Cell culture and detection device for rapidly screening antibodies

Abstract

The utility model discloses a cell culture and detection device for rapidly screening antibodies. The device comprises a detection capacitor chip, an accommodating structure and a culture room, the detection capacitor chip comprises a planar chip main body, a first electrode and a second electrode, and the surface of the first electrode is covered with a protein antigen layer; the accommodating structure is arranged on the first surface and is tightly combined with the non-detection area of the first electrode to form a culture cavity, and the protein antigen layer is exposed in the culture cavity; the culture chamber is arranged in the culture cavity, and at least part of the chamber wall is provided with micropores. According to the utility model, the flexible nano biochip sensing technology is utilized to realize high-precision synchronous real-time monitoring on the change of the capacitance value of the chip, and the detection sensitivity is high, so that cells secreting positive antibodies can be quickly identified and screened out; according to the pure electrical detection mode, a large amount of invalid waiting time and detection time caused by enzyme-linked immunosorbent assay in the hybridoma screening process in the traditional microscopic imaging technology are eliminated, and the antibody screening efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The utility model relates to the field of biopharmaceutical technology, especially to a cell culture and detection device for rapid screening of antibodies. BACKGROUND

[0002] Antibodies are widely used as highly specific diagnostic tools, and are studied as therapeutic drugs in aspects of viral infection, tumor, autoimmune disease, metabolic disease, nervous system disease and transplantation, etc., especially in the field of immunooncology, monoclonal antibodies and their variable fragment (ScFv), bispecific antibodies, antibody-drug conjugates (ADC), CAR-T, etc., as leading therapeutic biological drugs are increasingly important. Therefore, it is very important to quickly and effectively develop antibodies.

[0003] Currently, there are three main technologies for antibody discovery: hybridoma technology, phage display library technology and single B cell antibody preparation technology. The phage display technology has certain influence on the function and affinity of antibodies due to the limitation of library capacity and molecular diversity, and the non-natural pairing of heavy and light chains. The single B cell antibody preparation technology, especially the Beacon using microfluidic technology, directly separates and identifies isolated B cells, and performs sequencing expression and function verification in vitro, greatly shortening the cycle of antibody research and development. However, the equipment and experimental consumables for antibody screening are very expensive, and the cost of antibody screening increases. As a classic antibody screening technology, hybridoma technology is mature and has low requirements for equipment, and is widely used by some biological pharmaceutical companies. The disadvantage is that the cycle is long, and it is not easy to screen rare antibodies.

[0004] The main reason for the long cycle of antibody screening by hybridoma technology is that the culture cycle of the whole cell is too long from the fusion of positive hybridoma cells to the subcloning of monoclonal antibodies. The reason for the long cell culture cycle is that the existing detection methods, including enzyme-linked immunoassay (ELISA), cannot accurately predict whether the antibody secreted by each well can be detected. Just like in a dark box, it is impossible to predict the specific time when the antigen-antibody reaches the detection lower limit, so the overall cell culture time is lengthened to ensure that the positive antibody secreted by each well can be detected. But the only dimension that can be referred to when selecting clones is a numerical value that only reflects the number of positive antibodies combined at the end, and cannot reflect the physiological process of antigen-antibody reaction. Considering throughput and cost, the only way to reflect the antigen-antibody reaction process is to use surface plasmon resonance (SPR) or bio-layer interference (BLI) to test the affinity of the candidate clones selected at the end. These affinity tests only use optical methods to explore the biochemical process of antigen-antibody reaction of antibody binding and dissociation, and reflect the strength of the binding force of the antibody from the side, and cannot directly obtain more microscopic physicochemical properties generated by antigen-antibody reaction. In the development of antibody screening, especially antibody drugs, it is not necessarily that the higher the affinity of the antibody, the better. In order to balance the therapeutic effect, side effects, immunogenicity, and pharmacokinetics and other factors, in order to achieve the best therapeutic effect, an antibody with general affinity may also be selected. Therefore, it is very important to maintain the diversity of the screened antibodies. Therefore, the existing hybridoma screening technology has two disadvantages: one is that the antibody screening time is forced to be lengthened, and the other is that because of the single reference dimension of antibody screening, it is inevitable that some rare binding ability clones will be missed in the early screening. Limited by the single detection method, the method of enzyme-linked immunoassay (ELISA) for hybridoma antibody screening has been used until now.

