A method for high-throughput screening of human il17 inhibitors and use thereof

Abstract

This invention provides a method for high-throughput screening of human IL17 inhibitors and its application. The method comprises: Step 1, preparing streptavidin conjugated with rare earth metal elements, and then pre-binding it with biotinylated homodimer human IL17AA protein, biotinylated homodimer human IL17FF protein, or biotinylated heterodimer human IL17AF protein to form complexes respectively; Step 2, preparing human IL17RA protein conjugated with fluorescent dye; Step 3, adding a positive inhibitor to a microplate; Step 4, adding human IL17RA protein conjugated with fluorescent dye to the microplate; Step 5, adding the complexes obtained in Step 1 to the microplate respectively; Step 6, incubating the microplate and reading the fluorescence signal values. This invention also provides applications of this method. The method of this invention is simple to operate, involves homogeneous reactions, is easy to standardize, and is suitable for high-throughput screening of IL17 inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the establishment and application of a method for high-throughput screening of human IL17 inhibitors using TR-FRET detection technology, particularly a method for high-throughput screening of human IL17 inhibitors and its application. Background Technology

[0002] IL-17 (interleukin-17), also known as IL-17AA (homodimeric protein), is a cytokine belonging to the IL-17 family. Cytokines are protein-based signaling compounds and are major regulators of immune responses. They control a variety of cellular functions, including proliferation, differentiation, and cell survival / apoptosis, and are also involved in various pathophysiological processes, such as viral infections and autoimmune diseases. Cytokines are synthesized by various cells of the immune system in response to various stimuli, including monocytes, macrophages, and dendritic cells in the innate immune system, and T cells and B cells in the adaptive immune system. The IL-17 family of cytokines includes six members: IL-17 / IL-17A, IL-17B, IL-17C, IL-17D, IL-17E / IL-25, and IL-17F, which are produced by various cell types. IL-17 regulates the activity of NF-κB and mitogen-activated protein kinase. This cytokine can stimulate the expression of IL-6 and cyclooxygenase-2 (PTGS2 / COX-2) and enhance the production of nitric oxide (NO). High IL-17 levels are associated with a variety of chronic inflammatory diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis.

[0003] Interleukin-17F (IL-17F) is a cytokine with a sequence similar to IL-17. The most significant role of IL-17 is in inducing and mediating pro-inflammatory responses. IL-17 is commonly associated with allergic reactions. The homodimeric protein IL-17FF is expressed by activated T cells, specifically in activated CD4+ T cells and activated monocytes. IL-17F has been shown to stimulate the production of several other cytokines, including IL-6 and IL-8. This cytokine has also been found to inhibit angiogenesis in endothelial cells and induce the production of IL-2, TGF-β / TGFB, and monocyte chemoattractant protein-1 (MCP-1) from endothelial cells. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitor cells or the migration of mature leukocytes. However, it significantly inhibited angiogenesis in human endothelial cells and induced the production of IL-2, TGF-β, and MCP-1 from endothelial cells. IL-17F stimulates the production of other cytokines and granulocyte colony-stimulating factors and regulates the turnover of cartilage matrix. IL-17F stimulates the proliferation of peripheral blood mononuclear cells and T cells. It also inhibits angiogenesis through similarity, and plays a role in inducing neutrophilia in the lungs and exacerbating antigen-induced allergic inflammation in the lungs.

[0004] The IL-17AF heterodimer is a heterodimer of the cytokines IL-17A and IL-17F. Both IL-17A and IL-17F belong to the IL-17 cytokine family. The IL-17AF heterodimer, and the IL-17A and IL-17F homodimer, can be produced by differentiated Th17 cells. IL-17F is most similar to IL-17A (50% homology). Both IL-17A and IL-17F can induce antimicrobial peptides, cytokines (IL-6 and GM-CSF), chemokines (CCL2, CCL7, and CXCL1), and matrix metalloproteinases (MMP-1 and MMP13). IL-17A, IL-17F, and IL-17AF use the same receptor complex: the IL-17RA and IL-17RC heterodimers. They trigger signaling pathways with similar properties. The biological activity of IL-17AF is between that of IL-17A (the strongest) and IL-17F (the weakest).

