Methods for screening of fc gamma receptor i (fcγri) antagonists

Abstract

The present application belongs to the technical field of analytical chemistry, and particularly relates to a screening method for FcRn small molecule antagonists based on phase transition combined with FRET. In the present application, a fluorescent donor dye required in a FRET process is labeled on IgG, and a fluorescent acceptor dye is labeled on a phase transition material P4VP-SSO3Na-4VP; the phase transition material can reversibly change the phase when the environmental pH changes; when the material changes from a homogeneous system to a heterogeneous system, free IgG in the solution is embedded in the system, the distance between the fluorescent donor and the acceptor in the FRET process is shortened, and the fluorescent signal of the acceptor in the FRET system is detected; and further detection is made on whether the compound has antagonistic effect on FcRn, so as to screen FcRn small molecule antagonists. The present application overcomes the deficiency that some small molecule compounds have no suitable sites for dye labeling; has repeatability, low experimental cost, and can realize high-throughput and rapid screening of FcRn small molecule antagonists.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a screening method for FcRn small molecule antagonists based on phase transition binding FRET. Background Technology

[0002] Immunoglobulin G (IgG) is a fundamental component of the adaptive immune response and plays a crucial role in protecting against most infectious and autoimmune diseases. Compared to other types of immunoglobulins, IgG has attracted considerable attention due to its high concentration in the blood, long half-life, and unique ability to be directly passed from mother to offspring. These biological characteristics are primarily achieved through the interaction of IgG with the neonatal Fc receptor (FcRn). FcRn binds to the Fc region of IgG via a pH-dependent mechanism. This binding not only protects IgG from intracellular degradation but also enhances the cellular immune response after IgG forms immune complexes with its corresponding antigens. Furthermore, IgG antibody levels are closely related to the development of various autoimmune diseases. Therefore, blocking FcRn provides a novel strategy for treating IgG-mediated systemic immune diseases. By inhibiting FcRn function, the level of pathogenic IgG antibodies in the circulating system can be effectively reduced, thereby treating IgG-mediated diseases.

[0003] Currently, there are FcRn antagonist drugs on the market or in clinical trials worldwide, such as Efgartigimod (Efgartigimod α-fcab, Vyvgart). TM Efgartigimod is a first-line neonatal Fc receptor antagonist developed by Argenx for the treatment of autoimmune diseases, including myasthenia gravis. In December 2021, intravenous Efgartigimod was first approved in the United States for the treatment of adult generalized myasthenia gravis who is positive for anti-acetylcholine receptor (AChR) antibodies; Rozanolixizumab... Batoclimab is a high-affinity humanized immunoglobulin G4 monoclonal antibody that targets the neonatal Fc receptor (FcRn). Developed by UCB Pharma, this drug is intended to treat autoimmune diseases and was first approved in the United States on June 27, 2023, for the treatment of adult generalized myasthenia gravis (gMG) positive for anti-acetylcholine receptor (AChR) or anti-muscle-specific kinase (MuSK) antibodies. Batoclimab, from Harbour BioMed, is a fully humanized monoclonal antibody that binds to FcRn, blocking FcRn-IgG interaction to accelerate the degradation of autoantibodies and treating various pathogenic IgG-mediated autoimmune diseases. This product was previously included in the Breakthrough Therapy Program in China and is currently undergoing clinical trials for multiple indications.

[0004] It is not difficult to see that all currently marketed or investigational FcRn antagonists are macromolecules. Currently, there are no small molecule drugs or small molecule lead compounds used to develop FcRn antagonists, and there is also a lack of research and reports on small molecule FcRn antagonists, requiring further research to fill this gap. Small molecule drugs have inherent structural advantages over macromolecules, their preparation and production processes are simpler and lower in cost, and they do not induce immune inflammatory responses. Once the structure is determined, mass production can be achieved.

[0005] FRET (Fluorescence Resonance Energy Transfer) is a commonly used and mature technique for high-throughput screening. FRET offers advantages such as suitability for high throughput, ease of operation, high sensitivity, and high resolution. However, FRET has the following drawbacks for high-throughput screening: it is difficult to apply to the screening of small molecules. Small molecule compounds generally have simple structures and may lack labeling sites for fluorescent dyes; they lack signal amplification mechanisms, resulting in limited signal intensity. Traditional FRET relies on the intermolecular distance of interactions; if the molecule itself is large and structurally complex, the detection signal will be relatively weak; reagents are expensive, and most samples must be discarded after screening, making the screening cost high.

