Method, equipment and medium for determining binding activity of antibody and membrane protein

Abstract

The invention provides a method, a system, equipment, a medium and a program product for determining the binding activity of an antibody and a membrane protein, and relates to the technical field of experimental detection. The method comprises the following steps: preparing a to-be-bound antibody suspension and a to-be-bound cell suspension; the to-be-combined antibody suspension passes through the first microtube at a first flow rate and enters the single flow channel, and an antibody in the to-be-combined antibody suspension is fixed in the gold film area when the to-be-combined antibody suspension flows through the single flow channel; the to-be-combined cell suspension flows at a second flow rate, passes through the first microtube, enters the single flow channel, flows through the single flow channel for a second time, and is combined with the antibody fixed in the gold membrane area, so as to obtain measured data of the binding activity of the cells in the to-be-combined cell suspension and the antibody. According to the method disclosed by the invention, the binding activity of the antibody and the cell membrane protein can be accurately measured at high flux without expression and purification of the protein.

Description

Technical Field

[0001] The present invention relates to the field of experimental detection technology, and more specifically, to a method, device, medium and program product for determining the binding activity of an antibody to a membrane protein. Background Art

[0002] SPR technology is based on the physical phenomenon of the interaction between light waves and metal electrons. When the incident light interacts with the free electrons on the metal surface, under certain conditions, the incident light wave will excite the charge evanescent wave, resulting in a significant increase in the intensity of the transmitted light. This phenomenon is called surface plasmon resonance transmission enhancement, and the incident angle at this time is called the resonance angle. The resonance angle is related to the change in the refractive index of the material on the metal surface, and the change in the refractive index is related to the material adsorbed or specifically bound to the metal surface. Therefore, changes on the surface of the metal sensitive film will cause changes in the intensity and phase of the reflected light. By detecting changes in reflected light, the dynamic process of the biochemical reaction can be detected.

[0003] The determination of antibody-membrane protein binding activity is of great significance in drug development and drug efficacy determination. In the drug development stage, determining the binding efficiency of monoclonal antibodies to target membrane proteins helps to screen candidate drugs with high affinity and high specificity, thereby accelerating the discovery and optimization of new drugs; in drug efficacy determination, by evaluating the binding activity of monoclonal antibody drugs of different concentrations and categories, key pharmacodynamic parameters can be provided to help determine the optimal dose and dosing regimen, thereby improving the clinical efficacy of drugs. At the same time, the concentration and dose of the antibody will also affect the binding effect and the accuracy of the determination of binding activity. Traditional methods usually require protein expression and purification, which is not only complex and time-consuming, but may also cause conformational changes and activity loss of some membrane proteins. Therefore, accurate and high-throughput determination of the specific binding activity of antibodies to cell membrane proteins has important application value and research significance. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method, device, medium and program product for determining the binding activity of an antibody to a membrane protein; the method of the present invention can accurately and high-throughput determine the specific binding activity of an antibody to a cell membrane protein without the need for protein expression and purification.

[0005] The first aspect of the present application discloses a method for determining the binding activity of an antibody to a membrane protein, the method comprising: S1, preparing an antibody suspension to be bound and a cell suspension to be bound;

[0006] S2, the antibody suspension to be bound passes through the first microtube into the single flow channel at a first flow rate, the antibodies in the antibody suspension to be bound are fixed on the gold film region when flowing through the single flow channel, and the antibody suspension to be bound flows along the flow channel into the second microtube;

[0007] S3, the cell suspension to be bound flows through the first microtube at a second flow rate into a single flow channel, flows through the single flow channel and maintains for a second time, binds to the antibody fixed on the gold film area, obtains measurement data of the binding activity of cells in the cell suspension to be bound and the antibody, and the cell suspension to be bound flows into the second microtube along the flow channel;

[0008] The first microtube and the second microtube are respectively arranged at two ends of the flow channel; the flow channel is coupled with the gold film area, and the gold film area is coupled with the optical path of the SPR detection device.

[0009] In some embodiments, the inflow end of the first microtube and the outflow end of the second microtube are located on the upper surface of the fluid pool, and the outflow end of the first microtube and the inflow end of the second microtube are located on the lower surface of the fluid pool; the flow channel is a groove arranged on the upper surface of the fluid pool, and the outflow end of the first microtube and the inflow end of the second microtube are connected through the flow channel;

[0010] Optionally, the sizes of the first microtube, the second microtube and the flow channel are larger than the size of the cell;

[0011] Optionally, the flow channels are provided in a plurality, and the plurality of flow channels are not interconnected;

[0012] Optionally, a line connecting the center of the first microtube and the center of the second microtube in a single flow channel is used as the center line of the single flow channel, and the center lines of the plurality of flow channels are parallel to each other;

[0013] Optionally, the number of the first microtubes is consistent with the number of flow channels;

[0014] Optionally, the number of the second microtubes is consistent with that of the flow channels;

[0015] Optionally, the gold film is a substrate material used for drug fixation and cell binding, and the gold film is coupled to an SPR detection device to stimulate the SPR phenomenon.