[0005] With the growing autoimmune market, the increasing demand for treatment for niche autoimmune diseases, and, crucially, the lack of a clear leading product in the IL-17 field, making market substitutability readily available, this presents a significant opportunity for biotech companies. Traditional antibody immunoassay techniques such as ELISA (enzyme-linked immunosorbent assay), flow cytometry, and surface plasmon resonance (SPR) are costly and not routinely used. Therefore, there is an urgent need to develop a simple, direct, and efficient analytical method for IL-17 antibody drugs. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a TR-FRET detection method for high-throughput screening of human IL17 inhibitors and its application. This method is simple to operate, involves homogeneous reactions, is easy to standardize, and is suitable for high-throughput screening of IL17 inhibitors, thus overcoming the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention provides a method for high-throughput screening of human IL17 inhibitors, wherein the method comprises: Step 1, preparing rare earth metal-conjugated streptavidin, and then pre-binding it with biotinylated human IL17AA protein, biotinylated human IL17FF protein, or biotinylated human IL17AF protein to form complexes respectively; Step 2, preparing fluorescent dye-conjugated human IL17RA protein; Step 3, adding different concentrations of positive inhibitors to a microplate; Step 4, adding the fluorescent dye-conjugated human IL17RA protein obtained in Step 2 to the microplate; Step 5, adding the complexes formed by the rare earth metal-conjugated streptavidin obtained in Step 1 and the biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein pre-binding to the microplate respectively; Step 6, incubating the microplate and reading the fluorescence signal values.

[0008] The above-described high-throughput screening method for human IL17 inhibitors, wherein the sequence of human IL17AA protein is shown in SEQ ID NO.1, the sequence of human IL17FF protein is shown in SEQ ID NO.2, the sequence of human IL17AF protein is shown in SEQ ID NO.3, and the sequence of human IL17RA protein is shown in SEQ ID NO.4.

[0009] In the above-mentioned high-throughput screening method for human IL17 inhibitors, step 1 involves replacing streptavidin with carbonate buffer, then adding rare earth metal activated with NHS to the streptavidin solution to form a stable streptavidin-rare earth metal complex. The unchelated dye is then removed by gravity desalting column purification to obtain rare earth metal-coupled streptavidin. This streptavidin is then pre-bound with biotinylated human IL17AA protein, biotinylated human IL17FF protein, or biotinylated human IL17AF protein to form complexes.

[0010] In the above-described high-throughput screening method for human IL17 inhibitors, the rare earth metal element is europium or terbium, and the chelate formed therein can be excited by ultraviolet light.

[0011] In the above-described high-throughput screening method for human IL17 inhibitors, step 2 involves replacing human IL17-RA protein in carbonate buffer, adding an NHS-activated receptor fluorescent dye to the human IL17-RA solution to form a stable fluorescent dye-conjugated human IL17RA protein, and then purifying and removing unchelated dye using a gravity desalting column.

[0012] In the above-described high-throughput screening method for human IL17 inhibitors, the receptor fluorescent dye is a fluorescent dye capable of generating fluorescence resonance energy transfer with the chelate formed with the rare earth metal element used in step 1; it includes any one of Cy3, Cy5, Alexa Fluor 647, FITC, Alexa Fluor 488, BODIPY FL, APC, GFP and their respective derivatives.

[0013] In the above-described high-throughput screening method for human IL17 inhibitors, step 3 involves adding different concentrations of a positive inhibitor to a microplate. The positive inhibitor used is Bimekizumab, and the microplate is a white, opaque 384-well microplate.

[0014] In the above-described high-throughput screening method for human IL17 inhibitors, step 6 involves incubating the microplate at room temperature for 1-2 hours, then reading the fluorescence signal values ​​at 665 nm and 620 nm using a microplate reader. The IC50 of the bimekizumab inhibitor is then obtained by fitting the fluorescence signal values. 50 value.

[0015] The present invention also provides an application of the above-described high-throughput screening method for human IL17 inhibitors, wherein the method is used to prepare a TR-FRET detection kit for detecting IL17 inhibitors.

[0016] The application of the above-mentioned high-throughput screening method for human IL17 inhibitors, wherein the kit comprises: a complex formed by the pre-binding of rare earth metal-conjugated streptavidin with biotinylated human IL17AA protein, biotinylated IL17FF protein or biotinylated IL17AF protein, fluorescent dye-conjugated human IL17RA protein, positive inhibitor Bimekizumab, Assay dilution buffer, and Assay detection buffer.