[0006] To address the shortcomings of existing FRET techniques, this invention combines pH-responsive polymers with traditional FRET technology to develop a novel rapid screening method based on phase transition combined with FRET, termed PT-FRET. pH-responsive polymers possess pendant ionizable groups in their structure. These groups can alter the polymer's hydrodynamic volume, chain conformation, and solubility according to the ambient pH, enabling a generalized phase transition by adjusting the pH. Utilizing this property of pH-responsive polymers, the distance between fluorescent molecules within the system can be reduced through phase transitions, thereby achieving the FRET process. Summary of the Invention

[0007] The purpose of this invention is to provide a novel rapid and high-throughput screening method for FcRn small molecule antagonists based on phase transition binding FRET, so as to fill the gap in the research of FcRn antagonists in the field of small molecules.

[0008] The method for screening FcRn small molecule antagonists based on phase transition binding FRET provided by this invention includes the following specific steps:

[0009] (1) Labeling the fluorescent donor dye required for the FRET process onto IgG: Cy3 was used as the donor dye and labeled onto IgG in advance; since there is an amino group in the chemical structure of IgG, it was chosen to be linked to sulfonated Cy3-NHS activated ester; the entire labeling process was carried out in a PBS system with pH=5 to 7, the molar ratio of Cy3 to IgG was 1:5 to 1:10, and the mixture was mixed overnight in a mixer at 15 to 30°C in the dark. After labeling, the free dye was removed by ultrafiltration or dialysis.

[0010] (2) Labeling the acceptor dye onto the phase transition material P4VP-SSO3Na-4VP: Specifically, Cy 5-amine and P4VP-SSO3Na-4VP were selected for labeling; the entire labeling process was carried out in a PBS system with pH 5 to 7, the molar ratio of Cy 5 to P4VP-SSO3Na-4VP was 1:5 to 1:10, and the mixture was stirred overnight in a mixer at 15 to 30°C in the dark; after labeling, the free dye was removed by ultrafiltration or dialysis.

[0011] (3) Plate preparation: Prepare an appropriate amount of FcRn solution with a concentration of 400-500 nM and add it to a high-adsorption plate (transparent) using a pipette. Add 20-40 μL to each well. During the addition process, the pipette tip should be extended to the bottom of the plate. Shake well and ensure there is no residue on the plate. Place the plate at 4-10℃ overnight.

[0012] (4) Sealing: Discard the FcRn solution in the wells of the plate, do not wash the plate, immediately add 80-100 μL of PBST solution containing 1% BSA to each well, place at 30-37°C for at least 1 hour, then discard the solution in the wells. If it is not needed for immediate use, it can be temporarily stored at -20°C. If it is needed for immediate use, proceed to the next step.

[0013] (5) Washing the plate: Prepare a PBST solution with a pH of 5 to 7. Add 80 to 100 μL of the solution to each well, shake slightly, discard the solution, and then wash the plate until there is no solution residue in the well. Repeat this process 3 times.

[0014] (6) Add the compound to be screened: Mix the compound to be screened with IgG-Cy 3 in an equal proportion, add 30-40 μL of the mixed solution to each well, and incubate at 30-37°C for 1-1.5 h;

[0015] (7) Transfer the plate and add the phase transformation material P4VP-SSO3Na-4VP: Transfer 30-40 μL of solution per well in the transparent plate to the opaque black plate for fluorescence detection, and add 30-40 μL of P4VP-SSO3Na-4VP-Cy 5 solution with a concentration of 1-5 mg / mL to each well;

[0016] (8) Adjust pH to induce phase transition: Add 1-5 μL of 5% H3PO4 solution to each well to adjust pH so that P4VP-SSO3Na-4VP phase transition occurs. Do not shake. Let stand for at least 10 minutes in the dark until the phase transition is close to complete.

[0017] (9) Fluorescence detection: The fluorescence signal value of the corresponding well is detected using a microplate reader capable of detecting fluorescence. Multiple values ​​(≥5) are taken from each well, and the average value is taken as the final result for that point; Detection conditions: E x =500~550nm, E m =620~670nm.

[0018] Further:

[0019] In step (2), a phase transition material P4VP-SSO3Na-4VP is introduced based on the FRET principle, and donor or acceptor dyes are labeled on the phase transition material.