[0005] Flexible nanobiochip sensing technology combines microelectronic technology and biosensing technology, and provides a new tool for biomedical research. Its working principle is based on the combination of antigen-antibody immune reaction and flexible nanobiochip technology. The flexible nanobiochip is usually fixed with proteins on the surface of the gold layer of the chip, and is used for capturing biological macromolecules such as protein receptors and antibodies combined with the proteins. In the process of antigen binding to antibody, the antigen is often surrounded by the domain of the antibody, and the antigen is completely embedded in the antibody molecule. This binding mode makes the interaction between the antigen and the antibody more closely, and at the same time causes the conformational change and site exposure of the antibody molecule, so that the binding site is more easily recognized and acted by other molecules or cells. In addition, there are various interactions between the antibody and the antigen, such as van der Waals force, charge-charge interaction, hydrogen bond and the like, which will cause the change of the spatial structure of the two. The antigen will change the conformation after binding to the antibody, and the deformation will be generated on the nanofilm. The electrophysiological signal will be quickly captured by the capacitance chip, and the change of the capacitance is displayed. The technology directly converts the biochemical and physical signals of the antigen-antibody reaction into electrical signals, and is more sensitive than the enzyme-linked immunosorbent assay (ELISA) method. Therefore, the technology has been applied in early disease diagnosis and monitoring, but has not been well applied in the field of biological drug antibody discovery, and has not been applied in the screening of cell culture hybridoma clones. Practical new content

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a cell culture and detection device for rapid screening of antibodies.

[0007] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application comprises:

[0008] The present application provides a cell culture and detection device for rapid screening of antibodies, which comprises a detection capacitance chip, a containing structure and a culture chamber.

[0009] The detection capacitance chip comprises a planar chip body, a first electrode arranged on the first surface of the chip body and a second electrode arranged on the second surface of the chip body, and the surface of the first electrode is covered with a protein antigen layer; the containing structure is arranged on the first surface and is tightly combined with the non-detection area of the detection capacitance chip to form a culture cavity, and the protein antigen layer is exposed in the culture cavity; the culture chamber is arranged in the culture cavity, and at least part of the chamber wall of the culture chamber has a micropore, which allows the antibody to pass through but blocks the cells.

[0010] Based on the above technical scheme, compared with the prior art, the present application has the following beneficial effects:

[0011] The utility model discloses utilize flexible nanometer biochip sensing technology, realize high accuracy synchronous real -time monitoring to the change of chip capacitance value, and the detection sensitivity is high, and then the cell of secreting positive antibody is identified and screened out quickly, and this kind of pure electrical detection mode eliminates the large amount of invalid waiting time and detection time caused in hybridoma screening process based on enzyme -linked immunoassay of traditional microscopic image technology, greatly improves the efficiency of antibody screening.

[0012] In addition, since the flexible nanometer biochip is used, the pure electrical sensing technology is used, and the interaction force (binding force) between the two biomolecules when the antigen and antibody are specifically combined can be accurately characterized, thereby providing a new idea for antibody screening.

[0013] The above description is only a summary of the technical scheme of the utility model, in order to enable the person skilled in the art to more clearly understand the technical means of the application, and can be implemented according to the content of the specification, the following is the preferred embodiment of the utility model and the detailed description of the drawing as follows. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the cross-sectional structure schematic diagram of cell culture and detection device provided by a typical embodiment case of the utility model;

[0015] Figure 2 It is the whole structure schematic diagram of cell culture and detection device provided by a typical embodiment case of the utility model;

[0016] Figure 3 It is the local microstructure schematic diagram of cell culture and detection device provided by a typical embodiment case of the utility model;

[0017] Mark explanation:

[0018] 1, detection capacitance chip;2, containing structure;3, culture chamber;

[0019] 10, chip main body;11, first electrode;12, second electrode;13, protein antigen layer;

[0020] 111, insulating layer;112, metal layer;131, biotin sublayer;132, avidin sublayer;133, anti-His label protein antibody sublayer;134, target protein antigen sublayer;135, first electric contact;

[0021] 20, porous soft gel;21, elastic electrode;

[0022] 31, micropore;

[0023] 40, target antibody. DETAILED DESCRIPTION

[0024] In view of the deficiencies in the prior art, the present inventors have, through long-term research and a large number of practices, come up with the technical solution of the present application. The technical solution, its implementation process and principles will be further explained as follows.

[0025] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and therefore, the protection scope of the present application is not limited by the specific embodiments disclosed below.

[0026] Moreover, relational terms such as "first" and "second", and the like, are used merely to distinguish one from another of a similar nature, without necessarily requiring or implying any such actual relationship or order between such terms.

[0027] Referring to Figures 1-3 The present application provides a cell culture and detection device for rapid screening of antibodies, which comprises a detection capacitance chip 1, a containing structure 2 and a culture chamber 3. The detection capacitance chip 1 comprises a planar chip body 10, a first electrode 11 arranged on a first surface of the chip body 10 and a second electrode 12 arranged on a second surface of the chip body 10, and a protein antigen layer 13 is arranged on the surface of the first electrode 11. The containing structure 2 is arranged on the first surface and is tightly combined with the non-detection area of the detection capacitance chip 1 to form a culture cavity, and the protein antigen layer is exposed to the culture cavity. The culture chamber 3 is arranged in the culture cavity, and at least part of the chamber wall of the culture chamber 3 has micropores 31 which allow the target antibody 40 to pass through but block the cells.

