorescence quantitative detection method for protein antigens based on immune response, and the detection method comprises the following steps:

[0046] S1. Preparation of immune complex samples: preparing immune complex test samples containing the protein antigen to be tested and the fluorescently labeled antibody, and a series of immune complex standard samples containing the protein antigen and the fluorescently labeled antibody at different concentration gradients;

[0047] S2, construct a real-time fluorescence imaging system, specifically including an electrophoresis tank 1, used to load a flat gel 2 for electrophoresis and assist fluorescence imaging; an excitation light source 3, used to excite the fluorescent labeled antibody to generate a fluorescent signal; an imaging device 4, used to collect the fluorescent image on the flat gel 2; a bandpass filter 5 provided at the front end of the lens of the imaging device 4;

[0048] S3, electrophoretic separation of immune complexes: loading the prepared immune complex samples into sample wells of polyacrylamide gel, followed by electrophoretic separation;

[0049] S4, collecting electrophoresis fluorescence images: During the electrophoresis process, the real-time fluorescence imaging system constructed in S2 is used to collect fluorescence images, and the progress of immunoelectrophoresis is judged by observing the migration and separation of the fluorescence bands;

[0050] S5. Quantitative analysis of protein antigen concentration based on fluorescence images: Obtain the brightness information of the bands corresponding to the free fluorescently labeled antibodies (rather than the bands corresponding to the immune complexes), and quantitatively analyze the protein antigen concentration.

[0051] In some specific embodiments, in step S1, the specific method for preparing the immune complex sample is as follows: Mix the protein antigen and the fluorescently labeled antibody and incubate them to allow the protein antigen to bind to the fluorescently labeled antibody to form an immune complex; After cross-linking and fixing with formaldehyde, quench the excess formaldehyde with Tris buffer solution, and then mix the cross-linked immune complex with the loading buffer containing SDS (sodium dodecyl sulfate) and heat-treat it for use in SDS modification of the immune complex and proteins.

[0052] In some specific embodiments, in step S2, the flat gel 2 is fastened to the front end of the electrophoresis tank 1 through a transparent glass and a wedge.

[0053] In some specific embodiments, in step S2, the excitation light source 3 is an LED light board, and its central wavelength is adapted to the excitation wavelength of the fluorescently labeled antibody.

[0054] In some specific embodiments, the excitation light sources 3 are symmetrically arranged on both sides of the flat gel 2; the horizontal angles between the excitation light sources 3 and the flat gel 2 are both 10 - 70°.

[0055] In some specific embodiments, in step S2, the band-pass filter 5 is a filter that can transmit fluorescence with a wavelength of 520 - 560 nm.

[0056] In some specific embodiments, in step S2, the imaging device 4 includes a camera lens 41 with adjustable focal length and aperture and a CMOS image sensor 42; the distance between the camera lens 41 and the imaging surface of the flat gel 2 is 200 - 300 mm.

[0057] In some specific embodiments, in step S3, the voltage for electrophoresis separation is 80 - 200 V, and the total time for electrophoresis separation is 50 - 100 min.

[0058] In some specific embodiments, in step S4, the exposure time used for fluorescence image acquisition is 1000 - 9000 ms, and the gain parameter is 1 - 20.

[0059] In some specific embodiments, in step S5, the specific steps for quantitatively analyzing the protein concentration are as follows: Use image analysis software to measure the fluorescence intensity values of the bands corresponding to the free fluorescently labeled antibodies in each lane, and draw a standard curve of fluorescence intensity versus protein antigen concentration; Quantitatively estimate the protein antigen concentration in the test sample of the immune complex according to the standard curve.

[0060] The above embodiments can be implemented individually or in any combination of two or more. The above embodiments are described in more detail below with reference to specific examples.

[0061] Embodiment 1:

[0062] This embodiment provides an online fluorescence quantitative detection method for protein antigens based on immune response, and the detection method comprises the following steps:

[0063] S1. Preparation of immune complex samples: Prepare immune complex test samples containing the protein antigen to be tested and fluorescently labeled antibodies, as well as a series of immune complex standard samples containing protein antigens and fluorescently labeled antibodies at different concentration gradients.

[0064] The specific preparation method is as follows: the protein antigen and the fluorescently labeled antibody are mixed and then incubated to allow the protein antigen and the fluorescently labeled antibody to combine to form an immune complex; formaldehyde (1%, w / v) is used for cross-linking and fixation, and then excess formaldehyde is quenched with a Tris buffer solution (10%, w / v), and then the cross-linked immune complex is mixed with a loading buffer and heated for use.