[0020] In step (3), in order to make FcRn reusable, it was adsorbed onto the bottom of a transparent well plate by coating. After the experiment, the binding material on the FcRn on the bottom of the plate was removed by elution with pH adjusted to 6-8.

[0021] In step (8), in order to enhance the fluorescence signal and bring the donor and acceptor molecules closer together to achieve fluorescence resonance energy transfer, the pH is adjusted with 1-5 μL of 5% H3PO4 to induce a phase transition in P4VP-SSO3Na-4VP, and the mixture is left to stand for more than 10 minutes until the phase transition process tends to end.

[0022] After step (9), in order to achieve the recyclability of P4VP-SSO3Na-4VP, the pH is adjusted to the initial state, the pH is adjusted back, and P4VP-SSO3Na-4VP is separated by ultracentrifugation.

[0023] The mechanism of this invention is as follows: The fluorescent donor dye required for the FRET process is labeled on IgG, and the fluorescent acceptor dye is labeled on the phase transition material P4VP-SSO3Na-4VP. The phase transition material undergoes a reversible phase transition when the ambient pH changes. When the material transforms from a homogeneous system (solution) to a heterogeneous system (solid), it embeds the free IgG in the solution within the system, bringing the fluorescent donor and acceptor closer together in the FRET process, thereby detecting the fluorescent signal of the acceptor in the FRET system. FcRn is pre-coated on the bottom of the well plate. When the binding of IgG to FcRn is inhibited, IgG becomes free in the supernatant. Transferring the supernatant and adding P4VP-SSO3Na-4VP phase transition material and adjusting the pH to induce a phase transition causes the donor fluorescent group on IgG to undergo a FRET process with the acceptor fluorescent group on P4VP-SSO3Na-4VP. Conversely, compounds without antagonistic activity cannot undergo FRET. This principle can be used to detect whether a compound antagonizes FcRn, thus serving as a screening tool for small molecule FcRn antagonists.

[0024] The method for screening FcRn small molecule antagonists provided by this invention is an optimization and improvement of the original FRET technology, specifically including:

[0025] Traditional FRET technology is more suitable for screening macromolecular compounds. Small molecule compounds often lack suitable groups for labeling FRET dyes. This invention introduces phase transition materials such as P4VP-SSO3Na-4VP based on the original FRET technology. The groups of P4VP-SSO3Na-4VP can be modified according to different needs, so fluorescent dyes can be labeled on P4VP-SSO3Na-4VP, thereby making up for the shortcomings of traditional FRET technology for small molecule compounds.

[0026] Traditional FRET technology has a weak signal and lacks a signal amplification mechanism. After introducing the phase transition material P4VP-SSO3Na-4VP, the dyes can undergo a phase transition, which amplifies their fluorescence signal and makes the measured fluorescence signal stronger, thus solving the problem of low fluorescence signal intensity in FRET technology.

[0027] The P4VP-SSO3Na-4VP system changes with pH, ​​and the phase transition is reversible and does not alter other properties of P4VP-SSO3Na-4VP. Therefore, by properly adjusting the pH and correctly recovering the P4VP-SSO3Na-4VP solution, it can be reused, thereby reducing experimental costs. Furthermore, the binding ability of FcRn on the plate decreases significantly at pH 7–7.4, meaning that incubation with PBS solution at pH 7.4 can elute the bound compounds, enabling reuse and avoiding the problems of expensive reagents and non-reusability in traditional methods.

[0028] The experimental conditions and reagents have been optimized. Compared with the classic high-throughput screening ELISA method, the experimental time of this invention is shorter and the operation steps are more convenient and simpler, solving the problems of complex operation and long experimental time of traditional methods.

[0029] The method of this invention has stable detection results, simple experimental operation and short experimental time, and can be used as an ideal method for screening FcRn small molecule antagonists. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the experimental principle of the method of the present invention.

[0031] Figure 2 This is a schematic diagram of an experiment to verify the experimental principle of the method of the present invention.

[0032] Figure 3 This is a graph showing the optimization of various parameters in the method of the present invention. Among them, A is the optimization of the phase transition pH value. Since the tested system is only 80 μL, it is very difficult to obtain an accurate pH value, so the volume of 5% phosphoric acid added is used as a substitute; B is the optimization graph of the concentration of P4VP-SSO3Na-4VP phase transition material; C is the optimization of the phase transition time.

