An online fluorescence quantitative detection method for protein antigen based on immune reaction

Abstract

The present application relates to a kind of protein antigen online fluorescence quantitative detection method based on immune response.The detection method includes the following steps: preparation contains the immune complex sample of protein antigen to be measured to be measured, and the immune complex standard sample containing different concentration gradient protein antigen;Real-time fluorescence imaging system is constructed, specifically including electrophoresis tank, excitation light source, imaging device and band-pass filter;The immune complex sample is loaded into gel sample hole, and electrophoresis separation is carried out;During electrophoresis, fluorescence image is collected by real-time fluorescence imaging system, and the migration and separation of fluorescence band are observed to determine the progress of immune electrophoresis;The brightness information of the band corresponding to free fluorescently labeled antibody is obtained, and the concentration of protein antigen is quantitatively analyzed.Compared with prior art, the protein antigen online fluorescence quantitative detection method of the present application has the advantages of fast detection speed, simple operation, low cost, strong specificity and accurate quantification.

Description

Technical Field

[0001] This invention belongs to the field of protein detection technology, and in particular relates to an online fluorescence quantitative detection method for protein antigens based on immune response. Background Technology

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

[0003] Western blotting, a classic protein-specific detection technique, enables the identification and semi-quantitative analysis of specific proteins through a combination of electrophoretic separation and immunorecognition. This method first separates protein mixtures using polyacrylamide gel electrophoresis (PAGE), then transfers the separated proteins onto a membrane, where specific antibodies recognize the target protein and perform colorimetric detection. However, Western blotting has several limitations, such as cumbersome procedures, a long detection time (typically 2-3 days), poor reproducibility (unstable protein transfer efficiency after electrophoresis, leading to significant batch-to-batch variations in semi-quantitative results), inability to perform online detection (traditional chemiluminescence or colorimetric detection methods rely on endpoint detection, making real-time monitoring difficult), and its primary use for semi-quantitative protein analysis, making accurate quantitative results difficult, let alone absolute quantitative detection.

[0004] ELISA is another widely used immunoassay method for the quantitative detection of proteins and other proteins, offering high sensitivity and specificity. This method involves coating specific antibodies onto the surface of a solid-phase carrier, enabling the capture and detection of target proteins through antigen-antibody specific binding. However, ELISA also has significant drawbacks, such as high cost (commercially available ELISA kits are expensive, increasing testing costs); cumbersome operation (requiring multiple incubation and washing steps); high antibody quality requirements (antibody specificity and affinity directly affect the accuracy of results); and poor interference resistance (due to the lack of a separation step, interfering substances in complex matrices may affect detection accuracy).

[0005] Immunofixation electrophoresis is a widely used protein immunoassay method in clinical and laboratory settings, primarily for the typing analysis of serum immunoglobulin markers in myeloma patients. This method first separates immunoglobulin markers via electrophoresis, then applies specific antibodies directly to the surface of the electrophoresis gel. An antigen-antibody reaction forms a precipitate band, which is then detected by chemical staining. However, this method also has some limitations, such as low sensitivity and poor resistance to interference; it requires multiple manual steps, making it labor-intensive and time-consuming; result interpretation is susceptible to human experience, requiring professional personnel to interpret the results, thus introducing a degree of subjectivity; and it consumes a large amount of antibodies, resulting in high detection costs.

[0006] In recent years, with the deepening of biomedical research and the increasing demand for clinical diagnostics, the requirements for protein immunoassay methods have been continuously increasing. Especially in the fields of disease biomarker detection, drug screening, and biopharmaceutical quality control, there is an urgent need for a rapid, accurate, simple, and low-cost protein-specific quantitative detection method. Currently, although various detection technologies have been developed, it is still difficult to simultaneously meet the requirements of fast detection speed, ease of operation, low cost, high specificity, and accurate quantification. Therefore, developing a novel protein-specific quantitative detection method has significant theoretical and practical application value. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies in that they cannot simultaneously meet the requirements of fast detection speed, simple operation, low cost, high specificity and accurate quantification, and to provide an online fluorescence quantitative detection method for protein antigens based on immune response.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention provides an online fluorescence quantitative detection method for protein antigens based on immune responses, the detection method comprising the following steps:

[0010] S1. Preparation of immune complex samples: Prepare immune complex test samples containing the target protein antigen and fluorescently labeled antibody, as well as a series of immune complex standard samples containing protein antigen and fluorescently labeled antibody at different concentration gradients;

[0011] S2. Construct a real-time fluorescence imaging system, specifically including: an electrophoresis tank for loading slab gels for electrophoresis and assisting in fluorescence imaging; an excitation light source for exciting fluorescently labeled antibodies to generate fluorescence signals; an imaging device for acquiring fluorescence images on the slab gels; and a bandpass filter located at the front end of the lens of the imaging device.

