Method for detecting in-vitro binding activity of EPO and receptor EpoR thereof based on SPR (Surface Plasmon Resonance)

Abstract

The invention provides a method for detecting in-vitro binding activity of EPO and a receptor EpoR thereof based on SPR. The method comprises the following steps: (1) homogenizing a chip: homogenizing the chip; (2) chip coupling: coupling the chip according to an injection sequence of an activating reagent, a capture antibody and a sealing agent; (3) activity detection of the coupled chip: carrying out activity detection by using one or two of PEG-EPO and rhEPO protein as an analyte; and (4) binding activity detection: combining PEG-EPO and / or rhEPO with a receptor EpoR sample for 60-120s, and preferably combining the analyte with the receptor EpoR sample after gradient dilution. The method provided by the invention does not need to prepare cells, is simple to operate, and can rapidly, dynamically and quantitatively characterize the binding activity.

Description

Technical Field

[0001] The present invention relates to the technical field of binding activity detection, and more specifically, to a method for detecting binding activity based on SPR. Background Art

[0002] Erythropoietin (EPO), also known as erythrocyte stimulating factor and erythropoietin, is an endogenous glycoprotein hormone in the human body that can stimulate erythrocyte production. Recombinant human erythropoietin (rhEPO) is a glycoprotein hormone that can stimulate erythrocyte proliferation, differentiation and maturation. In the activity detection of rhEPO-related drugs, common methods include reticulocyte counting method and MTT method. The reticulocyte counting method requires the use of mice for experiments, which is complicated to operate, with an experimental cycle of at least 4 days and large variability in results. The MTT method uses EPO-dependent cell lines for experiments. Although the method is simpler than the animal method, the experimental cycle is still long, which is 3 days. With the increase in the production of rhEPO, in order to meet the needs of rapid batch detection of the biological activity of rhEPO, it is urgent to establish a simple, rapid and accurate activity detection method.

[0003] The primary condition for the biological effect of rhEPO to be exerted is the binding of EPO to its receptor (erythropoietin receptor, EpoR). When EpoR is not bound to EPO, it is distributed on the cell surface in the form of a dimer. When bound to EPO, the conformation of EpoR changes, activating the tyrosine kinase JAK2 (janus kinase 2, JAK2) that was previously bound to its intracellular segment. JAK2 then phosphorylates the tyrosine site of the intracellular segment of EpoR, providing an anchoring point for proteins containing the SH-2 (src homology domain 2, SH-2) domain, and continuing to trigger a series of downstream signal transduction processes. Based on this signal transduction mechanism, there is a reporter gene method based on the cell level to detect the biological activity of rhEPO. As a release detection method for rhEPO drugs, this method is simple and rapid, but it requires the construction of a reporter gene cell line that stably expresses EpoR. Therefore, a characterization method for in vitro detection of EPO and EpoR binding activity based on SPR (surface plasmon resonance technology) is established, which can be used as a simple and rapid supplementary method for the reporter gene method to provide a guarantee for the safe and effective use of rhEPO. At present, this method has not been applied to the rapid determination of EPO and EpoR binding activity.

[0004] Surface plasmon resonance (SPR) is a new technology developed in the 1990s. It uses the SPR principle to detect the interaction between ligands and analytes on biosensor chips and is widely used in various fields.

[0005] The contents of the background technology section are merely the technologies known to the inventors and do not necessarily represent the prior art in the field. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR, comprising the following steps:

[0007] (1) Chip homogenization: homogenize the chip;

[0008] (2) Chip coupling: The chip is coupled according to the injection order of activation reagent, capture antibody, and blocking agent;

[0009] (3) Activity detection on the chip after coupling: using one or both of PEG-EPO and rhEPO protein as analytes for activity detection;

[0010] (4) Binding activity detection: PEG-EPO and / or rhEPO are allowed to bind to the receptor EpoR sample, wherein the binding time is 60s-120s. Preferably, the analyte is bound to the receptor EpoR sample after gradient dilution.