[0065] Figure 1 The figure shows the formation of immune complexes between fluorescently labeled antibodies and target antigen proteins, as well as the cross-linking and fixation process mediated by formaldehyde molecules. Figure 2 The electrophoretic separation process of immune complexes in polyacrylamide gel according to molecular weight is demonstrated, and the quantitative relationship between band brightness and protein concentration is obtained through real-time fluorescence imaging, which explains the basic principle of this method to establish a standard curve to achieve quantitative detection.

[0066] S2. Construct a real-time fluorescence imaging system, which specifically includes an electrophoresis tank 1 , an excitation light source 3 , an imaging device 4 and a bandpass filter 5 .

[0067] Specifically, Figure 3 As shown, the electrophoresis tank 1 of this embodiment is used to load the flat gel 2 for electrophoresis and assist fluorescence imaging. The flat gel 2 is fastened to the front end of the electrophoresis tank 1 by transparent glass and wedges. The electrophoresis tank 1 is powered by a 10-400V stabilized power supply. The excitation light source 3 is used to excite the fluorescent labeled antibody to generate a fluorescent signal. The excitation light source 3 is an LED light board, and its central wavelength is adapted to the excitation wavelength of the fluorescent labeled antibody. The excitation light sources 3 of this embodiment are symmetrically arranged on both sides of the flat gel 2 and are about 30° with the horizontal angle of the flat gel 2.

[0068] The excitation light source 3 of this embodiment can use an LED light board with a central wavelength of 470nm, which can be adapted to the excitation wavelength of FTIC; or LED light boards with other central wavelengths can be used, which can be adapted to the excitation wavelength range of other fluorescent groups. Specifically, the LED light board with a central wavelength of 480-495nm can be adapted to the excitation wavelength of Cy3; the LED light board with a central wavelength of 630-640nm can be adapted to the excitation wavelength of Cy5; the LED light board with a central wavelength of 480-495nm can be adapted to the excitation wavelength of PE. The corresponding selection relationship between other fluorescent-labeled antibodies and LED light boards is similar. Those skilled in the art can make a choice according to the specific scenario, which will not be repeated here.

[0069] The imaging device 4 of this embodiment is used to collect the fluorescent image on the flat gel 2, and specifically includes a camera lens 41 with adjustable focal length and aperture and a CMOS image sensor 42 for real-time surface imaging of the detection object. The distance between the camera lens 41 and the imaging surface of the flat gel 2 is 200-300 mm, preferably 250 mm. The bandpass filter 5 is arranged at the front end of the camera lens 41 of the imaging device 4, and the bandpass filter 5 can be a filter that can transmit fluorescence with a wavelength of 520-560 nm.

[0070] S3. Electrophoretic separation of immune complexes: The prepared immune complex sample is loaded into the sample well of the polyacrylamide gel, and then electrophoretic separation is performed. The voltage of the electrophoretic separation is 80-200V, and the total time of the electrophoretic separation is 50-100 minutes. Specifically, the voltage of the electrophoretic separation can be increased in a gradient after a specific time interval, and the electrophoresis time of each gradient voltage can also be adjusted appropriately.

[0071] S4, collecting electrophoresis fluorescence images: During the electrophoresis process, the real-time fluorescence imaging system constructed in S2 is used to collect fluorescence images, and the progress of immunoelectrophoresis is judged by observing the migration and separation of the fluorescence bands. The exposure time used for fluorescence image collection is 1000-9000ms, and the gain parameter is 1-20.

[0072] S5. Quantitative analysis of protein antigen concentration based on fluorescence images: Obtain the brightness information of the corresponding bands of free fluorescent labeled antibodies and quantitatively analyze the protein antigen concentration.

[0073] The specific steps are: use image analysis software to measure the fluorescence intensity value of the corresponding band of the free fluorescent labeled antibody in each lane, draw a standard curve of fluorescence intensity and protein antigen concentration; quantitatively estimate the protein antigen concentration in the immune complex sample to be tested based on the standard curve.