[0028] As described above, the existing cell culture method such as hybridoma needs to reserve sufficient waiting time for the cell to secrete antibodies, for example, 7 days, and then the antibody detection is carried out to confirm whether the cultured cells are the required cells. However, the time for different cells to secrete antibodies is different, for example, some cells may secrete enough antibodies for detection on the second day, but in the above prior art, in order to avoid early detection, it is still necessary to wait for a long enough time, which increases a large amount of fixed time for the cell culture period, and seriously affects the efficiency of cell culture and screening. The present application combines the flexible nanobiological chip technology and the cell culture technology for the first time, and can monitor the secretion of antibodies in real time during the culture of hybridoma cells, without waiting for a fixed period of time. Moreover, the information of the hybridoma antibody, such as affinity, antibody titer and other physical and biochemical characteristics of the antibody, can be obtained from the experimental results by detecting the change of the capacitance value. The antibody screening technology has high sensitivity, short period (in some typical implementation cases, at least 2 / 3 of the waiting time is shortened), high diversity of screened antibodies, cost saving, and has a very broad application prospect.

[0029] The above embodiments are related to the basic structural features of the cell culture and detection device provided by the present application, and in actual application, in order to realize high-throughput culture and screening, the present application is preferably implemented in combination with the existing multi-well plate, that is, in some embodiments, the first electrode 11 and the culture cavity are both multiple and one-to-one corresponding.

[0030] More specifically, in some embodiments, the multiple first electrodes 11 are arranged in an array on the first surface, and the second electrode 12 continuously covers the second surface.

[0031] Especially referring to Figure 2 In some embodiments, the accommodation structure 2 includes an integrated porous soft gel 20, which is attached to the first surface, and multiple through-holes corresponding to the positions of the detection capacitor chip 1 constitute the culture cavities.

[0032] Continuing to refer to Figure 1 In some embodiments, the culture chamber 3 is an open container, which is detachably embedded in the through-hole, with the opening facing away from the detection capacitor chip 1, and the micropore 31 is arranged at the bottom of the culture chamber 3.

[0033] As for how to realize convenient electrical measurement in the structure of a multi-well array, that is, how to form an arrayed electrical conduction test for the change in capacitance, continuing to refer to Figure 2 In some embodiments, the first electrode 11 is divided into at least a detection area and a contact area, the detection area is covered with the protein antigen layer 13, and the contact area has a first electrical contact 135; the porous soft gel 20 is provided with multiple elastic electrodes 21, the first end of the elastic electrode 21 is exposed to the surface of the porous soft gel 20, and when the accommodation structure 2 cooperates with the detection capacitor chip 1, the second end of the elastic electrode 21 is in contact with the first electrical contact 135.

[0034] Figure 2 The electrical connection mode of the ends of the multiple elastic electrodes 21 exposed to the surface of the elastic soft gel 20 is shown in , for example, the ends of the elastic electrodes 21 can be connected to the positive electrode of an external capacitance detector, and the second electrode 12 is connected to the negative electrode of the capacitance detector, thereby completing the antibody detection of the corresponding cell culture sample; in actual application, the ends of the different elastic electrodes 21 can be connected one by one, or all the ends of the elastic electrodes 21 can be connected simultaneously through a multi-channel capacitance detector, and the specific mode is not limited.

[0035] In some embodiments, preferably, in the plane, the detection area is in the area where the through hole is located, the contact area extends beyond the area where the through hole is located; the elastic electrode 21 is located outside the area where the through hole is located and extends to the surface in the thickness direction of the porous soft gel 20.

[0036] In some embodiments, continuing to refer to Figure 3 As shown, in the detection area, the first electrode 11 includes an insulating layer 111 and a metal layer 112, the insulating layer 111 has opposite first and second faces, the first face is in direct contact with the metal layer 112, and the second face faces the second electrode 12, the second electrode 12 is planar, and the insulating layer 111 has a curvature protruding towards the first face to have a gap between the second electrode 12 and the second face.

[0037] As a typical example, in the detection area, the first electrode 11 (or referred to as "upper electrode plate") is a metal-insulator composite flexible film with a thickness of 30 nanometers to 10 micrometers, which is prepared by evaporating the metal layer 112 on the suspended insulating layer 111. Because the suspended insulating layer 111 is thin and soft, after evaporating the metal layer 112, the first electrode 11, i.e. the flexible composite film, naturally has a specific protruding curvature due to the residual stress of the metal layer 112 (high-temperature evaporation environment -> cooling to room temperature); the second electrode 12 (or referred to as "lower electrode plate") is a metal conductor layer prepared on a hard insulating substrate, and because the insulating substrate is not suspended, the metal conductor of the second electrode 12 is planar.

[0038] When the first electrode 11 and the second electrode 12 are assembled together (the first electrode is arranged above the second electrode), a variable capacitor is obtained at this time (according to the structure from top to bottom along the gravity direction: culture solution environment -> metal layer 112 of flexible composite film -> insulating layer 111 of flexible composite film -> gap, i.e. air layer, due to the protruding curvature of the flexible composite film -> metal conductor layer of second electrode 12 -> insulating substrate of second electrode). When a biological reaction occurs on the metal layer 112 of the first electrode 11 (substantially on the antigen protein layer 13 above the metal layer 112), the capacitance value of the entire capacitor will change. Therefore, when a high-precision capacitance detection device (such as a high-precision LCR bridge) is connected to the two electrode plates of the cell culture and detection device, the change of the capacitance value can be monitored in real time and continuously, and then whether a biological reaction occurs on the surface of the metal conductor layer of the first electrode and what kind of biological reaction occurs can be deduced in real time and with high precision.