[0017] Compared with the prior art, the high-throughput screening method for human IL17 inhibitors of the present invention and its application have the following beneficial effects: (1) The TR-FRET detection kit for detecting IL17 inhibitors provided by the present invention has simple operation steps and stable experimental system. It can be used to screen IL17 inhibitors in high throughput using 384-well plates, which helps pharmaceutical companies to quickly screen drugs for related targets and promote the research and development process of related drugs.

[0018] (2) The TR-FRET detection method of IL17 inhibitor used in this invention is a homogeneous system, and the antibody-antigen binding is not affected by steric hindrance, making it easy to establish a standardized analytical method.

[0019] (3) The application fields of this invention can connect basic research (signaling pathways) and applied pharmacy (drug development and quality control). The TR-FRET detection method for detecting IL17 inhibitors can be used as a core quality attribute (CQA) and critical quality study (CQS) in biosimilar drug development and comparability studies. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the TR-FRET human IL17AA / IL17RA binding detection principle of the present invention.

[0021] Figure 2 This is a schematic diagram illustrating the TR-FRET human IL17FF / IL17RA binding detection principle of the present invention.

[0022] Figure 3 This is a schematic diagram illustrating the binding detection principle of TR-FRET human IL17AF / IL17RA in this invention.

[0023] Figure 4 This is a dose-response curve of the IL17 inhibitor of the present invention against human IL17AA / IL17RA.

[0024] Figure 5 This is a dose-response curve of the IL17 inhibitor of the present invention on human IL17FF / IL17RA.

[0025] Figure 6This is a dose-response curve of the IL17 inhibitor of the present invention on human IL17AF / IL17RA. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. These drawings are only for illustrating the embodiments of the present disclosure and are not intended to limit the scope of the disclosure.

[0027] This invention provides a method for high-throughput screening of human IL17 inhibitors, comprising: Step 1, preparing rare earth metal-conjugated streptavidin, and then pre-binding it with biotinylated homodimer human IL17AA protein (Biotin-IL17AA), biotinylated homodimer human IL17FF protein (Biotin-IL17FF), or biotinylated heterodimer human IL17AF protein (Biotin-IL17AF) to form complexes respectively; Step 2, preparing fluorescent dye-conjugated human IL17RA protein (Dye-labeled human IL17RA protein). Step 3: Add different concentrations of positive inhibitors to the microplate; Step 4: Add the human IL17RA protein conjugated with the fluorescent dye obtained in Step 2 to the microplate; Step 5: Add the complex formed by the rare earth metal element conjugated streptavidin obtained in Step 1 and biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein pre-binding to the microplate; Step 6: Incubate the microplate and read the fluorescence signal value.

[0028] Preferably, in this method: The sequence of human IL17AA protein is Q16552, Gly 24-Ala 155, and the amino acid sequence is shown in SEQ ID NO.1.

[0029] The sequence of human IL17FF protein is NP_443104.1, Arg 31-Gln 163, and the amino acid sequence is shown in SEQ ID NO.2.

[0030] The sequence of human IL17AF protein is human IL17A (Q16552-1)(Gly24-Ala155) & human IL17F (NP_443104.1) (Arg31-Gln163), and the amino acid sequence is shown in SEQ ID NO.3.

[0031] The sequence of human IL17RA protein is NP_055154.3, Met 1-Trp 320, and the amino acid sequence is shown in SEQ ID NO.4.

[0032] Preferably, step 1 involves replacing streptavidin in a carbonate buffer solution, then adding rare earth metal activated with NHS (N-hydroxysuccinimide) to the streptavidin solution to form a stable streptavidin-rare earth metal complex. The unchelated dye is then removed by purification using a gravity desalting column to obtain rare earth metal-coupled streptavidin. This streptavidin is then pre-bound with biotinylated human IL17AA protein, biotinylated human IL17FF protein, or biotinylated human IL17AF protein to form complexes.

[0033] More preferably, the rare earth metal element is europium (Eu) or terbium (Tb), and the chelate formed therein can be excited by ultraviolet light.