[0033] Figure 4 This is a scatter plot created based on the screening results.

[0034] Figure 5 To evaluate the selected compounds using a competitive ELISA method, inhibition curves were plotted based on the OD signal values. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments and accompanying drawings, such as... Figure 1 As shown.

[0036] Example 1: A method and validation for screening small molecule FcRn antagonists using phase transition combined with FRET technology.

[0037] 1.1 Rapid and high-throughput screening of small molecule FcRn antagonists based on phase transition combined with FRET technology;

[0038] (1) Labeling of donor dye: Take 5 mg of sulfonated Cy 3-NHS activated ester and add it to a solution containing 6.6 × 10 -4 The nM IgG was placed in a PBS solution at pH 6 (dye:label = 10:1), shaken in a suspension apparatus, and incubated overnight at 25°C in the dark. A 10kd ultrafiltration tube was added to the solution and centrifuged. After removing the liquid outside the ultrafiltration tube, PBS solution at pH 6 was added to the system and centrifuged again. The above operation was repeated until the centrifuged liquid was colorless, thus removing the free dye.

[0039] (2) Labeling of receptor dye: Dissolve 12 mg of P4VP-SSO3Na-4VP in 4 mL of pure water, add 6 mg of EDC and 10.8 mg of sulfo-NHS, and place in a mixer at room temperature for 20 min to activate the carboxyl group on P4VP-SSO3Na-4VP. Then add 3.27 mg of Cy 5-amine in a PBS solution system with pH=6 to carry out the reaction. Place in a suspension apparatus and shake, and incubate overnight at 25°C in the dark. Take a 10 kDa ultrafiltration tube, add the solution and centrifuge. After removing the liquid outside the ultrafiltration tube, add PBS solution with pH=6 to the system and continue centrifugation. Repeat the above operation until the centrifuged liquid is colorless, which proves that the unlabeled dye and excess EDC and sulfo-NHS have been removed. Then add PBS solution (pH=6) to the P4VP-SSO3Na-4VP concentration to 3 mg / mL.

[0040] (3) Plate coating: Prepare 2 mL of 500 nM FcRn solution and add it to a 384-well (transparent) plate with high adsorption, 20 μL per well. In the example, 31 compounds were screened and each compound was replicated in 3 replicates. Therefore, 10 nM FcRn solution needs to be added to 93 wells, 20 μL per well. During the addition process, the pipette tip should be extended to the bottom of the plate, shake well and ensure no residue on the plate wall, so that the solution is completely spread on the bottom of each well. Place the plate at 4°C overnight to allow FcRn to bind tightly to the bottom of the transparent 384-well plate.

[0041] (4) Blocking: Prepare 8 mL of PBST solution containing 1% BSA in advance, discard the FcRn solution used for coating in the wells, and add the prepared PBST solution directly to each well using a pipette without washing the plate. Add 80 μL to each well and place it at 37°C for 1 hour to block the exposed parts of the bottom and sidewalls of each well that have not been adsorbed with FcRn. This will prevent the high adsorption of the exposed parts in the wells from affecting subsequent experiments. The well plate should be used as soon as possible after sealing. If it is not used temporarily, it can be stored at -40°C.

[0042] (5) Washing the plate: Prepare a sufficient amount of PBST solution with pH=6 in advance. When washing the plate, add 100μL of PBST solution to each well and gently tap the side of the plate. After discarding the PBST, tap the plate immediately until there is no liquid residue in the well of the plate. The washing operation should be repeated 3 times to ensure that the wells will not affect the subsequent experiments.

[0043] (6) Add the small molecule compound to be screened: Prepare a 10 nM solution for each small molecule compound to be screened in PBS with pH=6. If it does not dissolve, prepare a stock solution with a small volume of DMSO and then dilute it to 10 nM with PBS with pH=6. At the same time, prepare an IgG-Cy3 solution with a molar concentration of 10 nM. Mix the compound to be screened with IgG-Cy3 at a ratio of 1:1 and add 40 μL of the mixed solution to each well. Incubate at 37°C for 1.5 h to allow the compound to be screened to compete with IgG for a full reaction with FcRn.

[0044] (7) Transferring the plate and adding P4VP-SSO3Na-4VP: Since the transparent 384-well plate is not suitable for detecting fluorescence signals, in order to facilitate the subsequent detection of fluorescence signals, 40 μL of the solution in each well of the transparent 384-well plate was transferred to the corresponding black well plate, and 40 μL of P4VP-SSO3Na-4VP-Cy 5 solution with a concentration of 3 mg / mL was added to each well.