[0039] Of course, the specific way of detecting the above-mentioned capacitance value and the algorithm or software for analyzing the capacitance value change to infer the biological reaction are not the key of the utility model, and the skilled in the art can adopt various known or self-designed ways to achieve the above-mentioned purpose. The key technical contribution of the utility model is not how to analyze the biological reaction through the capacitance value change, but the structure of the above-mentioned cell culture and detection device, based on the above-mentioned structure, the cell culture and detection device can realize the cell culture and reflect the antibody secretion of the cell in real time through the capacitance value change.

[0040] In addition, in some embodiments, a second electrical contact is arranged on the second electrode 12 for electrical connection test of the second electrode 12.

[0041] In addition, regarding the specific microstructure of the antigen protein layer 13 and the first electrode 11, as shown in 3, in some embodiments, in the direction away from the first electrode 11, the protein antigen layer 13 comprises a biotin sublayer 131, a biotin-coupled avidin sublayer 132, an anti-His-tag protein antibody sublayer 133 and a target protein antigen sublayer with His tag 134 in sequence.

[0042] Or in some embodiments, in the direction away from the first electrode, the protein antigen layer 13 comprises a Ni-NTA sublayer and a target protein antigen sublayer with His tag in sequence.

[0043] Of course, the specific microstructure of the above-mentioned antigen protein layer and the first electrode 11 layer is not the only implementation way of the technical solution provided by the utility model, and the above-mentioned microstructure can be arranged and implemented by referring to many existing technical solutions, for example, referring to the existing technical solution of the flexible nanobiological chip proposed by the inventor of the utility model, which can be appropriately replaced or adjusted to meet the corresponding functional requirements, that is, the function of the first electrode 11 is to form a capacitor with the second electrode 12 to meet the requirement of capacitance test, and the above-mentioned preferred way cooperates with the higher sensitivity through chemical combination and physical change of the film, but if it is replaced by simple chemical combination or physical change, no matter what structure, after the antibody is combined, it can cause the capacitance change that can be detected; and the function of the antigen protein layer is to specifically combine the antibody to cause the change of the above-mentioned capacitance value.

[0044] In addition, in the utility model, the preferred way can also press the porous soft gel on the surface of the detection capacitance chip 1 through the pressing piece (not shown in the figure) to form a sealed chamber, but it is not limited to this, and the implementation means of combination such as cementing, welding and the like can achieve the purpose.

[0045] As some typical exemplary embodiments, the utility model discloses a flexible nanobiological chip for antibody screening and a 96-well cell culture plate using the flexible nanobiological chip (as a specific application of cell culture and detection device), each well of the cell culture plate is composed of three parts: a bottom detection capacitance chip 1, an upper cell culture chamber 3 (hereinafter referred to as Transwell) and a container (a multi-channel culture commonly used porous rubber ring as the containing structure 2) capable of containing cell culture fluid or sample, the three parts constitute a system compatible with cell culture and antibody detection. The detection capacitance chip 1 comprises a first electrode 11 (T-MCC) etched by ultraviolet lithography technology on the top and a second electrode 12 (B-MCC) integrated on the bottom, and the two square electrodes form a capacitor. The first electrode 11 is coated with a protein antigen layer 13 for specific binding of antibodies and capacitance detection. The T-MCC on the top contains a square insulating layer 111 with a structure recess, and a Cr / Au metal layer 112 is deposited on the surface to form the first electrode 11. An integrated rubber ring capable of loading liquid medium is connected to the top of the chip. A protruding contact is arranged beside each well to connect the elastic electrode 21 at the bottom of the cell culture plate, and the elastic electrode 21 can be connected to an external test bridge or any measurement device for measuring capacitance through a wire. The cell culture plate is matched with a Transwell chamber on the top, and the chamber is mainly used for cell culture, and the bottom is a polycarbonate PC membrane with a pore size of 0.4 μm. The membrane can prevent cells from entering the liquid environment of the lower chip detection, but cannot prevent proteins, antibodies and the like secreted by cells from entering the lower detection environment, so as to perform real-time detection.

[0046] The Transwell is detachable, and when used for real-time monitoring of cultured cells, it is placed above the detection capacitance chip. When not monitoring, it can be adapted to other cell culture plates without chips for cell culture. When only detecting supernatant samples, the Transwell component does not need to be placed on the chip, and the lower detection structure can also realize multi-channel detection.