[0074] Embodiment 2:

[0075] This embodiment provides an online fluorescence quantitative detection method for anti-human epidermal growth factor receptor 2 (anti-HER2) based on immune response, and the specific steps are as follows:

[0076] Immune complex samples with anti-HER2 concentrations of 0, 0.05, 0.25, 0.5, 1, 2.5, and 5 mg / L were prepared, respectively, among which FITC-anti-human (fluorescently labeled antibody, Fluorescein (FITC)-conjugated Goat Anti-HumanIgG (H+L / Wuhan Sanying Biotechnology Co., Ltd.) were all diluted at the same multiple (1:80). Incubate at room temperature for 3 h in a program mixer, then add formaldehyde solution (1%, w / v) to each reaction system for immune complex cross-linking, continue incubation at room temperature for 10 min, and add Tris solution (10%, w / v) to terminate the reaction. Finally, the sample was mixed with 5× protein loading buffer at a volume ratio of 4:1, heated at 95°C for 5 min, and the protein immune complex and protein were SDS-ylated.

[0077] Take 20 μL of the prepared immune complex sample and load it into the sample well of the polyacrylamide gel. Then, perform SDS-PAGE electrophoresis for 20 min at 80, 100, 120, 150, and 200 V voltages respectively. During the electrophoresis, the CMOS camera of the real-time fluorescence imaging system can be used at any time to collect fluorescence images, and the progress of electrophoretic separation can be monitored in real time by observing the migration and separation of the fluorescent bands. When the real-time fluorescence imaging system of this embodiment is used for fluorescence imaging, the exposure time is set to 3000 ms and the gain factor is 15.

[0078] like Figure 4 As shown in the figure, free anti-HER2 monoclonal antibody bands with different fluorescence intensities corresponding to each group of immune complex samples can be observed in the fluorescence spectrum. Fluorescence images were collected immediately at the end of electrophoresis for analysis, and the fluorescence intensity values ​​of the corresponding bands of free fluorescent labeled antibodies in each lane were measured using the image analysis software ImageJ, and a standard curve of fluorescence intensity and absolute anti-HER2 loading quality was drawn. Figure 4 From the standard curve, it can be seen that there is a good linear fitting result between the fluorescence intensity of free monoclonal antibody and the anti-HER2 content.

[0079] Whey diluted 1:200 was used as the matrix for the spike recovery experiment. Spiked immune complex samples with different absolute loading masses of anti-HER2mAb (2000, 200 and 20 ng) were prepared. They were run on immunoPAGE and the signal intensity of the free antibody band was measured. Combined with the standard curve, the recoveries at the three anti-HER2 mAb spike levels of 2000, 200 and 20 ng were calculated to be 104.9%, 96.0% and 96.4%, respectively (RSD<3.02%, n=3).

[0080] Embodiment 3:

[0081] This embodiment provides an online fluorescence quantitative detection method for APO recombinant human transferrin (TRF) based on immune response, and the specific steps are as follows:

[0082] TRF (recombinant human APO type TRF / purchased from Beijing Solebow Technology Co., Ltd., catalog number T8010-50mg) was prepared with immune complex samples of 0, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 mg / L, respectively, where FITC-anti-human was used at the same dilution multiple (1:100). The sample was processed using the same antigen-antibody incubation and formaldehyde cross-linking steps as in Example 1. Finally, the sample was mixed with 2× protein loading buffer at a volume ratio of 1:1, heated at 95°C for 5 min, and the protein immune complex and protein sample were SDS-treated.

[0083] Take 10 μL of the prepared immune complex sample and load it into the loading well of the polyacrylamide gel. Then, electrophoresis is performed at a voltage of 120V for 15 minutes, then at a voltage of 150V for 15 minutes, and finally at a voltage of 200V for 30 minutes. During the electrophoresis process, the CMOS camera of the real-time fluorescence imaging system can be used at any time to collect fluorescence images, and the progress of electrophoretic separation can be monitored in real time by observing the migration and separation of the fluorescent bands. When using the real-time fluorescence imaging system of this embodiment for fluorescence imaging, the exposure time is set to 3000ms and the gain factor is 20.

[0084] like Figure 5 As shown in the figure, bands with different fluorescence intensities corresponding to each group of immune complex samples can be observed in the fluorescence spectrum. Immediately after the electrophoresis, the fluorescence image was collected for analysis, and the image analysis software ImageJ was used to measure the fluorescence intensity values ​​of the bands corresponding to the free fluorescent labeled antibodies in each lane, and a standard curve of fluorescence intensity and TRF absolute loading mass was drawn. Figure 5 From the standard curve, it can be seen that there is a good linear fitting result between the fluorescence intensity of free antibody and the TRF content.