[0011] In the above embodiment, the binding time can reduce the risk of non-specific binding while ensuring a stronger binding signal, thereby ensuring a balance between a sufficiently strong binding signal and lower non-specific binding.

[0012] Furthermore, in the step (4), EPO-PEG is diluted with running buffer, and the dilution gradient is 16.48, 8.24, 4.12, 2.06, 1.03, 0.515, 0 nM, wherein the 2.06 nM concentration point sample is used as a repeated concentration point, and other detection parameters are the same as in step (3), and preferably the binding time is 100-120 s.

[0013] Furthermore, in step (4), rhEPO is diluted with running buffer, and the dilution gradient is 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 nM, wherein the 0.625 nM concentration point sample is used as a repeated concentration point, and other detection parameters are the same as in step (3), and preferably the binding time is 100-120 s.

[0014] In the above embodiment, by setting a series of detection objects with different concentrations, the change of the binding signal with the increase of concentration can be observed and understood, which is helpful to accurately evaluate the binding strength and saturation between the analyte and the ligand (receptor).

[0015] Furthermore, in a preferred embodiment of the present invention, by adopting the above-mentioned concentration gradient and the above-mentioned binding time, not only the balance between sufficiently strong binding signal and lower nonspecific binding is ensured, but also the binding strength and saturation between the analyte and the ligand (receptor) can be more accurately evaluated, so that the detection method of the present invention is not only faster, but also more sensitive and highly accurate.

[0016] Furthermore, the method of the present invention does not require cell preparation, is simple to operate, and can rapidly and dynamically quantitatively characterize binding activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 It is the binding affinity sensor graph of PEG-EPO sample to EpoR;

[0019] Figure 2 This is the binding affinity sensor diagram of rhEPO sample to EpoR. DETAILED DESCRIPTION

[0020] In the following description, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0021] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0022] One aspect of the present invention relates to a method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR, characterized in that it comprises the following steps:

[0023] (1) Chip homogenization: homogenize the chip;

[0024] (2) Chip coupling: The chip is coupled according to the injection order of activation reagent, capture antibody, and blocking agent;

[0025] (3) Activity detection on the chip after coupling: using one or both of PEG-EPO and rhEPO protein as analytes for activity detection;

[0026] (4) Binding activity detection: PEG-EPO and / or rhEPO are allowed to bind to the receptor EpoR sample, wherein the binding time is 60s-120s. Preferably, the analyte is bound to the receptor EpoR sample after gradient dilution.

[0027] In the above methods, surface plasmon resonance technology (SPR) is an optical phenomenon based on metal surfaces. It produces different resonance angles depending on whether the ligand coupled to the metal surface binds to the analyte. By monitoring the changes in the resonance angle, the affinity, affinity constant, binding kinetics and other information of the biomolecular interaction can be inferred.

[0028] EPO refers to erythropoietin, also known as erythrocyte stimulating factor and erythropoietin. It is a human endogenous glycoprotein hormone that can stimulate erythropoiesis. rhEPO refers to recombinant human erythropoietin, a glycoprotein hormone that can stimulate red blood cell proliferation, differentiation and maturation. PEG-EPO refers to EPO or rhEPO modified and repurified by polyethylene glycol (PEG), also known as polyethylene glycol erythropoietin or polyethylene glycol recombinant human erythropoietin. In this article, rhEPO and PEG-EPO are sometimes collectively referred to as EPO.

[0029] Furthermore, in step (1), the chip is homogenized using 70% glycerol and 50 mM NaOH.

[0030] Furthermore, the capture antibody in step (2) is prepared by diluting the anti-human Fc antibody to 25 μg / mL using a 10 mM sodium acetate buffer at pH 4.5-5.0; the activation reagent is prepared by mixing an EDC solution and an NHS solution at a molar ratio of 4:1; and the blocking agent is ETA.