[0047] Using the above cell culture and detection device, real-time antibody detection can be realized during cell culture, and when specific antibody secretion is detected, the cells in the culture window can be determined or tentatively determined as target cells. In addition, the change of capacitance can be continuously observed, which can reflect the various abilities of cells to analyze antibodies, and facilitate comprehensive judgment.

[0048] Specific cell culture methods are as follows:

[0049] 1. Preparation before cell plating:

[0050] Transwell chamber is mainly used for cell culture, coated with recombinant human fibronectin molecule rhFn (100 μg / mL) or poly-D-lysine PDL (100 μg / mL) to promote cell adhesion. The specific operation is to drop recombinant human fibronectin molecule rhFn (100 μg / mL) or poly-D-lysine PDL (100 μg / mL) on the bottom of the polycarbonate membrane, so that it is evenly covered on the membrane, incubated at room temperature for 1 hour or at 4℃ overnight, after the liquid is absorbed, it is placed in a clean bench for 15 minutes, and the remaining liquid is dried. Before inoculating cells, wash once with basal medium for standby.

[0051] 2. Chip pretreatment: fix the protein antigen on the gold layer surface of the chip to functionalize it:

[0052] First, wash the surface of the first electrode 11 with absolute ethanol, add Biotin-SAM (0.1 mmol / L), incubate at room temperature for 1 hour, then wash with absolute ethanol and sterile water, dry, then add streptavidin SA solution (100 μg / mL), incubate at room temperature for 30 minutes, then wash with sterile phosphate buffered saline PBS, dry, then add 1 μg / mL of biotinylated Anti-His tag antibody, incubate at room temperature for 30 minutes, then wash with phosphate buffered saline PBS, dry, then add 1 μg / mL of sterile protein antigen with His tag, incubate at room temperature for 30 minutes, and finally wash with phosphate buffered saline PBS and dry for standby.

[0053] Alternatively, the gold surface of the first electrode 11 is sterilized with an ultraviolet lamp for 1 hour, then washed with piranha solution (concentrated sulfuric acid solution mixed with 30% hydrogen peroxide solution at a volume ratio of 3:1), then added NTA-SAM (0.2 mmol / L), incubated at room temperature overnight, then washed with absolute ethanol and sterile water, dried, then added sterile 40 mmol / L Ni S O4 solution, incubated at room temperature for 5 minutes, then washed with 10 mmol / L HEPES buffer solution (pH 7.5) containing 0.05% TWEEN 20 (0.22 μm filtered and sterilized), dried, then added 1 μg / mL of sterile protein antigen with His tag, incubated at room temperature for 30 minutes, then washed with 10 mmol / L HEPES buffer solution (pH 7.5) containing 0.05% TWEEN 20, dried, and standby.

[0054] 3. Screening method of hybridoma antibody:

[0055] On the first day, the spleen cells of the immunized mouse and the mouse myeloma cells SP2 / 0 are fused, and the density of 5×10 4 / well is plated on the bottom of the Transwell of the cell culture plate, and the HAT screening medium is used for screening of the fusion clones.

[0056] On the 4th day, the Transwell cells were changed to HT medium, first washed 3 times in a culture plate containing 1x PBS, and then transferred to a culture plate containing HT medium for culture.

[0057] On the 5th day, 50 μL of supernatant from the fusion primary screening, secondary screening, and first subcloning was used for hybridoma positive clone screening using a culture room-free flexible nanobiochip. The results were obtained after 20 minutes of screening per chip. Note that the cell culture medium should be replenished in time after sampling to prevent cell death due to dehydration. Positive clones with good affinity were selected for subcloning, and were seeded onto Transwell in a cell culture plate containing a detection capacitance chip using gradient dilution. The medium used for the first subcloning was HT medium, and the volume was 50 μL.

[0058] On the 6th day, primary screening and second subcloning: due to the use of micro-culture and high-sensitivity detection by capacitance, the antibody secreted by the cells after one day of culture can be detected by the chip. Therefore, the second subcloning can be performed on the 2nd day of subcloning. The second subcloning used the limited dilution method to pick out clones with fewer and positive clones. At this time, it is estimated that the clone spheres have not yet formed, and there are generally between 2-10 clones. At this time, the clones were plated at a density of 1 per well. The medium used for the second subcloning was hybridoma culture medium SFM, and the volume was 50 μL.

[0059] On the 7th-10th day, secondary primary screening and cell expansion: according to the results of the 2nd subcloning, the hybridoma cells were expanded, one part was used for sequencing, and the remaining part was cultured and then strain-fixed.

[0060] This can save at least 2 / 3 of the time cost on the original basis, and the quality of the screened antibodies is higher.

[0061] 4. Data analysis of real-time monitored capacitance values:

[0062] The hybridoma cells are monitored for 16 hours, and the cell supernatant is detected for 20 minutes. The antibody in the culture supernatant binds to the protein antigen on the chip, and the force generated and the amount of antibody binding to the protein antigen affect the capacitance of the biochip. There are four parameters for result analysis. One is the peak value of the capacitance: C(Peak), which is the capacitance value when the antigen on the chip binds to the antibody to saturation, and indirectly reflects the titer of the antibody; two is the change value of the capacitance: ΔC, which reflects the combined result of the interaction force between the antigen and the antibody and the amount of antibody binding; three is the change rate of the capacitance: K, which indirectly reflects the affinity of the antibody under the condition that the hybridoma cells secrete the antibody at a consistent rate; and four is the time required to reach equilibrium: T, which can be compared with the change slope K. Generally, the greater the K, the smaller the T. If the antibody is negative, a straight line is basically presented.