[0016] In some embodiments, the inflow end of the first microtube and the outflow end of the second microtube are respectively provided with docking bosses, and a lumen for the flow of the suspension is left in the middle of the docking bosses; the docking bosses are pressed tightly against the second fluid control submodule;

[0017] Optionally, the surface of the gold film region includes a coupling layer containing carboxyl groups, and the antibody undergoes a condensation reaction with the carboxyl groups, and the antibody is fixed to the gold film region;

[0018] Optionally, the outflow end of the first microtube, the inflow end of the second microtube and the flow channel are in close contact with the gold film area;

[0019] Optionally, the docking boss is a cylinder;

[0020] Optionally, the second fluid control submodule is configured as a pressure block.

[0021] In some embodiments, the antibody suspension to be combined is an antibody suspension of different concentrations;

[0022] Optionally, the different concentrations are gradient concentrations, and the starting concentration is used as the reference concentration to establish a continuous dilution solution at point B;

[0023] Optionally, the S2 specifically includes:

[0024] S201, determining deionized water and a suspension of an antibody to be bound comprising a serial dilution of point B;

[0025] S202, the deionized water and the continuous dilution solution at point B are sequentially passed through the first microtube and B+1 flow channels at a first flow rate and maintained for a first time, the antibody is fixed to the gold film area, and then flows out of the second microtube along the flow channel;

[0026] Optionally, the point B serial dilution is established by consecutive A-fold serial dilutions;

[0027] Optionally, A is a positive number not equal to 1.

[0028] In some embodiments, S3 specifically includes: passing the cell suspension to be bound through the second microtube and B+1 flow channels in sequence at a second flow rate and maintaining for a second time, so that the cells in the cell suspension to be bound are bound to the antibodies in the gold film region, and obtaining the measurement data of the binding activity between the cells and the antibodies;

[0029] Optionally, the second time is greater than or equal to 3 minutes, preferably 5 minutes;

[0030] Optionally, the second flow rate is preferably 10 μL / min;

[0031] Optionally, after S3, the method further includes:

[0032] S4, obtaining cell count data of the cells in the cell suspension to be bound and the antibodies in the antibody suspension to be bound measured by a microscope;

[0033] S5, using the cell counting data as a control, establishing a model between the measurement data and the cell counting data, and calculating the specific binding activity between the cell membrane protein and the antibody.

[0034] In some embodiments, the interior of the second fluid control submodule is provided with a first channel connected to the first microtube, and a second channel connected to the second microtube; the first fluid control submodule is provided at the upper end of the second fluid control submodule, and the first fluid control submodule includes a first connecting tube connected to the first microtube, and a second connecting tube connected to the second microtube; the suspension sequentially passes through the first connecting tube, the first channel, the first microtube, the flow channel, the second microtube, the second channel, the second connecting tube, and enters other structures through the second connecting tube;

[0035] Optionally, the first connecting tube and the second connecting tube are both connected to the multi-channel injection pump. The first fluid control submodule is a pipeline connected to the multi-channel injection pump, and its inner diameter is similar to that of the microtube to achieve a relatively stable solution flow).

[0036] In some embodiments, the SPR detection device is used to detect the binding of cells and proteins on the surface of the gold film to obtain a detection result;

[0037] Optionally, the gold film is arranged on the upper end of the detection chip, and the detection chip is a pre-processed detection chip;

[0038] Optionally, the pretreatment steps include: washing, drug coating, and carboxyl activation treatment;

[0039] Optionally, the detection chip further comprises a prism coupled to the optical path of the SPR detection device, and incident light is reflected after irradiating the prism at a certain incident angle, thereby stimulating the SPR phenomenon.

[0040] The third aspect of the present application discloses a computer device, which includes: a memory and a processor; the memory is used to store a computer program; and the processor executes the computer program to implement the steps of the above method.

[0041] A fourth aspect of the present application discloses a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the steps of the above method when executed by a processor.

[0042] A fifth aspect of the present application discloses a computer program product, including a computer program, which implements the steps of the above method when executed by a processor.