[0031] Furthermore, in step (3), the analyte is diluted with running buffer, and the EpoR is diluted to 0.5-2 μg / mL; 0.5-2 μg / mL EpoR solution is injected into the detection channel, and the EpoR solution is captured at a flow rate of 5-10 μL / min, and the injection time is between 40-70 s so that the captured response level is between 60RU-200RU; the analyte is injected into the detection channel and the reference channel at a flow rate of 30-50 μL / min for 60-120 s, so that the analyte-receptor binding level is between 20-50RU (preferably about 33, 34, 35, 36RU), and optionally, finally 3 M MgCl2 regeneration solution is injected at a flow rate of 10-30 μL / min for 60-180 s to regenerate the detection channel.

[0032] In some embodiments, in step (3), the analyte is diluted with running buffer and the EpoR is diluted to 1 μg / mL; 1 μg / mL EpoR solution is injected into the detection channel, and the EpoR solution is captured at a flow rate of 5 μL / min, and the injection time is between 40-70 s so that the captured response level is between 120RU-200RU; the analyte is injected into the detection channel and the reference channel at a flow rate of 30 μL / min for 120 s, so that the analyte-receptor binding level is between 30-40RU (preferably about 33, 34, 35, 36RU), and optionally, finally 3M MgCl2 regeneration solution is injected at a flow rate of 30 μL / min for 60 s to regenerate the detection channel.

[0033] In some embodiments, in step (3), the analyte is PEG-EPO, and PEG-EPO is diluted to 16.48 nM.

[0034] In some embodiments, in step (3), the analyte is rhEPO, and the rhEPO protein is diluted to 5 nM.

[0035] In some embodiments, in step (4), EPO-PEG is diluted with running buffer, and the dilution gradient is 16.48, 8.24, 4.12, 2.06, 1.03, 0.515, 0 nM, wherein the 2.06 nM concentration point sample is used as a repeated concentration point, and other detection parameters are the same as in step (3), and preferably the binding time is 100-120 s.

[0036] In some embodiments, in step (4), rhEPO is diluted with running buffer, and the dilution gradient is 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 nM, wherein the 0.625 nM concentration point sample is used as a repeated concentration point, and other detection parameters are the same as in step (3), and preferably the binding time is 100-120 s.

[0037] In some embodiments, the chip has a carboxymethyl dextran matrix covalently attached to a gold surface.

[0038] Example

[0039] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0040] 1. Materials and Methods

[0041] 1.1 rhEPO and PEG-EPO samples: Human erythropoietin stock solution (hereinafter referred to as rhEPO stock solution) is obtained by cell culture, separation and purification of Chinese hamster ovary (CHO) cells that efficiently express human erythropoietin gene. The rhEPO stock solution is modified with polyethylene glycol (PEG) and repurified to obtain polyethylene glycol recombinant human erythropoietin stock solution (hereinafter referred to as PEG-EPO stock solution). PEG-EPO can be prepared by conventional methods in the art. For example, EPO or rhEPO glycoprotein is modified by covalently linking 1 to 3 lower alkoxy polyethylene glycol (PEG) groups, and the preparation is filled into PEG-EPO injection solutions of different specifications for standby use.

[0042] 1.2 EpoR (hFc Tag) receptor (sometimes referred to as "ligand" in this article): purchased from Sino Biological; Human antibody capture kit: purchased from Cytiva

[0043] 1.3 Reagents:

[0044] Biacore Maintenance Kit type 2, Cytiva, 29394519

[0045] Amine Coupling Kit, type 2, Cytiva, BR100050

[0046] HBS-EP buffer, 10X, Cytiva, BR100669

[0047] Sodium Acetate, pH 4.5, Cytiva, BR100350

[0048] NaOH (50 mM), Cytiva, BR100358

[0049] 1.4 Consumables:

[0050] CM5 chip, which has a carboxymethyl dextran matrix covalently linked to the gold surface, was purchased from Cytiva, 29149603.