[0063] 5. Antibody detection:

[0064] The antibody screened by the flexible nanobiochip is subjected to functional detection to verify the affinity and application of the antibody. After sequencing, the heavy and light chain sequences of the antibody are obtained, a plasmid is synthesized, PEI or electroporation is used to transfect cells to express the antibody, and then the antibody is subjected to affinity purification, and the purified antibody is subjected to antibody function detection.

[0065] The technical solutions of the utility model are further described in detail below by means of a plurality of embodiments and in combination with the drawings. However, the selected embodiments are only used for describing the utility model, and do not limit the scope of the utility model.

[0066] Embodiment 1: Preparation of the flexible nanobiochip

[0067] The flexible nanobiochip is prepared according to the Chinese invention patent CN107192747A "A variable capacitance type micro-nano bio-detection chip and a processing method thereof".

[0068] Embodiment 2: Protein fixation of the chip

[0069] The top chip is first cleaned with ethanol, Biotin-SAM (0.1 mmol / L) is added, and incubation is performed at room temperature for 1 hour. After cleaning with ethanol and sterile water and air drying, streptavidin SA solution (100 μg / mL) is added, and incubation is performed at room temperature for 0.5 hour. After cleaning with phosphate buffer PBS and air drying, biotinylated Anti-His tag antibody is added, the concentration of the antibody is 1 μg / mL, the volume is 50 μL, and incubation is performed at room temperature for 0.5 hour. After cleaning with phosphate buffer PBS and air drying, recombinant human killer cell lectin-like receptor G1 protein hKLRG1-His tag is added, the concentration of the protein is 1 μg / mL, the volume is 50 μL, and incubation is performed at room temperature for 0.5 hour. Finally, the chip is cleaned with phosphate buffer PBS and air dried for standby use.

[0070] Example 3: Immunization of mice and obtaining of mouse spleen cells

[0071] Four mice were immunized with hKLRG1-mFc protein using classical immunization technology. The first immunization was performed with 100 μg of protein emulsified with Freund's complete adjuvant in equal volume. The inguinal lymph nodes of Bal / c mice were immunized, and the subsequent immunization was performed every two weeks with 50 μg of protein emulsified with Freund's incomplete adjuvant in equal volume. After three immunizations, the antibody was detected by collecting blood from the inner canthus vein. After blood collection, centrifugation was performed, and the clear serum was transferred to a 0.5 mL EP tube. The serum was diluted at a ratio of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, and 1:729000. The diluted serum was detected by ELISA and the method of the above-mentioned embodiment (selecting the first three concentrations) of the capacitive biochip. It was found that the OD450 value and the change value of the capacitance value had a statistical difference (P<0.05), and the results of ELISA and the capacitive biochip were consistent. The mouse with a higher titer was selected, and the spleen cells were isolated by dissection.

[0072] Example 4: Preparation and screening of hybridoma antibodies

[0073] The isolated mouse spleen cells were counted, mixed with mouse myeloma cells SP2 / 0 at a ratio of 2:1 using an electrofusion buffer, and cell fusion was performed using a BTX ECM200 1 electrofusion instrument. The parameters for electrofusion were as follows: alternating parameters, high-frequency wave voltage of cell column was 70 V, pulse time was 30 s, post-fusion voltage was 7 V, and post-fusion time was 3 s; direct current parameters: direct current voltage for electroporation was 1000 V, time was 40 μs, and pulse number was 1. After fusion, the cells were left to stand for 10 minutes, and then diluted to a certain density (10 4 / well) using HAT complete medium, and uniformly plated on a 96-well plate. After 4 days of culture, the original culture medium was discarded and replaced with HT complete medium. After 1 day of culture, the cell supernatant was detected. The bridge was opened and preheated, and then the supernatant was transferred to a 96-well detection plate, 50 μL per well, and capacitive monitoring was performed for 20 minutes.

[0074] The positive clones screened by the detection capacitor chip were subcloned. The first subcloning used gradient dilution method. The specific operation process was as follows: the cells in the positive well were resuspended with 200 μL of culture medium, added to a sample groove containing 2.3 mL of HT medium, mixed, and then added to the last 4 columns of a 96-well plate, then added to the middle 4 columns, and then added to the front 4 columns. All clones were added to the Transwell cell culture chamber. Before plating, the bridge was opened and allowed to run for 1 minute. After adding, the culture plate was placed in a 37°C cell incubator for continuous observation. After 24 hours, the second subcloning was performed according to the results. The second subcloning used hybridoma culture medium and was plated at 1 / well. The positive single clone can be screened 24 hours after plating. At this time, the single clone was amplified, and after amplification to a certain amount, it was frozen (if sequencing is required, the clone can be collected to extract RNA for sequencing).