[0043] This application has the following beneficial effects:

[0044] 1. The present application innovatively discloses a method for determining the specific binding activity of monoclonal antibody drugs with receptors located on the cell membrane. The method is based on the in-situ real-time determination of the binding process of intact cells and antibodies fixed on the SPR chip substrate, which can achieve the determination of the specific binding activity of monoclonal antibodies and cell membrane receptor proteins, effectively improving the determination efficiency and accuracy, avoiding the expression and purification of membrane proteins, and possible structural changes and damage. Specifically, the binding process of specific binding receptors and monoclonal antibodies in cell membrane proteins is based on intact cells, without the need to destroy the membrane structure and its membrane proteins, ensuring the normal physiological activity of membrane proteins and improving the validity of the determination data. Then, in the embodiment of the present invention, the SPR method is used to detect the binding process of cells and antibodies, which has the characteristics of real-time and label-free, and is conducive to maintaining the biological activity of proteins and the effectiveness of detection effects. It is easy to understand that the embodiment of the present invention can simultaneously determine the binding activity of multiple different drugs or the same drug under different concentration conditions by means of a multi-channel microfluidic chip, and improve the detection efficiency by high-throughput detection means.

[0045] 2. The present application innovatively discloses a device for measuring the binding activity of antibodies and membrane proteins. By optimizing and improving the fluid pool, it effectively solves the current situation that traditional methods and devices cannot measure the binding status in real time when measuring the binding ability of membrane proteins and antibodies (the main feature of traditional equipment is that the membrane protein needs to be expressed and purified before the binding ability of membrane proteins and antibodies can be measured; another traditional method is to use endpoint methods such as ELISA, that is, to test the endpoint results of cell-monoclonal antibody binding (using the depth of color to represent different binding abilities), but it cannot measure the binding status in real time). It realizes real-time and in situ measurement of the binding activity of cell membrane proteins and antibody drugs, without the need for membrane protein expression and purification, and directly uses cells for measurement, avoiding conformational changes and activity loss of membrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0047] Figure 1 is a schematic diagram of a method flow chart provided by the first aspect of an embodiment of the present invention;

[0048] Figure 2 Schematic diagram of a system for measuring the binding activity of an antibody to a membrane protein provided in the second aspect of an embodiment of the present invention;

[0049] Figure 3is a schematic diagram of a computer device provided by an embodiment of the present invention;

[0050] Figure 4 is a schematic diagram of the architecture of an exemplary computing device provided by an embodiment of the present invention;

[0051] Figure 5 is a schematic diagram of a storage medium provided by an embodiment of the present invention;

[0052] Figure 6 is a schematic diagram of the overall structure of a microfluidic module provided in an embodiment of the present invention;

[0053] Figure 7 is a schematic diagram of the structure of a microfluidic chip provided by an embodiment of the present invention;

[0054] Figure 8 It is a schematic diagram of the combination of cells and monoclonal antibodies on the surface of the SPR detection chip provided by an embodiment of the present invention;

[0055] Fig. 9 It is a schematic diagram of the refractive index change of measuring the cell binding process when the refractive index is 52°, 54° and 56° provided by an embodiment of the present invention;

[0056] In the figure, 30, detection chip; 31, first fluid control submodule; 32, second fluid control submodule; 33, gold film; 40, fluid pool; 41, docking boss; 421, first microtube; 422, second microtube; 43, flow channel. DETAILED DESCRIPTION

[0057] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0058] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0060] Glossary:

[0061] Surface plasmon resonance (SPR): It is a physical optical phenomenon. When a laser of a specific wavelength is incident on the gold film 33 at a specific angle, the free electrons of the gold film 33 will couple with the evanescent wave at the solid-liquid interface on the other side of the gold film 33, which will cause the intensity and phase of the reflected light corresponding to the incident light to change. The refractive index of the solid-liquid interface between the gold film 33 and the solution will affect the properties of the reflected light. Therefore, the refractive index information of the solid-liquid interface can be obtained by solving the information related to the reflected light. The detection method based on the SPR principle can detect the dynamic binding process of antibodies and cell membrane protein receptors near the gold film 33 in a label-free and real-time manner, and obtain accurate and sensitive binding and dissociation curves.

[0062] Figure 1 1 is a flow chart of a method for determining the binding activity of an antibody to a membrane protein provided by an embodiment of the present invention. Specifically, the method comprises the following steps:

[0063] S1: preparing antibody suspension and cell suspension to be bound;

[0064] In some embodiments, the antibody suspension to be bound is an antibody suspension of different concentrations; the different concentrations are gradient concentrations, and the starting concentration is used as the base concentration to establish a continuous dilution at point B; optionally, the starting concentration is 0.956 mg / mL; optionally, the range of B is a natural number greater than 2, preferably 12; optionally, the volume of the dilution at each concentration is C; C is preferably 500 μL.