[0051] U-bottom 96-well plate, Cytiva, BR100503

[0052] 16mm glass bottle, Cytiva, BR100209

[0053] 1.5mL centrifuge tube, Axygen, MCT-150-CS

[0054] Filter, Corning, 431097

[0055] Sealing film, Cytiva, 28975816

[0056] Rubber caps, type 2, Cytiva, BR-1004-11

[0057] 1.5 Instruments:

[0058] Surface plasmon resonance instrument (Biacore T200), Cytiva, 28975001

[0059] 1.6 Experimental Procedure

[0060] 1.6.1 Solution preparation

[0061] (1) EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.4 M)

[0062] Dissolve the EDC dry powder in the Amine Coupling Kit in 10 mL of sterile water, cover the lid tightly and shake until the solid powder is completely dissolved, then dispense 100 μL into each tube and store at -20°C.

[0063] (2) NHS (N-hydroxysuccinimide, 0.1M)

[0064] Dissolve the NHS dry powder in the amino coupling kit in 10 mL of sterile water, cover the lid tightly and shake until the solid powder is completely dissolved, then dispense 100 μL into each tube and store at -20°C.

[0065] (3) ETA (1M ethanolamine hydrochloride-NaOH, pH 8.5)

[0066] Derived from the above mentioned amino coupling kit.

[0067] (4) Running buffer (1X HBS-EP)

[0068] Take 100 mL of 10×HBS-EP Buffer, add it to 900 mL of ultrapure water, mix well, then filter using a 500 mL or 1 L filter and store at 4°C.

[0069] 1.6.2 Chip preparation

[0070] (1) Chip homogenization: First, the CM5 chip was homogenized with 70% glycerol and 50 mM NaOH to eliminate non-experimental errors between measurements. The process is to put glycerol and NaOH into centrifuge tubes, and then put them into the corresponding positions of the test tube rack of the surface plasmon resonance instrument, and click the program to run.

[0071] (2) Chip coupling: Dilute the anti-human Fc antibody (human antibody capture Kit) to 25 μg / mL with 10 mM sodium acetate buffer at pH 4.5, and mix well in a 1.5 mL centrifuge tube as the capture antibody. Then mix 100 μL of EDC (0.4 M) and 100 μL of NHS (0.1 M) in a 1.5 mL centrifuge tube as the chip activation reagent. Prepare 160 μL of blocking reagent ETA and add it to a 1.5 mL centrifuge tube. All the above reagents need to be centrifuged and mixed to avoid bubbles. Mark the tube, cut off the cap of the centrifuge tube, and place the reagent in the test tube rack (Sample and Reagent Rack 1) of the surface plasmon resonance instrument. Use a flow rate of 10 μL / min, follow the injection order of activation reagent, capture antibody, and blocking agent, and inject each for 10 minutes to perform chip coupling.

[0072] (3) Chip activity detection after coupling: Use running buffer (HBS-EP Buffer) to dilute PEG-EPO and rhEPO proteins to 16.48nM and 5nM, respectively, and dilute EpoR solution to 1μg / mL. Prepare 3M MgCl2 (human antibody capture Kit) regeneration solution, remove bubbles after centrifugation, and place it in a test tube rack. Inject 1μg / mL EpoR solution on the detection channel (2 / 4) and capture EpoR solution at a flow rate of 5μL / min. The injection time is between 40-70s (seconds) to ensure that the captured response level is between 120RU-200RU. Then select channel 2-1 (or channel 4-3) to inject the analyte (16.48nM PEG-EPO or 5nM rhEPO) at a flow rate of 30μL / min for 120s (i.e., the binding time of PEG-EPO / rhEPO protein to the receptor is approximately 120s), so that the binding level of analyte (PEG-EPO or rhEPO)-ligand (EpoR) is between 20-50RU (for example, about 33, 34, 35, 36RU, etc.), and finally inject the regeneration solution at a flow rate of 30μL / min for 60s to regenerate channel 2-1 (or channel 4-3). During the detection process, the analyte can be selected to flow through the channel. The first step is to select detection channel 2 to flow through the ligand EpoR (reference channel 1 is used as a blank control and is not selected). Then, select the 2-1 series channel in the program, that is, channels 1 and 2 are connected in series, so that the analyte flows through the two channels. The EpoR captured on the detection channel binds to the analyte PEG-EPO and rhEPO. The reference channel theoretically has no binding, but is only a background value, which will eventually generate a signal value for channel 2-1. Repeat the surface test (the surface test results can examine whether the analyte and the ligand are bound, whether the injection dissociation time is appropriate, and the estimated KD value, etc.) - regeneration process. This operation is performed 3-4 times. Through the sensor graph results of the detection channel (for example, channel 2 or 4) subtracted from the reference channel (for example, channel 1, 3), it is examined whether the analyte (PEG-EPO or rhEPO) and the ligand (EpoR receptor) are bound, whether the binding response value is at an appropriate level, and the estimated Rmax value, etc. At the same time, after regeneration, the sensor curve can return to the baseline level before binding, and the surface binding ability to the analyte does not decrease too much.

