A method for detecting the occupancy rate of BTK receptor

Abstract

The present invention discloses a method for detecting the occupancy rate of BTK receptors, which relates to the technical field of BTK. The method uses the simoa detection method and includes the following steps: S1, connecting the capture antibody to the magnetic beads; S2, preparing the biotinylated detection antibody; S3, the detection method of simoa total BTK. In this method for detecting the occupancy rate of BTK receptors, for the first time, the ibrutinib biotin probe is applied to the simoa platform to replace the biotinylated detection antibody. Based on the principle of the traditional sandwich ELISA method on the simoa platform, small molecule biotin is used as the detection probe to broaden its scope of application. The traditional ELISA reaction takes 6-10 hours, while the present invention using the simoa technology only takes about 2 hours, which can greatly shorten the detection time, improve the detection efficiency, perform automatic analysis, improve the detection throughput, and have more controllable detection precision and accuracy. For the first time, the small molecule biotin probe is used to replace the biotinylated detection antibody and applied to the simoa platform, and this technology is used for the first time to detect the occupancy rate of BTK receptors.

Description

Technical Field

[0001] The present invention relates to the technical field of BTK, and specifically relates to a method for detecting the occupancy rate of BTK receptor. Background Art

[0002] Bruton's tyrosine kinase (BTK) is a member of the TEC family of non-receptor tyrosine kinases in the cytoplasm. It plays an important role in the signal transduction of B cell receptors, the growth, development and function regulation of B cells. The abnormal expression of this kinase usually leads to the occurrence of B lymphocyte malignancies and is one of the important therapeutic targets for B cell malignancies. Currently, 5 BTK inhibitors have passed the marketing application, and the research of other inhibitors is ongoing, showing good development prospects. BTK kinase is expressed in hematopoietic cells such as B cells, macrophages and monocytes, and plays an important role in the growth, development, proliferation and differentiation of B cells. It is a key kinase in the B cell surface antigen signaling pathway, and its abnormal expression is one of the important causes of B cell malignancies such as chronic lymphocytic leukemia (CLL), and is also an important therapeutic target for B cell malignancies. The discovery of this kinase has opened a new door for the research and treatment of B cell malignancies and provided an important theoretical basis for the research and development of BTK inhibitors.

[0003] In clinical studies related to BTK inhibitors, the occupancy rate of BTK receptor is usually used as a pharmacodynamic index to evaluate the inhibitory effect of drugs on this protein. Currently, many methods have been developed, among which the ELISA method is widely used. The detection of the occupancy rate of BTK receptor by the ELISA method usually consists of two parts: the detection of total BTK and the detection of free BTK. The detection of total BTK usually adopts the double antibody sandwich method; the detection principle of free BTK is to use a biotin-labeled irreversible BTK inhibitor to compete with the drug under study for binding to BTK, which is captured by an ELISA microplate coated with SA, and detected by a BTK antibody. By detecting the change in the ratio of free BTK to total BTK before and after drug administration, the receptor occupancy rate of the drug is calculated. The detection of the occupancy rate of BTK receptor by the ELISA method can achieve relatively accurate quantification through a standard curve. Compared with other methods, the ELISA method has a lower cost and can be carried out in most laboratories. However, the ELISA method has many operation steps, takes a long time and has a low throughput, which easily reduces the precision and accuracy of the detection results.

[0004] SiMoA (Single Molecule Array) is a multiplex immunoassay technology that is fast, flexible, robust, and sensitive. It has extremely high sensitivity for detecting low-concentration biomarkers, can measure biomarkers in various common samples with low sample requirements. The SiMoA method first conjugates capture antibodies to magnetic beads. The capture antibodies bound to the magnetic beads bind to the target protein and then bind to the detection antibody. In the case of extremely low antigen content, the magnetic beads are in excess. The target antibody binds to the detection antibody and is labeled; while the excess magnetic beads are unlabeled. When adding the enzyme reaction substrate, the magnetic beads with the added substrate are transferred to the SiMoA chip. The magnetic beads sink into the small holes in the chip, and digital interpretation is performed through fluorescence imaging. The magnetic beads containing the target protein carry the enzyme and emit fluorescence. Based on the proportion of the small holes that produce fluorescence in the entire chip and using the Poisson distribution principle, the concentration of the target protein in the sample is calculated. SiMoA technology has been widely used for the quantitative analysis of blood biomarkers and can also be applied to the customized detection of biomarkers other than test kits. It is an important technology for the detection and analysis of biological samples. Due to the high degree of automation and wide linear range of SiMoA technology, when using SiMoA technology to detect BTK receptor occupancy, experimental results with higher precision, better accuracy, and a wider linear range can be obtained. At the same time, due to the extremely high sensitivity of SiMoA technology, the usage amount of samples can be saved, which has important clinical significance. Therefore, we propose a method for detecting BTK receptor occupancy to solve the above problems. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for detecting BTK receptor occupancy to solve the problems raised in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solution: A method for detecting BTK receptor occupancy, and the method uses the simoa detection method.