[0065] S2: the antibody suspension to be bound passes through the first microtube 421 at a first flow rate and enters the single flow channel 43. When flowing through the single flow channel 43, the antibodies in the antibody suspension to be bound are fixed in the gold film 33 area, and the antibody suspension to be bound flows along the flow channel 43 into the second microtube 422;

[0066] In some embodiments, S2 specifically includes:

[0067] S201, determining deionized water and a suspension of an antibody to be bound comprising a serial dilution of point B;

[0068] S202, the deionized water and the continuous dilution solution at point B are sequentially passed through the first microtube 421 and B+1 flow channels 43 at a first flow rate and maintained for a first time, the antibody is fixed to the gold film 33 area, and then flows out from the second microtube 422 along the flow channel 43; the purpose of maintaining the fixed time is to ensure that the amino group on the antibody and the carboxyl group of the self-assembled layer undergo a condensation reaction. At this point, the carboxyl group that is not bound to the antibody protein is blocked, and a 1mg / ml BSA solution is used for blocking;

[0069] Optionally, the point B serial dilution is established by continuous A-fold serial dilution; A is a positive number not equal to 1;

[0070] In some embodiments, the first flow rate is preferably 10 μL / min.

[0071] S3: the cell suspension to be bound flows through the first microtube 421 at a second flow rate into the single flow channel 43, flows through the single flow channel 43 and maintains for a second time, binds to the antibody fixed on the gold film 33 area, obtains the measurement data of the binding activity between the cells in the cell suspension to be bound and the antibody, and the cell suspension to be bound flows along the flow channel 43 into the second microtube 422;

[0072] In some embodiments, the first microtube 421 and the second microtube 422 are respectively disposed at two ends of the flow channel 43; the flow channel 43 is coupled to the gold film 33 region, and the gold film 33 region is coupled to the optical path of the SPR detection device.

[0073] In some embodiments, the inflow end of the first microtube 421 and the outflow end of the second microtube 422 are located on the upper surface of the fluid pool 40, and the outflow end of the first microtube 421 and the inflow end of the second microtube 422 are located on the lower surface of the fluid pool 40; the flow channel 43 is a groove arranged on the upper surface of the fluid pool 40, and the outflow end of the first microtube 421 and the inflow end of the second microtube 422 are connected through the flow channel 43;

[0074] Optionally, the sizes of the first microtube 421, the second microtube 422 and the flow channel 43 are larger than the cell size; the microtube and the flow channel 43 are about one order of magnitude higher than the cell size to facilitate the effective passage of the cell and avoid blockage. The interface of the flow channel 43 is rectangular and the interface of the microtube is square.

[0075] Optionally, the flow channel 43 is provided in a plurality, and the plurality of flow channels 43 are not interconnected;

[0076] Optionally, a line connecting the center of the first microtube 421 and the center of the second microtube 422 in a single flow channel 43 is used as the center line of the single flow channel 43, and the center lines of several flow channels 43 are parallel to each other;

[0077] Optionally, the number of the first microtubes 421 is consistent with that of the flow channels 43;

[0078] Optionally, the number of the second microtubes 422 is consistent with that of the flow channels 43;

[0079] Optionally, the gold film 33 is a substrate material for drug fixation and cell binding, and the gold film 33 is coupled with an SPR detection device to stimulate the SPR phenomenon.

[0080] In some embodiments, a docking boss 41 is respectively provided at the inflow end of the first microtube 421 and the outflow end of the second microtube 422, and a tube cavity for the flow of suspension is left in the middle of the docking boss 41; the docking boss 41 is pressed tightly against the second fluid control submodule 32; the docking boss 41 is a cylinder, and since the material of the entire fluid pool 40 is PDMS (polydimethylsiloxane) polymer, the boss design is adopted in combination with the docking structure to increase the local pressure between the microtube and the second fluid control submodule 32 (external pressure block), thereby ensuring the airtightness between the fluid module and the fluid control module.

[0081] Optionally, the surface of the gold film 33 region includes a coupling layer containing carboxyl groups, and the antibody undergoes a condensation reaction with the carboxyl groups, and the antibody is fixed to the gold film 33 region;

[0082] Optionally, the outflow end of the first microtube 421, the inflow end of the second microtube 422 and the flow channel 43 are close to the gold film 33 area;

[0083] Optionally, the second fluid control submodule 32 is configured as a pressure block.

[0084] In some embodiments, S3 specifically includes: passing the cell suspension to be bound through the second microtube 422 and B+1 flow channels 43 at a second flow rate and maintaining the second time, so that the cells in the cell suspension to be bound are bound to the antibodies in the gold film 33 region, and obtaining the measurement data of the binding activity between the cells and the antibodies;

[0085] Optionally, the second time is greater than or equal to 3 minutes, preferably 5 minutes;

[0086] Optionally, the second flow rate is preferably 10 μL / min;

[0087] Optionally, after S3, the method further includes:

[0088] S4, obtaining cell count data of the cells in the cell suspension to be bound and the antibodies in the antibody suspension to be bound measured by a microscope;

[0089] S5, using the cell counting data as a control, establishing a model between the measurement data and the cell counting data, and calculating the specific binding activity between the cell membrane protein and the antibody.