[0012] S3. Electrophoretic separation of immune complexes: The prepared immune complex samples are loaded into the sample wells of a polyacrylamide gel and then separated by electrophoresis.

[0013] S4. Acquiring electrophoretic fluorescence images: During the electrophoresis process, fluorescence images are acquired using the real-time fluorescence imaging system constructed in S2, and the progress of immunoelectrophoresis is determined by observing the migration and separation of fluorescence bands.

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

[0015] Further, in step S1, the specific preparation method of the immune complex sample is as follows: after mixing the protein antigen with the fluorescently labeled antibody, incubate to allow the protein antigen and the fluorescently labeled antibody to combine and form an immune complex; after cross-linking and fixing with formaldehyde, quench excess formaldehyde with Tris buffer solution, and then mix the cross-linked immune complex with protein loading buffer and heat treatment to perform SDS-ization of the immune complex and protein for later use.

[0016] Furthermore, in step S2, the plate gel is secured 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 panel, the center wavelength of which is adapted to the excitation wavelength of the fluorescently labeled antibody.

[0018] The excitation source can be an LED panel with a center wavelength of 470 nm, which is compatible with the excitation wavelength of FTIC; or an LED panel with other center wavelengths, which is compatible with the excitation wavelength range of other fluorescent groups. Specifically, an LED panel with a center wavelength of 480-495 nm is compatible with the excitation wavelength of Cy3; an LED panel with a center wavelength of 630-640 nm is compatible with the excitation wavelength of Cy5; and an LED panel with a center wavelength of 480-495 nm is compatible with the excitation wavelength of PE. The selection relationship between other fluorescently labeled antibodies and LED panels is similar, and can be chosen according to specific scenarios and requirements.

[0019] The excitation light source is symmetrically arranged on both sides of the flat gel.

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

[0021] Furthermore, in step S2, the bandpass filter is a filter that can transmit fluorescence at 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 200-300mm.

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

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

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

[0027] Further, in step S5, the specific steps for quantitatively analyzing protein concentration are as follows: using image analysis software to measure the fluorescence intensity value of the corresponding band of the free fluorescently labeled antibody in each lane, and plotting a standard curve of fluorescence intensity versus protein antigen concentration; and quantitatively estimating the protein antigen concentration in the immune complex test sample based on the standard curve.

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

[0029] (1) The online fluorescence quantitative detection method for protein antigens of 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 through the principle of immune binding and online fluorescence imaging.

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

[0031] (3) The online fluorescence quantitative detection method for protein antigens of the present invention can monitor protein immunoelectrophoresis in real time without complicated and time-consuming steps such as gel disassembly, membrane transfer and washing, fixation and staining and scanning imaging, which effectively avoids the in-gel broadening of protein bands and improves the separation resolution.

[0032] (4) Compared with ELISA kits, the online fluorescence quantitative detection method for protein antigens 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) This invention constructs a standard curve of band fluorescence intensity and protein quantity through fluorescence spectrum analysis. The quantitative standard curve has a wide dynamic range and a good linear fitting coefficient, which can realize the accurate quantification of target proteins.

[0034] (6) This 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. Attached Figure Description

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

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

[0037] Figure 3 This is a schematic diagram of the optical system configuration and imaging optical path of the present invention.

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

[0039] Figure 5 The image shows the free antibody fluorescent bands and the protein antigen quantitative standard curve for Example 3.

[0040] Explanation of markings in the diagram:

[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 Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

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

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

[0045] This invention provides an online fluorescence quantitative detection method for protein antigens based on immune responses, the detection method comprising the following steps:

[0046] S1. Preparation of immune complex samples: Prepare immune complex test samples containing the target protein antigen and fluorescently labeled antibody, as well as a series of immune complex standard samples containing protein antigen and 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 plate gel 2 for electrophoresis and assist in fluorescence imaging; an excitation light source 3, used to excite fluorescently labeled antibodies to generate fluorescence signals; an imaging device 4, used to acquire fluorescence images on the plate gel 2; and a bandpass filter 5 located at the front end of the lens of the imaging device 4.