[0045] (8) Adjust pH: Add 4 μL of 5% H3PO4 solution to each well to adjust the pH, so that the P4VP-SSO3Na-4VP phase transition occurs. Do not shake. Let stand for 10 min in the dark until the phase transition is close to complete.

[0046] (9) Fluorescence detection: The fluorescence signal value of the corresponding well is detected using a microplate reader capable of detecting fluorescence. Multiple readings (≥5) are taken in a cross shape, and the average value is taken as the fluorescence signal intensity of each well. Then, the average value of three wells of the same sample is taken as the final result for the compound. Detection conditions: Ex = 550nm, Em = 664nm.

[0047] (10) Recovery of P4VP-SSO3Na-4VP-Cy 5 and FcRn: After detection, the solution in the plate was adjusted back to pH=6, collected, and added to a 100kd ultrafiltration tube for centrifugation and ultrafiltration. The filtrate was collected and then added to a 3kd ultrafiltration tube for ultrafiltration. The liquid in the filter tube was P4VP-SSO3Na-4VP-Cy 5. 80uL of PBS solution with pH=7.4 was added to the FcRn wells of the used plate, and eluted at 37℃ for 1h. The eluent was discarded, and the FcRn in the plate could be reused. Figure 2 As shown.

[0048] The structural formula of P4VP-SSO3Na-4VP is as follows:

[0049]

[0050] 1.2 Optimization of the developed method

[0051] After the method was fully established, a series of optimizations were performed on its parameters, such as... Figure 3 As shown: The volume of added 5% phosphoric acid was optimized by comparing the fluorescence signal intensity of the system when 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL were added, and 4 μL was finally selected for the next stage of formal screening; the concentration of the phase transition material P4VP-SSO3Na-4VP was optimized by testing the fluorescence intensity with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL, and 3 mg / mL was finally selected for the next stage of formal screening; the time required for phase transition in the experiment was optimized by comparing the changes in fluorescence intensity before phase transition and after 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min, and 10 min was finally selected for the next stage of formal screening.

[0052] 1.3 Processing the signal values ​​of the screened compounds

[0053] The 31 samples screened included 28 candidate compounds, 1 positive control group, and 2 blank control samples. Five points were evenly selected in a cross shape, and the average value was taken as the actual fluorescence signal for that well. The values ​​from the remaining three wells of the same sample were then averaged again to obtain the final result for that compound. A scatter plot was then created for each compound. The screening results are shown below. Figure 4 As shown, based on the screening results, three compounds with signals similar to the positive control were selected for ELISA verification.

[0054] 1.4 Competitive ELISA method was used to validate the screened compounds.

[0055] The scatter plot generated from the results showed that the fluorescence signal intensity of most compounds was near that of the blank control group. Some small molecule compounds showed results similar to the positive control group. In this experiment, the gold standard method, competitive ELISA, was selected to validate the compounds with similar positive results, and the IC50 was measured. 50 Values ​​were calculated, with 18 concentrations per sample and 2 replicates per concentration.

[0056] (1) Plate coating: Dilute IgG to 66 nM with PBS solution at pH=6, and coat the transparent 96-well plate with 66 nM IgG solution. The volume added to each well is 50 μL. During the addition process, the pipette tip should be extended to the bottom of the plate, shake well and avoid sticking to the wall, so that the solution is completely spread on the bottom of each well. Place at 4°C overnight.

[0057] (2) Blocking: Discard the IgG solution in the wells of the plate without washing the plate. Immediately add 200 μL of PBST solution containing 1% BSA to each well and incubate at 37°C for at least 1 hour. Then discard the solution in the wells. If the solution is to be used immediately, proceed to the next step.

[0058] (3) Washing the plate: Prepare a PBST solution with pH=6, add 250μL of the above solution to each well, gently tap and shake with your hand, discard the solution and tap the plate until there is no solution residue in the well. Repeat this process three times.

[0059] (4) Add primary antibody: Mix the test compound and FcRn in equal volume using a 96-well deep well. Keep the molar concentration of FcRn constant at 20 nM. Dilute the sample concentration three times (starting from 10000 nM). Incubate at 37°C for 1.5 hours. After incubation, add 100 μL of the mixed solution to each well. The final system in each well is 10 nM FcRn and the compound diluted three times (maximum concentration is 5000 nM).