[0007] A method for detecting BTK receptor occupancy includes the following steps:

[0008] S1. Connect the capture antibody to the magnetic beads;

[0009] S11. Take out the Bead Conjugation Buffer from the refrigerator at 2 - 8°C, aliquot 7 ml into a 15 ml centrifuge tube, and place it on ice.

[0010] S12. Take out the vial of beads from the storage at 2 - 8°C, vortex for at least 30 seconds, and then place it on a rotary mixer and mix for 10 minutes.

[0011] S13. Take 100 μg of BTK capture antibody in a 30 kDa ultrafiltration tube, and add the Bead Conjugation Buffer from step S1 to 500 μL;

[0012] S14. Invert the ultrafiltration tube into a clean centrifuge tube and centrifuge at 1000 xg for 2 minutes to collect the antibody;

[0013] S15. Wash both filter membranes 6 times each with 50 μL of Bead Conjugation Buffer;

[0014] S16. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and detect the antibody concentration;

[0015] S17. Vortex the antibody from step S16, place it on ice, and vortex the beads for 30 seconds;

[0016] S18. Gently open the lid, then place the EP tube in a magnetic rack and pipette off the excess liquid with a pipette tip;

[0017] S19. Add 300 μL of Bead Wash Buffer to the EP tube in step S18, vortex for 5 seconds, spin quickly, open the lid, place it in a magnetic rack, and pipette off the excess liquid with a pipette tip;

[0018] S191. Repeat step S19 to wash the magnetic beads 2 times;

[0019] S192. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, spin quickly, and place it in a magnetic rack;

[0020] S193. Repeat step S192 twice;

[0021] S194. Add 291 μL of Bead Conjugation Buffer, vortex for 5 seconds, spin quickly, and place it on ice;

[0022] S195. Add 1 mL of pre-cooled Bead Conjugation Buffer to 10 mg of EDC, vortex for 5 - 10 seconds until dissolved;

[0023] S196. Add 9 μL of pre-cooled EDC to the beads in step S203, vortex for 10 seconds, place it on a rotary mixer, and mix well;

[0024] S197. After mixing, spin quickly in a centrifuge, place it in a magnetic rack, and pipette off the excess liquid with a pipette tip;

[0025] S198. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0026] S199. Add 300 μL of the capture antibody from step S15, vortex for 10 seconds, place it on a rotary mixer, mix at 2 - 8 °C for 120 minutes, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0027] S20. Add 300 μL of Bead Wash Buffer, vortex for 5 seconds, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip. Repeat the washing twice.

[0028] S201. Add 300 μL of Bead Blocking Buffer, vortex for 5 seconds, place it on a rotary mixer, and mix at room temperature for 45 minutes.

[0029] S202. After the mixing is completed, rotate rapidly, place it in a magnetic stand, aspirate the excess liquid with a pipette tip, add 300 μL of Bead Wash Buffer, and vortex for 5 s.

[0030] S203. Rotate rapidly, place it in a magnetic stand, aspirate the excess liquid with a pipette tip, add 300 μL of Bead Diluent, and vortex for 5 s. Repeat twice.

[0031] S204. Rotate rapidly, measure the concentration, and store the magnetic bead-conjugated capture antibody in a 4 °C refrigerator for later use.

[0032] S2. Preparation of biotinylated detection antibody.

[0033] S21. Take 130 μg of BTK detection antibody in a 30 kDa ultrafiltration tube, and add Biotinylation Reaction Buffer to 500 μL.

[0034] S22. Invert the ultrafiltration tube in step S21 into a clean centrifuge tube, and centrifuge at 1000 xg for 2 minutes to collect the antibody.

[0035] S23. Wash both filter membranes 6 times each with 50 μL of Biotinylation Reaction Buffer.

[0036] S24. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and measure the antibody concentration.

[0037] S25. Measure the volume and antibody concentration: Adjust the concentration to 1 mg / mL with Biotinylation Reaction Buffer.