[0034] Preferably, step 2 involves replacing the human IL17-RA protein in the carbonate buffer, then adding the receptor fluorescent dye activated with NHS to the human IL17-RA solution to form a stable fluorescent dye-coupled human IL17RA protein, and then purifying it with a gravity desalting column to remove unchelated dye.

[0035] More preferably, the acceptor fluorescent dye is a fluorescent dye that can generate fluorescence resonance energy transfer (FRET) with the chelate formed with the rare earth metal element used in step 1; it includes any one of Cy3, Cy5, Alexa Fluor 647, FITC, Alexa Fluor 488, BODIPY FL, APC, GFP and their respective derivatives.

[0036] Preferably, step 3 involves adding different concentrations of positive inhibitors to the microplate. The positive inhibitor used is the commercially available Bimekizumab, and the microplate is a white, opaque 384-well microplate.

[0037] Preferably, step 6 involves incubating the microplate at room temperature for 1-2 hours, then reading the fluorescence signal values ​​at 665 nm and 620 nm using a microplate reader, and obtaining the IC50 of the Bimekizumab inhibitor by fitting the fluorescence signal values. 50 value.

[0038] This invention also provides an application of the high-throughput screening method for human IL17 inhibitors, which is used to prepare a TR-FRET detection kit for detecting IL17 inhibitors.

[0039] Preferably, the kit comprises: a complex formed by the pre-binding of rare earth metal-conjugated streptavidin with biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein; fluorescent dye-conjugated human IL17RA protein; the positive inhibitor Bimekizumab; Assay dilution buffer; Assay detection buffer; and a white opaque 384 microplate, etc.

[0040] This invention provides a method for high-throughput screening of human IL17 inhibitors and its applications. It relates to the development of a TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) detection method for high-throughput screening of human IL17 inhibitors. The main technical route is as follows: This invention utilizes Solar Eu... *1 The conjugated streptavidin (SA) and biotinylated Human IL17AA protein (Biotin-IL17AA), biotinylated Human IL17FF protein (Biotin-IL17FF), or biotinylated Human IL17AF protein (Biotin-IL17AF) are pre-conjugated into complexes (Biotin-IL17AA&SA-Solar Eu Mix, Biotin-IL17FF&SA-Solar Eu Mix, or Biotin-IL17AF&SA-Solar Eu Mix, respectively), while using LA *2 The IL17RA protein is conjugated with Tag 1 (Tag 1-IL17RA-LA). When IL17AA, IL17FF, or IL17AF binds to IL17RA, the tag antibodies conjugated with Solar Eu and LA, respectively, approach each other. Under excitation by an external light source, energy resonance transfer occurs between the donor and acceptor. The degree of interaction between IL17AA, IL17FF, or IL17AF and IL17RA proteins can be determined by detecting the signal intensity at a specific wavelength (665 nm). Therefore, compounds, peptides, or antibodies that block the interaction between IL17AA, IL17FF, or IL17AF and IL17RA will lead to a decrease in TR-FRET signal. For the TR-FRET Human IL17AA / IL17RA binding detection principle, please refer to [link to TR-FRET protocol description]. Figure 1 As shown, the detection principle of TR-FRET Human IL17FF / IL17RA combination is described in [reference needed]. Figure 2As shown, the TR-FRET Human (human-derived) IL17AF / IL17RA combined detection principle is described in [reference needed]. Figure 3 As shown in the figure. This method is characterized by its simplicity, speed, high accuracy, and good reproducibility, which helps pharmaceutical companies quickly screen drugs targeting relevant targets and facilitates the advancement of drug development.

[0041] Note: *1 Solar Eu: Fluorescence Resonance Energy Transfer Donor (TR-FRET Donor).

[0042] *2 LA (Luminescent Acceptor): Fluorescence resonance energy transfer acceptor (TR-FRETAcceptor).

[0043] To make the technical problems to be solved, the technical solutions, and the beneficial effects of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0044] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods, performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. All reagents or instruments used are conventional products that can be purchased through legitimate channels.

[0045] Example 1.