[0060] (5) Washing the plate: Same as step (3).

[0061] (6) Add secondary antibody: Select HRP labeled with streptavidin as secondary antibody, dilute it 1000 times and add 100 μL to each well, and incubate at 37°C for 1.5 hours.

[0062] (7) Washing the plate: Same as step (3).

[0063] (8) TMB color development: Add 100 μL of TMB color development solution to each well and wait for color development at room temperature, generally not exceeding 30 minutes.

[0064] (9) Termination of reaction: After the color has fully developed and a gradient change in color can be observed, immediately add 50 μL of stop solution (1M H2SO4) to each well, and immediately measure ABS at 450 nm and make IC. 50 curve.

[0065] ELISA data as follows Figure 5 As shown, the IC50 curves of the three compounds are located between the blank control group and the positive control group, meaning that compared with the blank control group, the three compounds have a certain inhibitory effect on FcRn, but their inhibitory effect is weaker than that of the positive control.

Claims

1. A method for screening FcRn small molecule antagonists based on phase transition binding FRET, characterized in that, The specific steps are as follows: (1) Label the fluorescent donor dye required for the FRET process onto IgG: Cy3 is used as the donor dye and it is labeled onto IgG; the entire labeling process is carried out in a PBS system with pH = 5~7, the molar ratio of Cy3 to IgG is 1:5~1:10, and the mixture is kept in the dark at 15~30℃ overnight. After labeling, the free dye is removed by ultrafiltration or dialysis. (2) The acceptor dye required for the FRET process was labeled on the phase transition material P4VP-SSO3Na-4VP; the entire labeling process was carried out in a PBS system with pH 5~7, the molar ratio of Cy 5 to P4VP-SSO3Na-4VP was 1:5~1:10, and the mixture was mixed overnight in a mixer at 15~30℃ in the dark; after labeling, the free dye was removed by ultrafiltration or dialysis. (3) Plate preparation: Prepare a 400-500 nM FcRn solution and add it to the high-adsorption plate using a pipette. Add 20-40 μL to each well. During the addition process, the pipette tip should be extended to the bottom of the plate. Shake well and ensure there is no residue on the plate. Place the plate at 4-10℃ overnight. (4) Sealing: Discard the FcRn solution in the wells of the plate, immediately add 80~100 μL of PBST solution containing 1% BSA to each well, place at 30~37 ℃ for at least 1 hour, then discard the solution in the well. If not used immediately, it can be temporarily stored at -20 ℃. If used immediately, proceed to the next step. (5) Washing the plate: Prepare a PBST solution with a pH of 5-7. Add 80-100 μL of the above solution to each well, shake slightly, discard the solution and pat the plate until there is no solution residue in the well. Repeat this process 3 times. (6) Add the compound to be screened: Mix the compound to be screened with IgG-Cy 3 in an equal proportion, add 30~40 μL of the mixed solution to each well, and incubate at 30~37 ℃ for 1~1.5 h; (7) Transfer the plate and add the phase transformation material P4VP-SSO3Na-4VP: Specifically, transfer 30~40 μL of solution per well in the plate to an opaque black plate for fluorescence detection, and add 30~40 μL of P4VP-SSO3Na-4VP-Cy 5 solution with a concentration of 1~5 mg / mL to each well; (8) Adjust pH to induce phase transition: Add 1-5 μL of 5% H3PO4 solution to each well to adjust pH so that P4VP-SSO3Na-4VP undergoes phase transition. Do not shake. Let stand for at least 10 min in the dark until the phase transition is close to complete. (9) Fluorescence detection: detect the fluorescence signal value of the corresponding well with a fluorescence detector, take multiple point values of each well and take the average value as the final result of the well; detection conditions: E x = 500~550 nm, E m = 620~670 nm.

2. The method for screening FcRn small molecule antagonists according to claim 1, characterized in that, In step (3), FcRn is adsorbed onto the bottom of a transparent plate by coating. After the experiment, the binding material on the FcRn at the bottom of the plate is removed by adjusting the pH to 6-8, so that FcRn can be reused.

3. The method for screening FcRn small molecule antagonists according to claim 1, characterized in that, After step (9), adjust the pH to the initial state, restore the pH, and use ultracentrifugation to separate P4VP-SSO3Na-4VP in order to recover P4VP-SSO3Na-4VP.

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