[0090] In some embodiments, the cell is required to be able to express the corresponding membrane protein, and the antibody is required to specifically bind to the membrane protein, such as the application case of the combination of α4β7 protein and vedolizumab in HUT78 cells mentioned below, where the two can specifically bind to each other;

[0091] In some embodiments, the interior of the second fluid control submodule 32 is provided with a first channel connected to the first microtube 421, and a second channel connected to the second microtube 422; the first fluid control submodule 31 is provided at the upper end of the second fluid control submodule 32, and the first fluid control submodule 31 includes a first connecting tube connected to the first microtube 421, and a second connecting tube connected to the second microtube 422; the suspension sequentially passes through the first connecting tube, the first channel, the first microtube 421, the flow channel 43, the second microtube 422, the second channel, the second connecting tube, and enters the other structures through the second connecting tube; optionally, the first connecting tube and the second connecting tube are both connected to the multi-channel injection pump, that is, the other structures are multi-channel injection pumps.

[0092] In some embodiments, the SPR detection device is used to detect the binding of cells and proteins on the gold film surface to obtain a detection result, and the detection result is in the form of a gold film surface cell-cell membrane protein binding curve; the method for detecting the binding of cells and proteins on the gold film surface includes: using phase detection and dual CCD differential phase interference imaging methods to achieve real-time, in-situ detection of the binding of cells and proteins on the gold film surface; optionally, the gold film 33 is arranged on the upper end of the detection chip 30, and the detection chip 30 is a pre-treated detection chip 30; optionally, the pre-treatment steps include: cleaning, drug coating, and carboxyl activation treatment;

[0093] In some embodiments, the detection chip 30 further includes a prism coupled to the optical path of the SPR detection device. After the incident light irradiates the prism at a certain incident angle, it is reflected on the surface of the gold film to stimulate the SPR phenomenon. The gold film 33 and the prism are an integrated structure. Figure 8 The gold film 33 exists in the form of a coating on the upper surface of the prism. The prism is coupled with the optical path in the SPR excitation and detection optical path. The incident light is taken in from one side of the prism, reflected by the gold film 33, the SPR phenomenon occurs, and then emitted from the other side of the prism.

[0094] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, such as Figure 3 As shown, the device may include: one or more processors, and one or more memories; wherein the memories store computer-readable codes, and when the computer-readable codes are run by the one or more processors, the method described above may be executed.

[0095] The processor in this embodiment can be an integrated circuit chip with signal processing capabilities. The above processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components. The disclosed methods, operations and logic block diagrams in the embodiments of the present disclosure can be implemented or executed. The general-purpose processor can be a microprocessor or the processor can also be any conventional processor, etc., which can be an X86 architecture or an ARM architecture.

[0096] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0097] For example, the method or device according to the embodiment of the present disclosure may also be implemented by Figure 4 The architecture of the computing device 3000 shown in FIG. Figure 4 As shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as ROM 3030 or hard disk 3070, may store various data or files used for processing and / or communication of the method provided by the present disclosure and program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 The architecture shown is only exemplary and can be omitted according to actual needs when implementing different devices. Figure 4 One or more components of a computing device are shown.

[0098] The embodiment of the present invention also provides a computer-readable storage medium, such as Figure 5As shown, it is a schematic diagram of a storage medium provided by an embodiment of the present invention, and a computer readable instruction 4010 is stored on the computer storage medium 4020. When the computer readable instruction 4010 is executed by the processor, the method according to the embodiment of the present disclosure described with reference to the above figures can be executed. The computer readable storage medium in the embodiment of the present disclosure can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM) or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDRSDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory. It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0099] The embodiments of the present disclosure further provide a computer program product or system, including a computer program, which implements the steps of the above method when executed by a processor.

[0100] In some embodiments, this embodiment also discloses a system for determining the binding activity of an antibody to a membrane protein, such as Figure 2 As shown, the system comprises:

[0101] Solution preparation module 201, used for or configured to prepare antibody suspension to be bound and cell suspension to be bound;

[0102] The antibody suspension to be bound flow module 202 is used or configured to allow the antibody suspension to be bound to pass through the first microtube 421 at a first flow rate into the single flow channel 43, and when flowing through the single flow channel 43, the antibodies in the antibody suspension to be bound are fixed in the gold film 33 area, and the antibody suspension to be bound flows along the flow channel 43 into the second microtube 422;

[0103] The cell suspension flow module 203 to be combined is used or configured to flow the cell suspension to be combined through the first microtube 421 at a second flow rate into the single flow channel 43, flow through the single flow channel 43 and maintain for a second time, combine with the antibody fixed on the gold film 33 area, obtain the measurement data of the cell-antibody binding activity in the cell suspension to be combined, and the cell suspension to be combined flows along the flow channel 43 into the second microtube 422;

[0104] The first microtube 421 and the second microtube 422 are respectively arranged at two ends of the flow channel 43; the flow channel 43 is coupled with the gold film 33 area, and the gold film 33 area is coupled with the optical path of the SPR detection device.