[0073] 1.6.3 In vitro binding activity detection of samples

[0074] (1) Sample preparation: Use running buffer (1X HBS-EP) to dilute EpoR (Fc tag) to 1 μg / mL, dilute PEG-EPO sample to 16.48 nM, and then use this as the starting concentration for 1:1 gradient dilution, i.e. 16.48, 8.24, 4.12, 2.06, 1.03, 0.515, 0 nM, with the 2.06 nM concentration point sample as a repeat concentration point. The dilution gradient of rhEPO is 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 nM, with the 0.625 nM concentration point sample as a repeat concentration point. The purpose of repeating the concentration point is to test whether the repeatability of the sample running process is good. If the curves of the same concentration point are basically the same when tested twice in a row, and the difference is not large, it means that there is no problem with the system operation.

[0075] (2) Detection template settings: Open the method setting template and set the detection parameters, including chip type, detection channel and detection mode, input ligand name, capture concentration, capture rate and time, sample binding time, flow rate, regeneration liquid name, regeneration time and flow rate. These detection parameters are the same as those in step (3) of 1.6.2. Then input sample name, molecular weight, running concentration gradient, arrange the samples in the order of running, check the sample running position, and finally check whether all input information is correct.

[0076] (3) Run the program: Pop up the test tube rack, place the sample, running buffer, and regeneration solution in the correct position of the selected test tube rack according to the settings in the detection program, save the running method and result file again, and the instrument starts running automatically.

[0077] 1.6.4 Data Analysis

[0078] The SPR experimental results were calculated using the Langmuir binding model (BIA analysis software) with a 1:1 binding to obtain the binding coefficient ka and dissociation coefficient kd. Affinity is conventionally expressed as the dissociation constant KD (KD = kd / ka [M]) rather than KA (KA = ka / kd [M-1]).

[0079] 2. Results Analysis

[0080] The results of this example are shown in Figure 1 , Figure 2 Among them, the binding affinity of PEG-EPO to EpoR reached 0.2056nM ( Figure 1 ), the affinity KD of rhEPO binding to EpoR is 0.2664nM ( Figure 2 ).

[0081] Among them, Figure 1In the figure, the colored curves represent the binding curves of PEG-EPO at concentrations of 16.48, 8.24, 4.12, 2.06, 1.03, and 0.515 nM, respectively, from top to bottom, and the sample at the concentration of 2.06 nM is used as a repeated concentration point. Figure 1 The curves of repeated concentration points in the figure basically overlap, which proves that the overlap is good. The black curve is the theoretical fitting curve. Figure 1 The curves shown in the figure are basically consistent with those at each concentration point.

[0082] exist Figure 2 In the figure, the colored curves represent the binding curves of rhEPO at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM from top to bottom, respectively, wherein the sample at the concentration point of 0.625 nM is used as a repeated concentration point. Figure 2 The curves of repeated concentration points in the figure basically overlap, which proves that the overlap is good. The black curve is the theoretical fitting curve. Figure 2 The curves shown in the figure are basically consistent with those at each concentration point.