[0038] S26. Take out a bottle of 2 mg NHS-PEG4-Biotin from a -20 °C refrigerator, and after equilibrating at room temperature, reconstitute it with 383 μL of deionized water;

[0039] S27. Add the reconstituted biotin to the detection antibody according to the ratio of 100 μL of 1 mg / ml detection antibody + 3 μL of Biotin;

[0040] S28. Vortex and mix, rotate rapidly, and incubate at room temperature for 30 minutes. After the incubation, transfer the biotinylated antibody to an ultrafiltration tube;

[0041] S29. Add Biotinylation Reaction Buffer to 500 μL, centrifuge at 14000 x g for 5 minutes, discard the liquid, and repeat the washing step 4 times;

[0042] S291. Invert the filter tube into a clean centrifuge tube and centrifuge at 1000 x g for 2 minutes to collect the antibody;

[0043] S292. Wash both sides of the filter membrane 6 times each with 50 μL of Biotinylation Reaction Buffer;

[0044] S293. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 x g for 2 minutes to collect the antibody, and detect the antibody concentration;

[0045] S294. After measuring the concentration and volume, store it in a 4 °C refrigerator;

[0046] S3. Detection method for total BTK of Simoa;

[0047] S31. Dilute the capture antibody and magnetic bead conjugate to 2*10 7 bead / mL with Bead Diluent Buffer;

[0048] S32. Dilute the biotinylated detection antibody to 0.3 μg·mL-1 with Detector Diluent Buffer;

[0049] S33. Dilute SBG to 0.15 nM with the corresponding diluent;

[0050] S34. Dilute the recombinant BTK standard curve to 1, 5, 10, 50, 100, 200, 500 ng / mL with cell lysate;

[0051] S35. Load the reagents and samples into the corresponding sample and reagent compartments of simoa HD-X;

[0052] S36. All sample incubation, washing, and detection steps are carried out according to the "3-step homebrew assay".

[0053] S4. Detection method for Simoa free BTK;

[0054] S41. The capture antibody-conjugated magnetic beads are diluted to 2*10 7 bead / mL with Bead Diluent Buffer;

[0055] S42. The biotinylated antibody is diluted to 7 μM with Detector Diluent Buffer using Ibrutinib-biotin.

[0056] S43. SBG is diluted to 0.15 nM with the corresponding diluent.

[0057] S44. The recombinant BTK standard curve is diluted to 1, 5, 10, 50, 100, 200, 500 ng / mL with cell lysate.

[0058] S45. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the simoa HD-X instrument.

[0059] To further optimize this technical solution, the receptor occupancy calculation formula is: relative%occupancy = (1 - (Free BTK / totalBTK) / (Free BTK predose / totalBTK predose)) * 100.

[0060] To further optimize this technical solution, all sample incubation, washing, and detection steps are carried out according to the "3-step homebrew assay".

[0061] To further optimize this technical solution, in step S16, the antibody is diluted to 0.2 mg / mL with Bead Conjugation Buffer.

[0062] To further optimize this technical solution, after vortexing in step S17, transfer 4.2×10 8 beads to a 2 mL EP tube.

[0063] To further optimize this technical solution, in step S192, use a pipette tip to aspirate the excess liquid in the EP tube.

[0064] To further optimize this technical solution, the mixing treatment in step S196 is to mix at 2 - 8 °C for 30 minutes.

[0065] Further optimize this technical solution. The ultrafiltration tube in step S21 is centrifuged at 14,000 xg for 5 minutes, the liquid is discarded, and the washing is repeated twice.

[0066] Compared with the prior art, the present invention provides a method for detecting the occupancy rate of BTK receptors, having the following beneficial effects:

[0067] 1. For this method for detecting the occupancy rate of BTK receptors, the detection method prepared by the present invention first applies the ibrutinib biotin probe to the simoa platform, replacing the biotinylated detection antibody. Based on the principle of the traditional sandwich ELISA method on the simoa platform, a small molecule biotin is used as the detection probe to broaden its scope of application. The traditional ELISA reaction takes 6 - 10 hours, while the present invention using the simoa technology only takes about 2 hours, which can greatly shorten the detection time, improve the detection efficiency, and perform fully automatic analysis. While improving the detection throughput, it has more controllable detection precision and accuracy.

[0068] 2. For this method for detecting the occupancy rate of BTK receptors, the present invention first applies a small molecule biotin probe to replace the biotinylated detection antibody on the simoa platform, and for the first time uses this technology to detect the occupancy rate of BTK receptors, improving the scope of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is a schematic flow chart of a method for detecting the occupancy rate of BTK receptors proposed by the present invention;

[0070] Figure 2 is the total BTK standard curve of a method for detecting the occupancy rate of BTK receptors proposed by the present invention;

[0071] Figure 3 is the free BTK standard curve of a method for detecting the occupancy rate of BTK receptors proposed by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0073] Example 1: Please refer to Figures 1 to 3 As shown, the present invention discloses a method for detecting the occupancy rate of BTK receptors, and the method is the simoa detection method;

[0074] A method for detecting the occupancy rate of BTK receptors includes the following steps:

[0075] S1. The capture antibody is conjugated to magnetic beads;

[0076] S11. Take out the Bead Conjugation Buffer from the 3°C refrigerator, aliquot 7 ml into a 15-ml centrifuge tube, and place it on ice;