[0048] S3. Electrophoretic separation of immune complexes: The prepared immune complex samples are loaded into the sample wells of a polyacrylamide gel and then separated by electrophoresis.

[0049] S4. Acquiring electrophoretic fluorescence images: During the electrophoresis process, fluorescence images are acquired using the real-time fluorescence imaging system constructed in S2, and the progress of immunoelectrophoresis is determined by observing the migration and separation of fluorescence bands.

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

[0051] In some specific embodiments, in step S1, the specific preparation method of the immune complex sample is as follows: after mixing the protein antigen with the fluorescently labeled antibody, the mixture is incubated to allow the protein antigen and the fluorescently labeled antibody to bind and form an immune complex; after cross-linking and fixing with formaldehyde, excess formaldehyde is quenched with Tris buffer solution, and then the cross-linked immune complex is mixed with a loading buffer containing SDS (sodium dodecyl sulfate) and heated to perform SDS-coating of the immune complex with the protein for later use.

[0052] In some specific embodiments, in step S2, the flat gel 2 is secured to the front end of the electrophoresis tank 1 by transparent glass and wedges.

[0053] In some specific implementations, in step S2, the excitation light source 3 is an LED light panel, the center wavelength of which is adapted to the excitation wavelength of the fluorescently labeled antibody.

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

[0055] In some specific embodiments, in step S2, the bandpass filter 5 is a filter that can transmit fluorescence at 520-560nm.

[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 200-300mm.

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

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

[0059] In some specific implementations, step S5 involves the following steps for quantitatively analyzing protein concentration: using image analysis software to measure the fluorescence intensity of the corresponding bands of the free fluorescently labeled antibody in each lane, and plotting a standard curve of fluorescence intensity versus protein antigen concentration; and quantitatively estimating the protein antigen concentration in the immune complex sample based on the standard curve.

[0060] Each of the above embodiments can be implemented individually, or in any combination of two or more. The following detailed description of specific examples will further illustrate these embodiments.

[0061] Example 1:

[0062] This embodiment provides an online fluorescence quantitative detection method for protein antigens based on immune responses. The detection method includes the following steps:

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

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

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

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

[0067] Specifically, such as Figure 3 As shown, in this embodiment, the electrophoresis tank 1 is used to load the slab gel 2 for electrophoresis and to assist in fluorescence imaging. The slab gel 2 is secured to the front end of the electrophoresis tank 1 by transparent glass and wedges. The electrophoresis tank 1 is powered by a 10-400V regulated power supply. The excitation light source 3 is used to excite the fluorescently labeled antibody to generate a fluorescence signal. The excitation light source 3 is an LED light panel, and its center wavelength is adapted to the excitation wavelength of the fluorescently labeled antibody. In this embodiment, the excitation light source 3 is symmetrically arranged on both sides of the slab gel 2, and the horizontal angle between it and the slab gel 2 is approximately 30°.

[0068] In this embodiment, the excitation light source 3 can be an LED panel with a center wavelength of 470 nm, which is compatible with the excitation wavelength of FTIC; or an LED panel with other center wavelengths can be used, which is compatible with the excitation wavelength range of other fluorescent groups. Specifically, for example, an LED panel with a center wavelength of 480-495 nm is compatible with the excitation wavelength of Cy3; an LED panel with a center wavelength of 630-640 nm is compatible with the excitation wavelength of Cy5; and an LED panel with a center wavelength of 480-495 nm is compatible with the excitation wavelength of PE. The corresponding selection relationship between other fluorescently labeled antibodies and LED panels is similar, and those skilled in the art can select according to the specific scenario, which will not be elaborated here.

[0069] The imaging device 4 in this embodiment is used to acquire fluorescence images on the flat gel 2. Specifically, it includes a camera lens 41 with adjustable focal length and aperture, and a CMOS image sensor 42 for real-time surface imaging of the object being detected. The distance between the camera lens 41 and the imaging surface of the flat gel 2 is 200-300 mm, preferably 250 mm. A bandpass filter 5 is disposed at the front end of the camera lens 41 of the imaging device 4. 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 wells of a polyacrylamide gel and then separated by electrophoresis. The electrophoretic separation voltage is 80-200V, and the total electrophoretic separation time is 50-100min. Specifically, the electrophoretic separation voltage can be gradually increased at specific time intervals, and the electrophoresis time for each voltage gradient can also be adjusted appropriately.