[0046] A high-throughput screening method for human IL17 inhibitors, using the following main reagents and consumables: streptavidin conjugated with rare earth metals, biotinylated human IL17AA protein (Biotin-IL17AA), biotinylated human IL17FF protein (Biotin-IL17FF), biotinylated human IL17AF protein (Biotin-IL17AF), fluorescent dye conjugated human IL17RA protein (Dye-labeled IL17RA), positive inhibitor bimekizumab, Assay dilution buffer, Assay detection buffer, and white opaque 384 microplates, etc.

[0047] The sequence of human IL17AA protein is Q16552, Gly 24-Ala 155, and the amino acid sequence is shown in SEQ ID NO.1.

[0048] The sequence of human IL17FF protein is NP_443104.1, Arg 31-Gln 163, and the amino acid sequence is shown in SEQ ID NO.2.

[0049] The sequence of human IL17AF protein is human IL17A (Q16552-1) (Gly24-Ala155) & human IL17F (NP_443104.1) (Arg31-Gln163), and the amino acid sequence is shown in SEQ ID NO.3.

[0050] The sequence of human IL17RA protein is NP_055154.3, Met 1-Trp 320, and the amino acid sequence is shown in SEQ ID NO.4.

[0051] This method mainly includes the following steps: S1. Prepare streptavidin coupled with rare earth metal elements, and then pre-bind it with Biotin-IL17AA, Biotin-IL17FF or Biotin-IL17AF to form complexes.

[0052] Preferably, the rare earth metal element is europium (Eu), and the chelate formed therein can be excited by ultraviolet light.

[0053] Streptavidin (SA) was replaced in carbonate buffer, and then rare earth metals activated with NHS were added to the streptavidin solution to form a stable streptavidin-rare earth metal complex. The unchelated dye was then removed by gravity desalting column purification to obtain rare earth metal-coupled streptavidin. This complex was then pre-bound with Biotin-IL17AA, Biotin-IL17FF, or Biotin-IL17AF to form complexes, namely Biotin-IL17AA&SA-EuMix, Biotin-IL17FF&SA-Eu Mix, or Biotin-IL17AF&SA-Eu Mix.

[0054] S2. Prepare fluorescent dye-conjugated human IL17RA protein, i.e., Dye-labeled IL17RA.

[0055] Preferably, the human IL17-RA protein is replaced in carbonate buffer, and then the receptor fluorescent dye activated with NHS is added to the human IL17-RA solution to form stable dye-labeled IL17RA. The unchelated dye is then removed by purification using a gravity desalting column.

[0056] The acceptor fluorescent dye is a fluorescent dye that can produce fluorescence resonance energy transfer (FRET) through a chelate formed with the rare earth metal element used in S1. A derivative of Cy5 is preferred.

[0057] S3. Add different concentrations of the positive inhibitor Bimekizumab to the well plate.

[0058] S4. Add the Dye-labeled IL17RA obtained from S2 into the orifice plate.

[0059] S5. Add the Biotin-IL17AA&SA-Eu Mix, Biotin-IL17FF&SA-Eu Mix, or Biotin-IL17AF&SA-Eu Mix obtained in S1 to the orifice plate, respectively.

[0060] S6. After incubating at room temperature for 1 hour, the fluorescence signal value is read using an ELISA reader.

[0061] Example 2.

[0062] Application of a high-throughput screening method for human IL17 inhibitors in Example 1, which is used to prepare a TR-FRET assay kit for detecting IL17 inhibitors.

[0063] Preferably, the kit comprises: a complex formed by the pre-binding of rare earth metal-conjugated streptavidin with biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein (Biotin-IL17AA&SA-Eu Mix, Biotin-IL17FF&SA-Eu Mix, or Biotin-IL17AF&SA-Eu Mix), fluorescent dye-conjugated human IL17RA protein (Dye-labeled IL17RA), positive inhibitor bimekizumab, Assay dilution buffer, Assay detection buffer, and white opaque 384 microplate, etc.

[0064] More preferably, the fluorescent dye is a derivative of Cy5.

[0065] The Assay dilution buffer is Binding Assay Diluent Buffer, sourced from Pharmaron, catalog number A1010001S.

[0066] The Assay detection buffer is Solar Eu Detection Buffer (UV-excited europium detection buffer), sourced from Pharmaron, catalog number A1010002S.

[0067] Example 3.