[0077] S12. Take out the vial of beads from the 3°C storage, vortex for at least 30 seconds, and then place it on a rotary mixer and mix for 10 minutes;

[0078] S13. Take 100 μg of the BTK capture antibody in a 30 kDa ultrafiltration tube, and add the Bead Conjugation Buffer from step S1 to 500 μL;

[0079] S14. Invert the ultrafiltration tube into a clean centrifuge tube and centrifuge at 1000 xg for 2 minutes to collect the antibody;

[0080] S15. Wash both sides of the filter membrane 6 times with 50 μL of Bead Conjugation Buffer;

[0081] S16. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, measure the antibody concentration, and dilute the antibody to 0.2 mg / mL with Bead Conjugation Buffer;

[0082] S17. Vortex the antibody from step S16, place it on ice, vortex the beads for 30 seconds. After vortexing, transfer 4.2×10 8 beads to a 2-ml EP tube;

[0083] S18. Gently open the lid, then place the EP tube in a magnetic stand, and use a pipette tip to aspirate the excess liquid;

[0084] S19. Add 300 μL of Bead Wash Buffer to the EP tube in step S18, vortex for 5 seconds, rotate quickly, open the lid, place it in a magnetic stand, and use a pipette tip to aspirate the excess liquid;

[0085] S191. Repeat step S19 to wash the magnetic beads 2 times;

[0086] S192. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, place it in a magnetic stand, and use a pipette tip to aspirate the excess liquid in the EP tube;

[0087] S193. Repeat step S192 twice;

[0088] S194. Add 291 μL of Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, and place on ice;

[0089] S195. Add 1 mL of pre-cooled Bead Conjugation Buffer to 10 mg of EDC, vortex for 5 - 10 seconds until dissolved;

[0090] S196. Add 9 μL of pre-cooled EDC to the beads in step S203, vortex for 10 seconds, place on a rotary mixer, and mix. The mixing process is to mix at 2 - 8 °C for 30 minutes;

[0091] S197. After mixing, spin quickly in a centrifuge, place in a magnetic stand, and aspirate the excess liquid with a pipette tip;

[0092] S198. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, place in a magnetic stand, and aspirate the excess liquid with a pipette tip;

[0093] S199. Add 300 μL of the capture antibody in step S15, vortex for 10 seconds, place on a rotary mixer, mix at 4 °C for 120 minutes, rotate quickly, place in a magnetic stand, and aspirate the excess liquid with a pipette tip;

[0094] S20. Add 300 μL of Bead Wash Buffer, vortex for 5 seconds, rotate quickly, place in a magnetic stand, and aspirate the excess liquid with a pipette tip. Repeat the washing twice;

[0095] S201. Add 300 μL of Bead Blocking Buffer, vortex for 5 seconds, place on a rotary mixer, mix at room temperature for 45 minutes, centrifuge the ultrafiltration tube at 14000 xg for 5 minutes, discard the liquid, and repeat the washing twice;

[0096] S202. After mixing, spin quickly, place in a magnetic stand, and aspirate the excess liquid with a pipette tip. Add 300 μL of Bead Wash Buffer and vortex for 5 s;

[0097] S203. Spin quickly, place in a magnetic stand, and aspirate the excess liquid with a pipette tip. Add 300 μL of Bead Diluent and vortex for 5 s. Repeat twice;

[0098] S204. Spin quickly, measure the concentration, and store the magnetic bead-conjugated capture antibody in a 4 °C refrigerator for later use;

[0099] S2. Preparation of biotinylated detection antibody;

[0100] S21. Take 130 μg of the BTK detection antibody in a 30 kDa ultrafiltration tube, and add Biotinylation Reaction Buffer to 500 μL.

[0101] S22. Invert the ultrafiltration tube in step S21 into a clean centrifuge tube, and centrifuge at 1000 xg for 2 minutes to collect the antibody.

[0102] S23. Wash both sides of the filter membrane 6 times each with 50 μL of Biotinylation Reaction Buffer.

[0103] S24. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and detect the antibody concentration.

[0104] S25. Measure the volume and antibody concentration: Adjust the concentration to 1 mg / mL with Biotinylation Reaction Buffer.

[0105] S26. Take out a bottle of 2 mg of NHS-PEG4-Biotin from the -20 °C refrigerator, equilibrate at room temperature, and reconstitute with 383 μL of deionized water.

[0106] S27. Add the reconstituted biotin to the detection antibody according to the ratio of 100 μL of 1 mg / ml detection antibody + 3 μL of Biotin.

[0107] S28. Vortex mix, spin rapidly, and incubate at room temperature for 30 minutes. After incubation, transfer the biotinylated antibody to an ultrafiltration tube.