[0083] The above results also show that the binding time of the sample and the receptor established in the present invention, and the gradient dilution of the analyte, enable the detection method of the present invention to not only ensure the balance between sufficiently strong binding signals and low nonspecific binding, but also to more accurately evaluate the binding strength and saturation between the analyte and the ligand (receptor), making the detection method of the present invention not only faster, but also more sensitive and highly accurate.

[0084] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. In addition, although the elements of the present invention may be described or required in individual form, it is also conceivable to have multiple elements unless explicitly limited to a single element.

Claims

1. A method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR, characterized in that: The following steps are involved: (1) Chip homogenization: homogenize the chip; (2) Chip coupling: The chip is coupled according to the injection order of activation reagent, capture antibody, and blocking agent; (3) Activity detection on the chip after coupling: using one or both of PEG-EPO and rhEPO protein as analytes for activity detection; (4) Binding activity detection: PEG-EPO and / or rhEPO are allowed to bind to the receptor EpoR sample, wherein the binding time is 60s-120s. Preferably, the analyte is bound to the receptor EpoR sample after gradient dilution.

2. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to claim 1, characterized in that: In the step (1), the chip was homogenized using 70% glycerol and 50 mM NaOH.

3. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to claim 1 or 2, characterized in that: The capture antibody in step (2) is prepared by diluting the anti-human Fc antibody to 25 μg / mL using a 10 mM sodium acetate buffer at pH 4.5-5.0; the activation reagent is prepared by mixing an EDC solution and an NHS solution at a molar ratio of 4:1; and the blocking agent is ETA.

4. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to any one of claims 1 to 3, characterized in that: In step (3), the analyte is diluted with running buffer, and the EpoR is diluted to 0.5-2 μg / mL; 0.5-2 μg / mL EpoR solution is injected into the detection channel, and the EpoR solution is captured at a flow rate of 5-10 μL / min, and the injection time is between 40-70 s so that the captured response level is between 60RU-200RU; the analyte is injected into the detection channel and the reference channel at a flow rate of 30-50 μL / min for 60-120 s, so that the analyte-receptor binding level is between 20-50RU; optionally, 3M MgCl2 regeneration solution is injected at a flow rate of 10-30 μL / min for 60-180 s to regenerate the detection channel.

5. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to claim 4, characterized in that: In step (3), the analyte is diluted with running buffer, and the EpoR is diluted to 1 μg / mL; 1 μg / mL EpoR solution is injected into the detection channel, and the EpoR solution is captured at a flow rate of 5 μL / min, and the injection time is between 40-70 s so that the captured response level is between 120RU-200RU; the analyte is injected into the detection channel and the reference channel at a flow rate of 30 μL / min for 120 s, so that the analyte-receptor binding level is between 30-40RU (preferably about 33, 34, 35, 36RU); optionally, 3M MgCl2 regeneration solution is injected at a flow rate of 30 μL / min for 60 s to regenerate the detection channel.

6. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to claim 4 or 5, characterized in that: In the step (3), the analyte is PEG-EPO, and PEG-EPO is diluted to 16.48 nM.

7. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to claim 4 or 5, characterized in that: In the step (3), the analyte is rhEPO, and the rhEPO protein is diluted to 5 nM.

8. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to any one of claims 1 to 7, characterized in that: In the step (4), EPO-PEG is diluted with running buffer, and the dilution gradient is 16.48, 8.24, 4.12, 2.06, 1.03, 0.515, 0 nM, wherein the 2.06 nM concentration point sample is used as a repeated concentration point, and the other detection parameters are the same as in step (3), and the binding time is preferably 100-120 s.

9. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to any one of claims 1 to 7, characterized in that: In the step (4), rhEPO is diluted with running buffer, and the dilution gradient is 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 nM, wherein the 0.625 nM concentration point sample is used as a repeated concentration point, and the other detection parameters are the same as in step (3), and the binding time is preferably 100-120 s.

10. The method for detecting the in vitro binding activity of EPO and its receptor EpoR based on SPR according to any one of claims 1 to 9, characterized in that: The chip has a carboxymethyl dextran matrix covalently attached to a gold surface.

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