[0068] A high-throughput TR-FRET detection method for screening human IL17 inhibitors mainly includes the following steps: S1. Prepare rare earth metal-coupled streptavidin. Displace streptavidin into carbonate buffer, and then add rare earth metal activated with NHS to the streptavidin solution to form a stable streptavidin-rare earth metal complex. Purify and remove unchelated dye using a gravity desalting column to obtain rare earth metal-coupled streptavidin. Then, pre-bind it with biotinylated human IL17AA protein, biotinylated human IL17FF protein, or biotinylated human IL17AF protein (AVI-Biotin) to form complexes, namely Biotin-IL17AA&SA-Solar Eu Mix, Biotin-IL17FF&SA-Solar Eu Mix, or Biotin-IL17AF&SA-Solar Eu Mix.

[0069] S2. Prepare fluorescent dye-conjugated human IL17RA protein by replacing human IL17-RA protein in carbonate buffer, and then adding Alexa Fluor 647 derivative activated with NHS to human IL17-RA solution to form stable fluorescent dye-conjugated human IL17RA protein, i.e., Dye-labeled IL17RA. Finally, remove unchelated dye by purifying with a gravity desalting column.

[0070] S3. Add different concentrations of the positive inhibitor Bimekizumab to the 384-well plate.

[0071] The positive inhibitor is the commercially available Bimekizumab.

[0072] S4. Add the Dye-labeled IL17RA obtained in S2 to a 384-well plate.

[0073] S5. Add the Biotin-IL17AA&SA-Solar Eu Mix, Biotin-IL17FF&SA-Solar Eu Mix, or Biotin-IL17AF&SA-Solar Eu Mix obtained in S1 to the 384-well plate, respectively.

[0074] S6. After incubating the 384-well plate at room temperature for 1 hour, read the fluorescence signals at 665 nm and 620 nm using a microplate reader. Obtain the IC50 of the Bimekizumab inhibitor by fitting the fluorescence signal values. 50 value.

[0075] Example 4.

[0076] Application of a high-throughput screening TR-FRET detection method for human IL17 inhibitors in Example 3, which is used to prepare a TR-FRET detection kit for detecting IL17 inhibitors.

[0077] Preferably, the kit comprises: a complex formed by the pre-binding of rare earth metal-conjugated streptavidin with biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein (Biotin-IL17AA&SA-Solar Eu Mix, Biotin-IL17FF&SA-Solar Eu Mix, or Biotin-IL17AF&SA-SolarEu Mix), fluorescent dye-conjugated human IL17RA protein (Dye-labeled IL17RA), positive inhibitor Bimekizumab, Assay dilution buffer, Assay detection buffer, and white opaque 384 microplate, etc.

[0078] More preferably, the fluorescent dye is an Alexa Fluor 647 derivative.

[0079] The positive control was bimekizumab monoclonal antibody.

[0080] The Assay dilution buffer is Binding Assay Diluent Buffer, sourced from Pharmaron, catalog number A1010001S.

[0081] The Assay detection buffer is Solar Eu Detection Buffer (UV-excited europium detection buffer), sourced from Pharmaron, catalog number A1010002S.

[0082] Example 5.

[0083] A high-throughput screening method for human IL17 inhibitors is disclosed, comprising the following main reagents and consumables: rare earth metals, streptavidin, biotinylated human IL17AA protein, biotinylated human IL17FF protein, biotinylated human IL17AF protein, fluorescent dye, human IL17RA protein, positive inhibitor bimekizumab, assay dilution buffer, assay detection buffer, and white opaque 384 microplates. The specific details of this method are as follows.

[0084] Preparation of standards and test samples.

[0085] To reduce matrix effect interference and obtain more accurate sample back-calculation concentration, it is preferable to use a solution with the same matrix as the sample to be tested to prepare the standard curve.

[0086] In this embodiment, the sample to be tested is cell supernatant in DMEM medium (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum), and the standard is diluted using a medium with the same matrix.

[0087] The standards and test samples were serially diluted threefold using Assay dilution buffer. The positive control was bimekizumab monoclonal antibody, with initial and final antibody concentrations of 30 μg / mL, 10 μg / mL, 3.33 μg / mL, 1.111 μg / mL, 0.370 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.014 μg / mL, 0.005 μg / mL, 0.002 μg / mL, and 0 μg / mL. 10 μL of each solution was added to a 384-well plate using a power pipette and centrifuged at 1000 rpm for 1 min.