[0108] S29. Add Biotinylation Reaction Buffer to 500 μL, centrifuge at 14000 xg for 5 minutes, discard the liquid, and repeat the washing step 4 times.

[0109] S291. Invert the filter tube into a clean centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody.

[0110] S292. Wash both sides of the filter membrane 6 times each with 50 μL of Biotinylation Reaction Buffer.

[0111] S293. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and detect the antibody concentration.

[0112] S294. After measuring the concentration and volume, store in a 4 °C refrigerator.

[0113] S3. Detection method for Simoa total BTK

[0114] S31. Dilute the capture antibody - magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 bead / mL;

[0115] S32. Dilute the biotinylated detection antibody with Detector Diluent Buffer to 0.3 μg·mL-1;

[0116] S33. Dilute SBG with the corresponding diluent to 0.15 nM;

[0117] S34. Dilute the recombinant BTK standard curve with cell lysate to 1, 5, 10, 50, 100, 200, 500 ng / mL;

[0118] S35. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the simoa HD-X;

[0119] S36. Perform all sample incubation, washing, and detection steps according to the "3-step homebrew assay";

[0120] S4. Detection method for Simoa free BTK;

[0121] S41. Dilute the capture antibody - magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 bead / mL;

[0122] S42. Dilute the biotinylated antibody with Ibrutinib-biotin and Detector Diluent Buffer to 7 μM;

[0123] S43. Dilute SBG with the corresponding diluent to 0.15 nM;

[0124] S44. Dilute the recombinant BTK standard curve with cell lysate to 1, 5, 10, 50, 100, 200, 500 ng / mL;

[0125] S45. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the simoa HD-X instrument.

[0126] Perform all sample incubation, washing, and detection steps according to the "3-step homebrew assay", and the formula for calculating the receptor occupancy is: relative%occupancy=(1-(Free BTK / total BTK) / (FreeBTKpredose / totalBTK predose))*100.

[0127] Example 2: Please refer to Figures 1 to 3 As shown, the present invention discloses a method for detecting the occupancy rate of BTK receptors, and the method is the simoa detection method.

[0128] A method for detecting the occupancy rate of BTK receptors includes the following steps:

[0129] S1. Connect the capture antibody to the magnetic beads;

[0130] S11. Take out the Bead Conjugation Buffer from the refrigerator at 6°C, dispense 7 ml into a 15-ml centrifuge tube, and place it on ice;

[0131] S12. Take out the vial of beads from the storage at 6°C, vortex for at least 30 seconds, and then place it on a rotary mixer and mix for 10 minutes;

[0132] S13. Take 100 μg of the BTK capture antibody in a 30 kDa ultrafiltration tube, and add the Bead Conjugation Buffer in step S1 to 500 μL;

[0133] S14. Invert the ultrafiltration tube into a clean centrifuge tube, and centrifuge at 1000 xg for 2 minutes to collect the antibody;

[0134] S15. Wash both sides of the filter membrane 6 times with 50 μL of Bead Conjugation Buffer;

[0135] S16. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, detect the antibody concentration, and dilute the antibody to 0.2 mg / mL with Bead Conjugation Buffer;

[0136] S17. Vortex the antibody in step S16, place it on ice, vortex the beads for 30 seconds. After the vortex ends, transfer 4.2×10 8 beads to a 2-ml EP tube;

[0137] S18. Gently open the lid, then place the EP tube in a magnetic rack, and use a pipette tip to aspirate the excess liquid;

[0138] S19. Add 300 μL of Bead Wash Buffer to the EP tube in step S18, vortex for 5 seconds, rotate quickly, open the lid, place it in a magnetic rack, and use a pipette tip to aspirate the excess liquid;

[0139] S191. Repeat step S19 to wash the magnetic beads 2 times;

[0140] S192: Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, place in a magnetic stand, and pipette off the excess liquid in the EP tube with a pipette tip.

[0141] S193: Repeat step S192 twice.

[0142] S194: Add 291 μL of Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, and place on ice.

[0143] S195: Add 1 mL of pre-cooled Bead Conjugation Buffer to 10 mg of EDC, vortex for 5 - 10 seconds until dissolved.

[0144] S196: Add 9 μL of pre-cooled EDC to the beads in step S203, vortex for 10 seconds, place on a rotary mixer, and mix. The mixing process is to mix at 2 - 8 °C for 30 minutes.

[0145] S197: After mixing, centrifuge quickly in a centrifuge, place in a magnetic stand, and pipette off the excess liquid with a pipette tip.

[0146] S198: Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate quickly, place in a magnetic stand, and pipette off the excess liquid with a pipette tip.

[0147] S199: Add 300 μL of the capture antibody in step S15, vortex for 10 seconds, place on a rotary mixer, mix at 2 - 8 °C for 120 minutes, rotate quickly, place in a magnetic stand, and pipette off the excess liquid with a pipette tip.