[0075] In the antibody screening process, the antibodies produced by the cells bind to the protein or cell antigen at the bottom, and the force and the number of antibodies binding to the protein antigen will affect the capacitance of the biochip. There are four parameters that can be used for result analysis. One is the peak value of the capacitance: C(Peak), which represents the capacitance value reached when the antigen on the chip binds to the antibody to saturation, which indirectly reflects the titer of the antibody; two is the change value of the capacitance: AC, which reflects the combined result of the force between the antigen and the antibody and the number of antibody binding; three is the change rate of the capacitance: K, which considers the case where the hybridoma cells secrete antibodies at a consistent rate, and indirectly reflects the affinity of the antibody. The time required to reach equilibrium: T, this parameter can be compared with the change slope K, generally the larger the K, the smaller the T. If the secreted antibody is negative, it basically presents a straight line. These parameters provide a new way of thinking for multi-dimensional screening of antibodies.

[0076] Example 5: Evaluation of antibodies with different affinities at the same concentration using the system, comparison of antibody screening parameters for reference, statistical analysis:

[0077] Experimental scheme: Different affinity antibodies screened by SPR technology were added to the capacitance biochip according to 3 different concentrations, and the real-time monitoring results were analyzed. The consistency of the data was compared. The specific operation was as follows: antibodies: Ab1 (Kd = 2 x 10 -11 ), Ab2 (Kd = 7 x 10 -10 ), Ab3 (Kd = 2 x 10 -9), the concentration of the antibody: 0.1 μg / mL, 1 μg / mL, 5 μg / mL. The amount of antigen coated on the chip: 1 μg / mL, 50 μL. According to the concentration, three groups of experiments are divided, and the chip monitors the capacitance change after adding three antibodies under each concentration in real time. In addition, two independent experiments are added: Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, also with the same concentration gradient. The experimental data is statistically analyzed by Prim8. The results show that the affinity and concentration can cause the capacitance of the chip. At the same concentration, the affinity and the reaction parameter K present a positive correlation (P<0.05).

[0078] Example 6: Quality verification of the screened antibody - immunocytochemical staining (ICC) and DAB staining

[0079] The utility model adopts the screened antibody 8B6 immunocytochemical staining ICC and DAB staining to verify the specificity of the antibody

[0080] Immunocytochemical staining ICC: the circular small wave sheet is treated with 100 μg / mL of polylysine PDL, and the cells 293T-Hklrg1 are spread on the polylysine treated circular small glass sheet at a concentration of 4×10 5 / mL. After the cells are cultured for 2-3 days and the cells are completely expanded, the circular cover glass with the cells is taken out with tweezers and placed in a new 24-well plate. Wash with PBST liquid (containing 0.1% Tween 20) for 3 times, 5 minutes each time. Add 4% polyformaldehyde, and fix at room temperature for 10 minutes. After the polyformaldehyde is absorbed, 5% goat serum is added and blocked at room temperature for 1 hour. Discard the serum, add the primary antibody diluted with 5% goat serum: 8B6 antibody (10 μg / mL x 0.2 mL / glass sheet), put it into the glass box, and incubate overnight in the refrigerator at 4°C. Take out the glass box, preheat at room temperature for 30 minutes, then wash with PBST liquid, and then add the fluorescent secondary antibody mixture diluted with goat serum: goat anti-mouse Alex488 (1:2000), incubate at room temperature for 1 hour. After washing with PBST liquid, add 1x DAPI nuclear staining (200 μL / glass sheet), and incubate at room temperature for 1 minute. After washing with ddH2O, seal the glass sheet with a fluorescence quenching containing sealing agent, and place it in a dark box. Observe and take pictures under a fluorescence microscope.

[0081] DAB staining: the circular small wave sheet is treated with 100 μg / mL of polylysine PDL, and the cells 293T-Hklrg1 are spread on the polylysine treated circular small glass sheet at a concentration of 4×10 5 / mL concentration on polylysine coated round glass slides and spread apart. After the cells were cultured for 2-3 days in a fully developed state, the cell culture round coverslips were taken out with tweezers and placed in a new 24-well plate. Washed with PBST solution (containing 0.1% Tween 20) for 3 times, 5 minutes each time. Add 4% polyformaldehyde, fix for 10 minutes at room temperature. Polyformaldehyde is absorbed, 5% goat serum is added to block for 1 hour at room temperature. Discard the serum, add the first antibody diluted with 5% goat serum: 8B6 monoclonal antibody (10 μg / mL x 0.2 mL / slide), put it into the slide box, incubate overnight in the refrigerator at 4°C. Take out the slide box, preheat for 30 minutes at room temperature, then wash with PBST solution and add the second antibody diluted with goat serum: goat anti-mouse IgG-HRP (Ab6789, 1:2000, lot#GR3282513-4). Incubate for 1 hour at room temperature. Wash with PBST solution and add DAB Substrate kit (Abeam, ab64238), observe and incubate for 1-5 minutes at room temperature. The positive reference presents brownish yellow. Add Hematoxylin staining solution (Shanghai Shenguo, E607317-0100, lot#H603FA0002) and react for 0.5-1 minute at room temperature, wash with ddH2O for 3 times, incubate for 1 minute at room temperature in the dark. Dehydrate the slides by gradient, drop 95% alcohol into the slides, dry, drop 100% alcohol, dry again, and put into 100% alcohol. Drop the resin onto the glass slide for mounting, and observe and take pictures under the fluorescence microscope. The experimental results show that the antibody 8B6 screened by the capacitive biochip is strongly positive on the membrane of the stable cell line 293T-Hklrg1.