[0071] S4. Acquiring Electrophoretic Fluorescence Images: During electrophoresis, fluorescence images are acquired using the real-time fluorescence imaging system constructed in S2. The progress of immunoelectrophoresis is determined by observing the migration and separation of fluorescence bands. The exposure time for acquiring fluorescence images 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 fluorescently labeled antibodies and quantitatively analyze the protein antigen concentration.

[0073] The specific steps are as follows: use image analysis software to measure the fluorescence intensity of the corresponding bands of the free fluorescently labeled antibody in each lane, and plot a standard curve of fluorescence intensity versus protein antigen concentration; based on the standard curve, quantitatively estimate the protein antigen concentration in the test sample of the immune complex.

[0074] Example 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. The specific steps are as follows:

[0076] Immunocomplex samples with anti-HER2 concentrations of 0, 0.05, 0.25, 0.5, 1, 2.5, and 5 mg / L were prepared. FITC-anti-human (fluorescently labeled antibody, Fluorescein (FITC) – conjugated Goat Anti-Human IgG (H+L / Wuhan Sanying Biotechnology Co., Ltd.)) were all diluted at the same factor (1:80). The samples were incubated at room temperature for 3 h in a programmed mixer. Formaldehyde solution (1%, w / v) was then added to each reaction system to cross-link the immunocomplexes, and incubation continued at room temperature for 10 min. The reaction was then terminated by adding Tris solution (10%, w / v). Finally, the samples were mixed with 5× protein loading buffer at a 4:1 volume ratio and heated at 95 °C for 5 min to SDS-pair the protein immunocomplexes with the protein.

[0077] 20 μL of the prepared immune complex sample was loaded into the sample wells of a polyacrylamide gel. Subsequently, SDS-PAGE electrophoresis was performed sequentially at voltages of 80, 100, 120, 150, and 200 V for 20 min each. During electrophoresis, fluorescence images could be acquired at any time using the CMOS camera of the real-time fluorescence imaging system, and the progress of electrophoretic separation could be monitored in real time by observing the migration and separation of fluorescence bands. When using the real-time fluorescence imaging system of this embodiment, the exposure time was set to 3000 ms and the gain factor to 15.

[0078] like Figure 4 As shown, the fluorescence spectrum reveals free anti-HER2 monoclonal antibody bands with varying fluorescence intensities corresponding to different groups of immune complex samples. Fluorescence images were acquired and analyzed immediately after electrophoresis. The fluorescence intensity of the corresponding bands of the free fluorescently labeled antibodies in each lane was measured using ImageJ image analysis software, and a standard curve was plotted comparing fluorescence intensity with the absolute loading quality of anti-HER2. Figure 4 The standard curve shows that the fluorescence intensity of the free monoclonal antibody has a good linear fit with the anti-HER2 content.

[0079] Whey diluted 1:200 was used as the matrix for spiked recovery experiments. Spiked immune complex samples with different absolute loading masses of anti-HER2 mAb (2000, 200, and 20 ng) were prepared. Immunoplasty was performed on these samples, and the signal intensity of the free antibody bands was measured. 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% (RSD < 3.02%, n = 3), respectively, based on the standard curve.

[0080] Example 3:

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

[0082] Immunoconjugate samples with TRF (recombinant human APO-type TRF / purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number T8010-50mg) concentrations of 0, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 mg / L were prepared, with all FITC-anti-human samples using the same dilution factor (1:100). Samples were treated using the same antigen-antibody incubation and formaldehyde cross-linking steps as in Example 1. Finally, the samples were mixed with 2× protein loading buffer at a 1:1 volume ratio and heated at 95°C for 5 min to SDS-pair the protein immune complexes and protein samples.

[0083] 10 μL of the prepared immune complex sample was loaded into the sample wells of a polyacrylamide gel. Electrophoresis was then performed first at 120 V for 15 min, then at 150 V for 15 min, and finally at 200 V for 30 min. During electrophoresis, fluorescence images could be acquired at any time using the CMOS camera of the real-time fluorescence imaging system, and the progress of electrophoretic separation could be monitored in real time by observing the migration and separation of the fluorescence bands. When using the real-time fluorescence imaging system of this embodiment, the exposure time was set to 3000 ms and the gain factor to 20.