[0148] S20: Add 300 μL of Bead Wash Buffer, vortex for 5 seconds, rotate quickly, place in a magnetic stand, and pipette off the excess liquid with a pipette tip. Repeat the washing twice.

[0149] S201: Add 300 μL of Bead Blocking Buffer, vortex for 5 seconds, place on a rotary mixer, mix at room temperature for 45 minutes, centrifuge the ultrafiltration tube at 14000 xg for 5 minutes, discard the liquid, and repeat the washing twice.

[0150] S202: After mixing, rotate quickly, place in a magnetic stand, and pipette off the excess liquid with a pipette tip. Add 300 μL of Bead Wash Buffer and vortex for 5 s.

[0151] S203: Rotate quickly, place in a magnetic stand, and pipette off the excess liquid with a pipette tip. Add 300 μL of Bead Diluent, vortex for 5 s, and repeat twice.

[0152] S204. Spin rapidly. After measuring the concentration, store the magnetic bead-conjugated capture antibody in a refrigerator at 4°C for future use.

[0153] S2. Preparation of biotinylated detection antibody

[0154] S21. Take 130 μg of BTK detection antibody in a 30 kDa ultrafiltration tube, and add Biotinylation Reaction Buffer to 500 μL.

[0155] S22. Invert the ultrafiltration tube in step S21 into a clean centrifuge tube, and centrifuge at 1000 xg for 2 minutes to collect the antibody.

[0156] S23. Wash both filter membranes 6 times each with 50 μL of Biotinylation Reaction Buffer.

[0157] S24. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and measure the antibody concentration.

[0158] S25. Measure the volume and antibody concentration: Adjust the concentration to 1 mg / mL with Biotinylation Reaction Buffer.

[0159] S26. Take out a 2 mg bottle of NHS-PEG4-Biotin from the refrigerator at -20°C. After equilibration at room temperature, reconstitute it with 383 μL of deionized water.

[0160] S27. Add the reconstituted biotin to the detection antibody according to the ratio of 100 μL of 1 mg / ml detection antibody + 3 μL of biotin.

[0161] S28. Vortex mix, spin rapidly, and incubate at room temperature for 30 minutes. After incubation, transfer the biotinylated antibody to an ultrafiltration tube.

[0162] S29. Add Biotinylation Reaction Buffer to 500 μL, centrifuge at 14000 xg for 5 minutes, discard the liquid, and repeat the washing step 4 times.

[0163] S291. Invert the filter tube into a clean centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody.

[0164] S292. Wash both filter membranes 6 times each with 50 μL of Biotinylation Reaction Buffer.

[0165] S293. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 x g for 2 minutes to collect the antibody, and detect the antibody concentration;

[0166] S294. After measuring the concentration and volume, store it in a refrigerator at 4 °C;

[0167] S3. Detection method for total BTK in Simoa;

[0168] S31. Dilute the capture antibody - magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 bead / mL;

[0169] S32. Dilute the biotinylated detection antibody with Detector Diluent Buffer to 0.3 μg·mL-1;

[0170] S33. Dilute SBG with the corresponding diluent to 0.15 nM;

[0171] S34. Dilute the recombinant BTK standard curve with cell lysate to 1, 5, 10, 50, 100, 200, 500 ng / mL;

[0172] S35. Load the reagents and samples into the corresponding sample chamber and reagent chamber positions of the simoa HD-X;

[0173] S36. All sample incubation, washing and detection steps are carried out according to the "3-step homebrew assay";

[0174] S4. Detection method for free BTK in Simoa;

[0175] S41. Dilute the capture antibody - magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 bead / mL;

[0176] S42. Dilute the biotinylated antibody with Ibrutinib-biotin, using Detector Diluent Buffer to 7 μM;

[0177] S43. Dilute SBG with the corresponding diluent to 0.15 nM;

[0178] S44. Dilute the recombinant BTK standard curve with cell lysate to 1, 5, 10, 50, 100, 200, 500 ng / mL;

[0179] S45. Load the reagents and samples into the corresponding sample chamber and reagent positions of the simoa HD-X instrument.

[0180] All sample incubation, washing, and detection steps were carried out according to the "3-step homebrew assay", and the formula for calculating receptor occupancy is: relative%occupancy=(1-(Free BTK / total BTK) / (FreeBTKpredose / total BTK predose))*100.

[0181] Example 3: Please refer to Figures 1 to 3 As shown, the present invention discloses a method for detecting the occupancy rate of the BTK receptor, and the method is the simoa detection method.