[0105] In some embodiments, the system further comprises:

[0106] A control data acquisition module, used for or configured to acquire cell count data of the cells in the cell suspension to be combined with the antibodies in the antibody suspension to be combined measured by a microscope;

[0107] The binding activity calculation module is used for or configured to establish a model between the measurement data and the cell counting data using the cell counting data as a control, and calculate the specific binding activity between the cell membrane protein and the antibody.

[0108] In some embodiments, a method and apparatus for determining the specific binding activity of a monoclonal antibody to a cell membrane receptor protein are provided, which can determine the specific binding activity of a HUT-78 cell membrane protein receptor to the vitrolizumab monoclonal antibody under intact cell conditions.

[0109] In the specific implementation process, the method of binding activity determination includes five parts: chip pretreatment, antibody fixation, cell binding, binding curve determination, signal analysis and binding activity calculation:

[0110] Among them, chip pretreatment, cleaning and drug coating pretreatment of the detection chip 30 coupled with the SPR system in the device, and coating the chip used for antibody fixation and cell binding with carboxyl and activation; specifically including the following steps, a) Carboxyl coupling: The chip with a gold film 33 thickness of 32nm is rinsed with acetone, ethanol and deionized water in turn, dried with nitrogen, and immersed in 1mM11-MUA ethanol solution for 24 hours, and a coupling layer containing carboxyl groups is self-assembled on the surface of the gold film 33; b) Carboxyl activation: After the self-assembly is completed, the sensor chip is taken out, rinsed with ethanol and deionized water, and dried with nitrogen. Next, 100mMNHS and 400mMEDC solutions are mixed in equal volumes, and then covered on the chip surface for 30 minutes to activate the carboxyl groups on the self-assembled layer;

[0111] The antibody fixation method uses the microfluidic chip in the device to fix antibodies of different concentrations on the activated detection chip 30; specifically, the following steps are included: a) gradient drug configuration: the antibody is diluted 2 times starting from a concentration of 0.956 mg / mL to establish a 12-point continuous dilution solution, and each dilution solution retains about 500 μL; b) assembly: the SPR chip treated with carboxyl groups is assembled with the microfluidic module to ensure that the PDMS flow path and the SPR gold film 33 are closely fitted; c) antibody fixation: deionized water and 12-point continuous dilution solutions are respectively introduced into the flow channels 43 numbered from #1 to #13 at a flow rate of 10 μL / min, and maintained for 1 hour to ensure that the amino groups on the antibody undergo condensation reaction with the carboxyl groups of the self-assembled layer to complete the fixation of the antibody; d) carboxyl group blocking of unbound antibody protein: 1 mg / mL BSA solution is used for blocking;

[0112] Cell binding, using the first flow control submodule, the second flow control submodule and the fluid pool 40 in the flow system, the cell suspension flows through the antibody surface, and the cells in the cell suspension are specifically bound to the antibodies fixed on the detection chip 30; specifically, the following steps are included: a) using a multi-channel precision injection pump, on the SPR chip, injecting a cell suspension with a concentration of 3×10 cell / mL into the #1 to #13 flow channels 43 at a flow rate of 10 μL / mL for 5 minutes each, and injecting sterile culture fluid at the same speed for flushing; b) observing and recording the binding of cells in the suspension with antibodies of different concentrations on the surface of the glass chip in the ROI area corresponding to each SPR flow channel 43;

[0113] Signal analysis: Analyze the SPR curve obtained by the measurement, compare it with the cell count of cell-antibody binding measured by microscope, establish a mathematical model between the SPR curve and the counting results of the cell binding effect, and thus calculate the specific binding activity of the membrane protein receptor and the antibody.

[0114] Reference Figure 8 The binding of HUT-78 cells to vedolizumab is shown in Figure 8 As shown, HUT-78 cells flow into the microchannel 43 with the microfluid flow, approaching the vedolizumab fixed on the surface of the gold film 33 of the SPR sensor chip, and specifically bind to the α4β7 receptor protein on the cell membrane of the HUT-78 cells, causing the cells to be close to the surface of the gold film 33 and enter the sensitive range of the SPR method, thereby being dynamically observed by the SPR detection method.