[0088] Preparation of rare earth metal-coupled proteins / fluorescent dye-coupled proteins and detection of IL17 inhibitors.

[0089] Step 1: Replace streptavidin in carbonate buffer, then add rare earth metal activated with NHS to the streptavidin solution to form a stable streptavidin-rare earth metal complex. Purify with a gravity desalting column to remove unchelated dye. Then, pre-bind the rare earth metal-coupled streptavidin with biotinylated human IL17AA protein, human IL17FF protein, or human IL17AF protein (AVI-Biotin) to form complexes, namely Biotin-IL17AA&SA-Solar Eu Mix, Biotin-IL17FF&SA-Solar Eu Mix, or Biotin-IL17AF&SA-Solar Eu Mix.

[0090] Step 2: Replace the human IL17-RA protein in the carbonate buffer, and then add the NHS-activated AlexaFluor 647 derivative (fluorescent dye) to the human IL17-RA solution to form a stable fluorescent dye-conjugated human IL17RA protein, i.e., Dye-labeled IL17RA. Purify the protein using a gravity desalting column to remove unchelated dye.

[0091] Step 3: Add 5 μL of Dye-labeled IL17RA (IL17RA-LA) to the 384 shallow well plates obtained in (I) above, which contain the standard and the test sample respectively, and centrifuge at 1000 rpm for 1 min.

[0092] Step 4: Add 5 μL of Biotin-IL17AA&SA-Solar EuMix with a final concentration of 2.5 nM, 5 μL of Biotin-IL17FF&SA-Solar Eu Mix with a final concentration of 10 nM, or 5 μL of Biotin-IL17AF&SA-Solar Eu Mix with a final concentration of 5 nM to the 384 shallow well plate, and centrifuge at 1000 rpm for 1 min.

[0093] Step 5: Seal the 384 shallow well plate with sealing film, and incubate at room temperature for 2 hours. Then, read the signals at 665 and 620 nm using a microplate reader, calculate the 665 nm / 620 nm ratio (TR-FRET Ratio) for each well, and then perform nonlinear four-parameter fitting using GraphPad Prism 8 software.

[0094] The test results are as follows: Using the human IL17AA / IL17RA binding assay kit provided by this invention, the dose-response curves of the obtained IL17 inhibitors against human IL17AA / IL17RA are shown in the figure. Figure 4 As shown.

[0095] Using the Human IL17FF / IL17RA binding assay kit provided by this invention, the dose-response curves of the obtained IL17 inhibitors against Human IL17FF / IL17RA are shown in the figure. Figure 5 As shown.

[0096] Using the Human IL17AF / IL17RA binding assay kit provided by this invention, the dose-response curve of the obtained IL17 inhibitor against Human IL17AF / IL17RA is shown in the figure. Figure 6 As shown.

[0097] The sequences involved in this invention are as follows.

[0098] SEQ ID NO.1, amino acid sequence of human IL17AA protein (Recombinant Human IL17 Protein, His & AVI Tag, Biotinylated).

[0099] GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA SEQ ID NO.2, amino acid sequence of human IL-17FF protein (Recombinant Human IL-17F Protein, His & AVI Tag, Biotinylated, HPLC-verified; recombinant human interleukin-17F protein, tagged with His and AVI, biotinylated, verified by high performance liquid chromatography).

[0100] RKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMSNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ SEQ ID NO.3, amino acid sequence of human IL17AF protein (Recombinant Human IL17A&IL17FHeterodimer Protein, Strep II&His-AVI tag, Biotinylated, HPLC-verified; Recombinant human interleukin-17AF protein, with streptavidin II and His and AVI tags, biotinylated, verified by high performance liquid chromatography).

[0101] GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA& RKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMSNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHRVQ SEQ ID NO.4, amino acid sequence of human IL-17RA protein (Recombinant Human IL-17RA Protein, ECD, hFc Tag; recombinant human interleukin-17 receptor α protein, extracellular domain, with human Fc tag).