[0084] like Figure 5 As shown, the fluorescence spectrum reveals bands with different fluorescence intensities corresponding to each group of immune complex samples. Fluorescence images were acquired and analyzed immediately after electrophoresis. The fluorescence intensity values ​​of the bands corresponding to the free fluorescently labeled antibodies in each lane were measured using ImageJ image analysis software, and a standard curve was plotted between fluorescence intensity and the absolute sample loading mass of the TRF. Figure 5 The standard curve shows that there is a good linear fit between the fluorescence intensity of the free antibody and the TRF content.

[0085] A complex multi-component protein sample containing TRF, BSA, C-PC, Mb, and Cyt-C was prepared, with TRF concentration gradients of 200, 100, and 50 mg / L. These components were mixed with the same concentration of FITC-anti-human to prepare a spiked immune complex sample. Immunoplasty was then performed to measure the signal intensity of the free antibody band. The recoveries at the three TRF spike levels of 200, 100, and 50 mg / L were calculated to be 98.2%, 104.8%, and 105.0% (RSD < 3.30%, n = 3), indicating that this invention can achieve specific quantitative detection of target proteins in complex samples.

[0086] In summary, this invention eliminates the need for complex gel disassembly and time-consuming fixation and staining steps. It achieves specific quantitative detection of target proteins in complex samples through the principle of immunobinding and online fluorescence imaging, enabling rapid detection within 1.5 hours and real-time fluorescence imaging monitoring of the electrophoresis process. This method offers advantages such as simplicity, speed, low cost, high specificity, high sensitivity, and accurate quantification, providing a new option for protein quantification and demonstrating significant application potential.

[0087] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope 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 is used for the specific quantitative detection of low-abundance proteins, and includes the following steps: S1. Preparation of immune complex samples: Prepare immune complex test samples containing the target protein antigen and fluorescently labeled antibody, as well as a series of immune complex standard samples containing protein antigen and fluorescently labeled antibody at different concentration gradients; S2. Construct a real-time fluorescence imaging system, specifically including: An electrophoresis tank (1) is used to load a plate of gel (2) for electrophoresis and to assist in fluorescence imaging; Excitation light source (3) is used to excite fluorescently labeled antibodies to generate fluorescence signals; Imaging device (4) is used to acquire fluorescence images on the plate gel (2); A bandpass filter (5) is located at the front end of the lens of the imaging device (4). S3. Electrophoretic separation of immune complexes: The prepared immune complex samples are loaded into the sample wells of a polyacrylamide gel and then separated by electrophoresis. S4. Acquiring electrophoretic fluorescence images: During the electrophoresis process, fluorescence images are acquired using the real-time fluorescence imaging system constructed in S2, and the progress of immunoelectrophoresis is determined by observing the migration and separation of fluorescence bands. S5. Quantitative analysis of protein antigen concentration based on fluorescence images: Obtain the brightness information of the corresponding bands of free fluorescently labeled antibodies and quantitatively analyze the protein antigen concentration; In step S1, the specific preparation method of the immune complex sample is as follows: after mixing the protein antigen with the fluorescently labeled antibody, the mixture is incubated to allow the protein antigen and the fluorescently labeled antibody to combine and form an immune complex; after cross-linking and fixing with formaldehyde, excess formaldehyde is quenched with Tris buffer solution; then the cross-linked immune complex is mixed with protein loading buffer and heated to perform SDS-addition of the immune complex and protein for later use. In step S5, the specific steps for quantitatively analyzing protein concentration are as follows: using image analysis software to measure the fluorescence intensity value of the corresponding band of the free fluorescently labeled antibody in each lane, and plotting a standard curve of fluorescence intensity versus protein antigen concentration; and quantitatively estimating the protein antigen concentration in the immune complex test sample based on the standard curve.

2. 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 gel (2) is fastened to the front end of the electrophoresis tank (1) by transparent glass and wedges.

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 excitation light source (3) is an LED light panel, whose center wavelength is adapted to the excitation wavelength of the fluorescently labeled antibody.

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

5. 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, 520-560 nm fluorescence.

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 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.

7. 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 for electrophoretic separation is 80-200 V, and the total time for electrophoretic separation detection is 50-100 min.

8. 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 acquiring the fluorescence image is 1000-9000 ms, and the gain parameter is 1-20.

Images