[0182] A method for detecting the occupancy rate of the BTK receptor includes the following steps:

[0183] S1. Connect the capture antibody to the magnetic beads;

[0184] S11. Take out the Bead Conjugation Buffer from the refrigerator at 2-8°C, dispense 7 ml into a 15-ml centrifuge tube, and place it on ice;

[0185] S12. Take out the vial of beads from the storage at 8°C, vortex for at least 30 seconds, and then mix on a rotary mixer for 10 minutes;

[0186] S13. Take 100 μg of the BTK capture antibody in a 30 kDa ultrafiltration tube, and add the BeadConjugationBuffer from step S1 to 500 μL;

[0187] S14. Invert the ultrafiltration tube into a clean centrifuge tube and centrifuge at 1000 xg for 2 minutes to collect the antibody;

[0188] S15. Wash both sides of the filter membrane 6 times with 50 μL of Bead Conjugation Buffer;

[0189] S16. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, detect the antibody concentration, and dilute the antibody to 0.2 mg / mL with Bead Conjugation Buffer;

[0190] S17. Vortex the antibody from step S16, place it on ice, vortex the beads for 30 seconds, and after vortexing, transfer 4.2×10 8 beads to a 2-ml EP tube;

[0191] S18. Gently open the lid, then place the EP tube in a magnetic rack, and use a pipette tip to suck off the excess liquid;

[0192] S19. Add 300 μL of Bead Wash Buffer to the EP tube in step S18, vortex for 5 seconds, rotate rapidly, open the lid, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0193] S191. Repeat step S19 to wash the magnetic beads twice.

[0194] S192. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid in the EP tube with a pipette tip.

[0195] S193. Repeat step S192 twice.

[0196] S194. Add 291 μL of Bead Conjugation Buffer, vortex for 5 seconds, rotate rapidly, and place it on ice.

[0197] S195. Add 1 mL of pre-cooled Bead Conjugation Buffer to 10 mg of EDC, vortex for 5 - 10 seconds until dissolved.

[0198] S196. Add 9 μL of pre-cooled EDC to the beads in step S203, vortex for 10 seconds, place it on a rotary mixer, and mix evenly. The mixing process is to mix evenly at 2 - 8 °C for 30 minutes.

[0199] S197. After the mixing is completed, centrifuge it rapidly in a centrifuge, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0200] S198. Add 300 μL of pre-cooled Bead Conjugation Buffer, vortex for 5 seconds, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0201] S199. Add 300 μL of the capture antibody in step S15, vortex for 10 seconds, place it on a rotary mixer, mix evenly at 8 °C for 120 minutes, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip.

[0202] S20. Add 300 μL of Bead Wash Buffer, vortex for 5 seconds, rotate rapidly, place it in a magnetic stand, and aspirate the excess liquid with a pipette tip, and repeat the washing twice.

[0203] S201. Add 300 μL of Bead Blocking Buffer, vortex for 5 seconds, place it on a rotary mixer, mix evenly at room temperature for 45 minutes, centrifuge the ultrafiltration tube at 14000 xg for 5 minutes, discard the liquid, and repeat the washing twice.

[0204] S202. After mixing is completed, rotate rapidly, place it in a magnetic stand, aspirate the excess liquid with a pipette tip, add 300 μL of Bead Wash Buffer, and vortex for 5 s;

[0205] S203. Rotate rapidly, place it in a magnetic stand, aspirate the excess liquid with a pipette tip, add 300 μL of Bead Diluent, vortex for 5 s, and repeat twice;

[0206] S204. Rotate rapidly, measure the concentration, and store the magnetic bead-conjugated capture antibody in a 4 °C refrigerator for later use;

[0207] S2. Preparation of biotinylated detection antibody;

[0208] S21. Take 130 μg of BTK detection antibody in a 30 kDa ultrafiltration tube, and add Biotinylation Reaction Buffer to 500 μL;

[0209] S22. Invert the ultrafiltration tube in step S21 into a clean centrifuge tube, and centrifuge at 1000 xg for 2 minutes to collect the antibody;

[0210] S23. Wash both filter membranes 6 times each with 50 μL of Biotinylation Reaction Buffer;

[0211] S24. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 xg for 2 minutes to collect the antibody, and measure the antibody concentration;

[0212] S25. Measure the volume and antibody concentration: Adjust the concentration to 1 mg / mL with Biotinylation Reaction Buffer;

[0213] S26. Take out a bottle of 2 mg of NHS-PEG4-Biotin from the -20 °C refrigerator, equilibrate at room temperature, and reconstitute it with 383 μL of deionized water;

[0214] S27. Add the reconstituted biotin to the detection antibody according to the ratio of 100 μL of 1 mg / ml detection antibody + 3 μL of biotin;

[0215] S28. Vortex and mix, rotate rapidly, and incubate at room temperature for 30 minutes. After the incubation is completed, transfer the biotinylated antibody to an ultrafiltration tube;

[0216] S29. Add Biotinylation Reaction Buffer to 500 μL, centrifuge at 14000 xg for 5 minutes, discard the liquid, and repeat the washing step 4 times;

[0217] S291. Invert the filter tube into a clean centrifuge tube and centrifuge at 1000 x g for 2 minutes to collect the antibody.