[0102] MGAARSPPSAVPGPLLGLLLLLGVLAPGGASLRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYE VTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYM This invention provides a streptavidin protein-labeled rare earth metal element method with high efficiency and good specific binding ability. The high-throughput screening method for human IL17 inhibitors employed in this invention is simple to operate, involves homogeneous reactions, is easy to standardize, and is suitable for high-throughput screening of IL17 inhibitors.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application without departing from the principles of the present invention should be included within the protection scope of this application.

Claims

1. A method for high-throughput screening of human IL17 inhibitors, characterized in that, The method includes: Step 1: Prepare streptavidin coupled with rare earth metal elements, and then pre-bind it with biotinylated human IL17AA protein, biotinylated human IL17FF protein or biotinylated human IL17AF protein to form complexes respectively. Step 2: Prepare fluorescent dye-conjugated human IL17RA protein; Step 3: Add positive inhibitors to the microplate; Step 4: Add the human IL17RA protein conjugated with the fluorescent dye obtained in step 2 to the microplate. Step 5: Add the rare earth metal element-coupled streptavidin obtained in step 1 to the microplate to form a complex with biotinylated human IL17AA protein, biotinylated IL17FF protein or biotinylated IL17AF protein pre-bound. Step 6: After incubating the microplate, read the fluorescence signal value.

2. The method for high-throughput screening of human IL17 inhibitors according to claim 1, characterized in that, In the method described, the sequence of human IL17AA protein is shown in SEQ ID NO.1, the sequence of human IL17FF protein is shown in SEQ ID NO.2, the sequence of human IL17AF protein is shown in SEQ ID NO.3, and the sequence of human IL17RA protein is shown in SEQ ID NO.

4.

3. The method for high-throughput screening of human IL17 inhibitors according to claim 1, characterized in that, Step 1 involves replacing streptavidin in a carbonate buffer solution, then adding rare earth metals activated with NHS to the streptavidin solution to form a stable streptavidin-rare earth metal complex. The unchelated dye is then removed by purification using a gravity desalting column to obtain rare earth metal-coupled streptavidin. This streptavidin is then pre-bound with biotinylated human IL17AA protein, biotinylated human IL17FF protein, or biotinylated human IL17AF protein to form complexes.

4. The method for high-throughput screening of human IL17 inhibitors according to claim 3, characterized in that, The rare earth metal element is europium or terbium, and the chelate formed therein can be excited by ultraviolet light.

5. The method for high-throughput screening of human IL17 inhibitors according to claim 1, characterized in that, Step 2 involves replacing the human IL17-RA protein in a carbonate buffer, then adding the receptor fluorescent dye activated with NHS to the human IL17-RA solution to form a stable fluorescent dye-conjugated human IL17RA protein, followed by purification using a gravity desalting column to remove unchelated dye.

6. The method for high-throughput screening of human IL17 inhibitors according to claim 5, characterized in that, The receptor fluorescent dye is a fluorescent dye that can produce fluorescence resonance energy transfer with the chelate formed with the rare earth metal element used in step 1. It includes any one of Cy3, Cy5, Alexa Fluor 647, FITC, Alexa Fluor 488, BODIPY FL, APC, GFP and their respective derivatives.

7. The method for high-throughput screening of human IL17 inhibitors according to claim 1, characterized in that, In step 3, different concentrations of positive inhibitors are added to the microplate. The positive inhibitor used is Bimekizumab, and the microplate is a white, opaque 384 microplate.

8. The method for high-throughput screening of human IL17 inhibitors according to claim 1, characterized in that, In step 6, after the microplate is incubated at room temperature for 1-2 hours, the fluorescence signal values ​​at 665 nm and 620 nm are read using a microplate reader. The IC50 of the Bimekizumab inhibitor is obtained by fitting the fluorescence signal values. 50 value.

9. The application of a method for high-throughput screening of human IL17 inhibitors according to any one of claims 1 to 8, characterized in that, The method described herein is used to prepare a TR-FRET assay kit for detecting IL17 inhibitors.

10. The application of the high-throughput screening method for human IL17 inhibitors according to claim 9, characterized in that, The kit comprises: a complex formed by the pre-binding of rare earth metal-conjugated streptavidin with biotinylated human IL17AA protein, biotinylated IL17FF protein, or biotinylated IL17AF protein; a fluorescent dye-conjugated human IL17RA protein; a positive inhibitor Bimekizumab; an Assay dilution buffer; and an Assay detection buffer.

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