[0085] A multi-component protein complex sample containing TRF, BSA, C-PC, Mb and Cyt-C was prepared, wherein the concentration gradient of TRF was 200, 100 and 50 mg / L, respectively. They were mixed and incubated with FITC-anti-human of the same concentration to prepare spiked immune complex samples, and then immuno-PAGE was run to determine the signal intensity of the free antibody band. The recoveries at the three TRF spiked levels of 200, 100 and 50 mg / L were calculated based on the standard curve and were 98.2%, 104.8% and 105.0% (RSD<3.30%, n=3), respectively, indicating that the present invention can achieve specific quantitative detection of target proteins in complex samples.

[0086] In summary, the present invention does not require complicated gel disassembly and time-consuming fixation and staining steps. It can achieve specific quantitative detection of target proteins in complex samples through the immune binding principle and online fluorescence imaging. The detection can be completed quickly within 1.5 hours, and the electrophoresis process can be monitored by real-time fluorescence imaging. This method has the advantages of simplicity, rapidity, low cost, strong specificity, high sensitivity and accurate quantification, providing a new option for protein quantitative detection and has important application prospects.

[0087] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for online fluorescence quantitative detection of protein antigens based on immune response, characterized in that: The detection method comprises the following steps: S1. Preparation of immune complex samples: preparing immune complex test samples containing the protein antigen to be tested and the fluorescently labeled antibody, and a series of immune complex standard samples containing the protein antigen and the fluorescently labeled antibody at different concentration gradients; S2. Build a real-time fluorescence imaging system, including: An electrophoresis tank (1) for loading a flat plate gel (2) for electrophoresis and assisting in fluorescence imaging; An excitation light source (3) for exciting the fluorescently labeled antibody to generate a fluorescent signal; An imaging device (4) for collecting a fluorescent image on the flat gel (2); A bandpass filter (5) disposed at the front end of the lens of the imaging device (4); S3, electrophoretic separation of immune complexes: loading the prepared immune complex samples into sample wells of polyacrylamide gel, followed by electrophoretic separation; S4, collecting electrophoresis fluorescence images: During the electrophoresis process, the real-time fluorescence imaging system constructed in S2 is used to collect fluorescence images, and the progress of immunoelectrophoresis is judged by observing the migration and separation of the fluorescence bands; S5. Quantitative analysis of protein antigen concentration based on fluorescence images: Obtain the brightness information of the corresponding bands of free fluorescent-labeled antibodies and quantitatively analyze the protein antigen concentration.

2. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S1, the specific method for preparing the immune complex sample is: The protein antigen and the fluorescently labeled antibody are mixed and then incubated to allow the protein antigen and the fluorescently labeled antibody to bind to form an immune complex; After cross-linking and fixation with formaldehyde, excess formaldehyde is quenched with Tris buffer solution, and then the cross-linked immune complex is mixed with protein loading buffer and heated to perform SDS treatment on the immune complex and protein for use.

3. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S2, the flat plate gel (2) is fixed to the front end of the electrophoresis tank (1) by means of transparent glass and wedges.

4. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S2, the excitation light source (3) is an LED light board, the central wavelength of which is adapted to the excitation wavelength of the fluorescent-labeled antibody.

5. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 4, characterized in that: The excitation light sources (3) are symmetrically arranged on both sides of the flat gel (2); The horizontal angles between the excitation light source (3) and the flat gel (2) are both 10-70°.

6. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S2, the bandpass filter (5) is a filter that can transmit, but is not limited to, 520-560nm fluorescence.

7. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S2, the imaging device (4) comprises a camera lens (41) with adjustable focal length and aperture and a CMOS image sensor (42); The distance between the camera lens (41) and the imaging surface of the flat gel (2) is 200-300 mm.

8. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S3, the voltage of the electrophoretic separation is 80-200 V, and the total time of the electrophoretic separation detection is 50-100 min.

9. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S4, the exposure time used for the fluorescence image acquisition is 1000-9000 ms, and the gain parameter is 1-20.

10. The method for online fluorescence quantitative detection of protein antigens based on immune response according to claim 1, characterized in that: In step S5, the specific steps of quantitatively analyzing the protein concentration are: Use image analysis software to measure the fluorescence intensity of the corresponding band of the free fluorescent-labeled antibody in each lane, and draw a standard curve between fluorescence intensity and protein antigen concentration; The protein antigen concentration in the immune complex test sample is quantitatively estimated based on the standard curve.

Images