[0218] S292. Wash both sides of the filter membrane 6 times with 50 μL of Biotinylation Reaction Buffer.

[0219] S293. Invert the filter tube into the same centrifuge tube, centrifuge at 1000 x g for 2 minutes to collect the antibody, and measure the antibody concentration.

[0220] S294. After measuring the concentration and volume, store it in a refrigerator at 4°C.

[0221] S3. Detection method for total BTK in Simoa

[0222] S31. Dilute the capture antibody - magnetic bead conjugate to 2*10 7 bead / mL with Bead Diluent Buffer.

[0223] S32. Dilute the biotinylated detection antibody to 0.3 μg·mL-1 with Detector Diluent Buffer.

[0224] S33. Dilute SBG to 0.15 nM with the corresponding diluent.

[0225] S34. Dilute the recombinant BTK standard curve to 1, 5, 10, 50, 100, 200, 500 ng / mL with cell lysate.

[0226] S35. Load the reagents and samples into the corresponding sample and reagent compartments of simoa HD - X.

[0227] S36. All sample incubation, washing, and detection steps are carried out according to the "3 - step homebrew assay".

[0228] S4. Detection method for free BTK in Simoa

[0229] S41. Dilute the capture antibody - magnetic bead conjugate to 2*10 7 bead / mL with Bead Diluent Buffer.

[0230] S42. Dilute the biotinylated antibody with Ibrutinib - biotin to 7 μM with Detector Diluent Buffer.

[0231] S43. Dilute SBG to 0.15 nM with the corresponding diluent.

[0232] S44. Dilute the recombinant BTK standard curve with cell lysate to 1, 5, 10, 50, 100, 200, 500 ng / mL;

[0233] S45. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the simoaHD-X instrument.

[0234] All sample incubation, washing and detection steps are carried out according to the "3-step homebrew assay". The formula for calculating the receptor occupancy is: relative% occupancy = (1 - (Free BTK / total BTK) / (Free BTK pre-dose / total BTK pre-dose)) * 100.

[0235] Judgment criterion: By comparing three examples, the best one is Example 2. Therefore, Example 2 is selected as the best example. Specific changes in quantity also fall within the scope of protection of this technical solution.

[0236] Advantages of the present invention: For the detection method of the BTK receptor occupancy, the detection method prepared by the present invention first applies the ibrutinib biotin probe to the simoa platform, replacing the biotinylated detection antibody. Based on the principle of the traditional sandwich ELISA method on the simoa platform, a small molecule biotin is used as the detection probe to broaden its application range. The traditional ELISA reaction takes 6 - 10 hours, while the present invention only takes about 2 hours using the simoa technology, which can greatly shorten the detection time and improve the detection efficiency. With automatic analysis, while improving the detection throughput, it has more controllable detection precision and accuracy; the present invention first applies a small molecule biotin probe to replace the biotinylated detection antibody on the simoa platform, and first uses this technology to detect the BTK receptor occupancy, which improves the scope of use.

[0237] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the occupancy rate of BTK receptor, characterized in that, The method uses the SIMOA detection method; It includes the following steps: S1. Connect the capture antibody to the magnetic beads, and couple the capture antibody to the magnetic beads through EDC; S2. Preparation of biotinylated detection antibody, and use NHS-PEG4-Biotin to prepare the biotinylated detection antibody; S3. Detection method of total BTK by SIMOA; S31. Dilute the capture antibody and magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 beads / mL; S32. Dilute the biotinylated detection antibody with Detector Diluent Buffer to 0.3 μg·mL -1 ; S33. Dilute SBG to 0.15 nM with the corresponding diluent; S34. Dilute the recombinant BTK standard curve to 1, 5, 10, 50, 100, 200, 500 ng / mL with cell lysate; S35. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the SIMOA HD-X; S36. All sample incubation, washing and detection steps are carried out according to the "3-step homebrew assay"; S4. Detection method of free BTK by SIMOA; S41. Dilute the capture antibody and magnetic bead conjugate with Bead Diluent Buffer to 2*10 7 beads / mL; S42. Dilute the biotinylated antibody with Ibrutinib-biotin to 7 μM with the Detector Diluent Buffer; S43. Dilute SBG to 0.15 nM with the corresponding diluent; S44. Dilute the recombinant BTK standard curve to 1, 5, 10, 50, 100, 200, 500 ng / mL with cell lysate; S45. Load the reagents and samples into the corresponding sample compartments and reagent compartments of the SIMOA HD-X instrument.

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