[0115] Reference Figure 6 , 7 In this specific embodiment, a device for determining the specific binding activity of a monoclonal antibody and a cell membrane receptor protein is shown in the figure. Specifically, referring to Figure 6, the left side is a composition diagram, the right side is an exploded diagram, the left side diagram specifically includes the first fluid control submodule 31, the second fluid control submodule 32 and the detection chip 30 from top to bottom, and the right side diagram specifically includes the first fluid control submodule 31, the second fluid control submodule 32, the fluid pool 40, the gold film 33 and the detection chip 30 from top to bottom; the specific structure of the fluid pool 40 is referred to Figure 7 The first microtube 421 and the second microtube 422 are respectively located at two ends of the single flow channel 43. Figure 7 13 single flow channels 43 were drawn, Figure 7 The upper end of the middle fluid pool is the lower surface of the fluid pool. Figure 7 The lower end of the fluid pool is the upper surface of the fluid pool; In the embodiment of the present invention, the binding activity determination device includes three parts: an SPR detection module, a detection chip 30, and a flow path module:

[0116] SPR detection module, the SPR module implements the SPR detection method, and uses phase detection and dual CCD differential phase interference imaging to achieve real-time, in-situ detection of cell-protein binding on the gold film surface, and forms a cell membrane protein-antibody binding curve; SPR detection module, implements the SPR detection method, and uses phase detection and dual CCD differential imaging to achieve real-time, in-situ detection of cell-protein binding on the gold film surface. Among them, when the detection module excites the SPR phenomenon, a 632 nanometer (nm) laser is incident on the detection chip. The incident laser within a certain range can excite the SPR phenomenon. Generally, sensitive phase detection of the refractive index can be achieved within the range of 52° to 56°. Furthermore, for the detection of the cell binding process, since the refractive index of the culture medium and the extracellular fluid is about 1.35RIU, the refractive index of the intracellular fluid is about 1.37RIU, and the refractive index of the cell membrane is about 1.46RIU, the thickness of the cell membrane is small and can be approximately ignored. Taking all factors into consideration, SPR can mainly distinguish the difference in refractive index between the intracellular fluid and the extracellular fluid. The incident angle is preferably set to 56°, which can most sensitively measure the refractive index change caused by the cell binding process. The specific refractive index change is as follows: Fig. 9 shown.

[0117] The detection chip 30 is connected to the optical path of the SPR detection module, and includes components such as a gold film 33 and a prism. The gold film 33 is a base material for drug fixation and cell binding. The prism and the gold film 33 are coupled to the optical path of the SPR module at the same time to stimulate the SPR phenomenon at a specific incident angle.

[0118] The flow path module includes a fluid control submodule and a fluid pool 40, wherein the fluid control submodule is connected to a multi-channel programmable injection pump and is connected to the fluid pool 40; the fluid pool 40 includes a docking boss 41 to ensure the airtightness of the docking with the fluid control submodule, and includes a plurality of parallel flow channels 43 to achieve the fixation of antibodies in designated areas and the binding of cells and antibodies. Antibodies or cell suspensions containing a certain concentration enter the fluid pool 40 through the fluid control submodule and enter the flow channel 43 through a microtube. The flow channel 43 is tightly coupled with the gold film 33 area of ​​the detection chip 30, and finally the microfluid flows through the SPR detection chip 30 to achieve the detection functions of antibody fixation and cell binding.

[0119] Among them, the basic principle of fluid control is to utilize the dynamic characteristics of the fluid, and through the design and control of the shape, structure, position and control method of flow channels, valves, pumps and other components, make the fluid move along a predetermined path and speed. Commonly used control parameters include flow, pressure, speed, state, etc.

[0120] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architecture, functions and operations of the systems, methods and computer program products according to various embodiments of the present disclosure. In this regard, each box in the flowchart or block diagram can represent a module, a program segment, or a part of a code, and the module, program segment, or a part of the code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in a different order from the order marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart, and the combination of boxes in the block diagram and / or flowchart can be implemented with a dedicated hardware-based system that performs a specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0121] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0122] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0123] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0124] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0125] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0126] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. It should be understood by those skilled in the art that various modifications and combinations may be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A method for determining the binding activity of an antibody to a membrane protein, It is characterized in that The method comprises: S1, preparing an antibody suspension to be bound and a cell suspension to be bound; S2, the antibody suspension to be bound passes through the first microtube into the single flow channel at a first flow rate, the antibodies in the antibody suspension to be bound are fixed on the gold film region when flowing through the single flow channel, and the antibody suspension to be bound flows along the flow channel into the second microtube; S3, the cell suspension to be bound flows through the first microtube at a second flow rate into a single flow channel, flows through the single flow channel and maintains for a second time, binds to the antibody fixed on the gold film area, obtains measurement data of the binding activity of cells in the cell suspension to be bound and the antibody, and the cell suspension to be bound flows along the flow channel into the second microtube; The first microtube and the second microtube are respectively arranged at two ends of the flow channel; the flow channel is coupled with the gold film area, and the gold film area is coupled with the optical path of the SPR detection device.

2. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that The inflow end of the first microtube and the outflow end of the second microtube are located on the upper surface of the fluid pool, and the outflow end of the first microtube and the inflow end of the second microtube are located on the lower surface of the fluid pool; the flow channel is a groove arranged on the upper surface of the fluid pool, and the outflow end of the first microtube and the inflow end of the second microtube are connected through the flow channel; Optionally, the sizes of the first microtube, the second microtube and the flow channel are larger than the size of the cell; Optionally, the flow channels are provided in a plurality, and the plurality of flow channels are not interconnected; Optionally, a line connecting the center of the first microtube and the center of the second microtube in a single flow channel is used as the center line of the single flow channel, and the center lines of the plurality of flow channels are parallel to each other; Optionally, the number of the first microtubes is consistent with that of the flow channels; Optionally, the number of the second microtubes is consistent with that of the flow channels; Optionally, the gold film is a substrate material used for drug fixation and cell binding, and the gold film is coupled to an SPR detection device to stimulate the SPR phenomenon.

3. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that The inflow end of the first microtube and the outflow end of the second microtube are respectively provided with docking bosses, and a lumen for the flow of the suspension is left in the middle of the docking bosses; the docking bosses are pressed tightly against the second fluid control submodule; Optionally, the surface of the gold film region includes a coupling layer containing carboxyl groups, and the antibody undergoes a condensation reaction with the carboxyl groups, and the antibody is fixed to the gold film region; Optionally, the outflow end of the first microtube, the inflow end of the second microtube and the flow channel are in close contact with the gold film area; Optionally, the docking boss is a cylinder; Optionally, the second fluid control submodule is configured as a pressure block.

4. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that The antibody suspensions to be combined are antibody suspensions of different concentrations; Optionally, the different concentrations are gradient concentrations, and the starting concentration is used as the reference concentration to establish a continuous dilution solution at point B; Optionally, the S2 specifically includes: S201, determining deionized water and a suspension of an antibody to be bound comprising a serial dilution of point B; S202, the deionized water and the continuous dilution solution at point B are sequentially passed through the first microtube and B+1 flow channels at a first flow rate and maintained for a first time, the antibody is fixed to the gold film area, and then flows out of the second microtube along the flow channel; Optionally, the point B serial dilution is established by consecutive A-fold serial dilutions; Optionally, A is a positive number not equal to 1.

5. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that Specifically, S3 includes: passing the cell suspension to be bound through the second microtube and B+1 flow channels in sequence at a second flow rate and maintaining the flow for a second time, so that the cells in the cell suspension to be bound are bound to the antibodies in the gold film region, and obtaining the measurement data of the binding activity between the cells and the antibodies; Optionally, the second time is greater than or equal to 3 minutes, preferably 5 minutes; Optionally, the second flow rate is preferably 10 μL / min; Optionally, after S3, the method further includes: S4, obtaining cell count data of the cells in the cell suspension to be bound and the antibodies in the antibody suspension to be bound measured by a microscope; S5, using the cell counting data as a control, establishing a model between the measurement data and the cell counting data, and calculating the specific binding activity between the cell membrane protein and the antibody.

6. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that The second fluid control submodule is provided with a first channel connected to the first microtube and a second channel connected to the second microtube inside; the first fluid control submodule is provided at the upper end of the second fluid control submodule, and the first fluid control submodule includes a first connecting tube connected to the first microtube and a second connecting tube connected to the second microtube; The suspension sequentially passes through the first connecting tube, the first channel, the first microtube, the flow channel, the second microtube, the second channel, the second connecting tube, and enters other structures through the second connecting tube; Optionally, both the first connecting tube and the second connecting tube are connected to a multi-channel injection pump.

7. The method for determining the binding activity of an antibody to a membrane protein according to claim 1, It is characterized in that The SPR detection device is used to detect the binding of cells and proteins on the surface of the gold film to obtain the detection results; Optionally, the gold film is arranged on the upper end of the detection chip, and the detection chip is a pre-processed detection chip; Optionally, the pretreatment steps include: washing, drug coating, and carboxyl activation treatment; Optionally, the detection chip further comprises a prism coupled to the optical path of the SPR detection device, and incident light is reflected after irradiating the prism at a certain incident angle, thereby stimulating the SPR phenomenon.

8. A computer device, It is characterized in that The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 1 to 7.

9. A computer-readable storage medium, It is characterized in that A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 7 are implemented.

10. A computer program product comprising a computer program, It is characterized in that When the computer program is executed by a processor, the steps of the method described in any one of claims 1 to 7 are implemented.

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