[0082] Based on the above implementation cases, it can be clear that the embodiments of the present application provide a cell culture and detection device for rapid screening of antibodies. The device comprises a detection capacitive chip, a containing structure, and a culture chamber. The detection capacitive chip is composed of an upper chip (i.e. a first electrode) aligned on a lower chip (i.e. a second electrode). The first electrode is a composite film of a flexible metal conductor and an insulating material, and a protein antigen layer is covered on the metal conductor layer. The containing structure refers to the combination of a sealing gasket layer with a certain thickness (a plurality of holes exist on the layer) and the planar part (non-detection area) of the first electrode, forming a plurality of culture cavities capable of containing a certain amount of liquid. The detection area of the first electrode covered with the protein antibody layer will be exposed to the center of each culture cavity. The cell culture chamber is placed in the culture cavity, and the bottom is above the upper chip (i.e. the first electrode) of the detection capacitive chip. The bottom and part of the chamber wall have micropores (pore size is smaller than the cell, the cell cannot pass through, but the antibody secreted by the cell can penetrate and specifically adsorb with the protein antigen on the first electrode of the lower detection capacitive chip).

[0083] The utility model discloses a flexible nanometer biochip sensing technology is utilized to realize high-precision synchronous real-time monitoring to the change situation of multiple flexible nanometer biochip capacitance, and the detection sensitivity is high, and then the cell of secreting positive antibody is quickly identified and screened out. This pure electrical detection mode eliminates the large amount of invalid waiting time and detection time caused by enzyme-linked immunoassay based on hybridoma screening process in traditional microscopic image technology, and greatly improves the efficiency of antibody screening.

[0084] In addition, since the flexible nanometer biochip, a brand-new pure electrical sensing technology, is used, the size of the interaction force (binding force) between the two biomolecules when the antigen and antibody specifically combine can also be characterized with high precision, providing a new idea for antibody screening.

[0085] It should be understood that the above embodiments are only for illustrating the technical concept and characteristics of the utility model, and the purpose is to enable those skilled in the art to understand the content of the utility model and implement it, and cannot limit the protection scope of the utility model. Any equivalent change or modification according to the spirit and essence of the utility model should be covered within the protection scope of the utility model.

Claims

1. A cell culture and detection device for rapid antibody screening, characterized in that, This includes the detection capacitor chip, the accumulator structure, and the culture chamber; The detection capacitor chip includes a planar chip body, a first electrode disposed on a first side of the chip body, and a second electrode disposed on a second side of the chip body, wherein the surface of the first electrode is covered with a protein antigen layer; the accommodating structure is disposed on the first side and tightly combined with the non-detection area of ​​the detection capacitor chip to form a culture chamber, wherein the protein antigen layer is exposed in the culture chamber; the culture chamber is disposed in the culture chamber, and at least a portion of the chamber wall of the culture chamber has micropores, wherein the micropores allow antibodies to pass through but block cells from passing through.

2. The cell culture and detection device according to claim 1, characterized in that, There are multiple first electrodes and culture chambers, and they correspond one-to-one.

3. The cell culture and detection device according to claim 2, characterized in that, Multiple first electrodes are arrayed on the first surface, and second electrodes continuously cover the second surface.

4. The cell culture and detection device according to claim 3, characterized in that, The accommodating structure shown includes an integrated porous soft gel, which is attached to the first surface, and multiple through holes corresponding to the positions of the detection capacitor chip form the culture chamber.

5. The cell culture and detection device according to claim 4, characterized in that, The culture chamber is an open container that is detachably embedded in the through hole, with the opening facing away from the detection capacitor chip, and the micropores are located at the bottom of the culture chamber.

6. The cell culture and detection device according to claim 4, characterized in that, The first electrode is divided into at least a detection area and a contact area, the detection area is covered with the protein antigen layer, and the contact area has a first electrical contact. The porous soft gel is provided with a plurality of elastic electrodes. The first end of the elastic electrode is exposed on the surface of the porous soft gel, and when the accommodating structure is engaged with the detection capacitor chip, the second end of the elastic electrode abuts against the first electrical contact.

7. The cell culture and detection device according to claim 6, characterized in that, On a plane, the detection area is located in the area where the through hole is located, and the contact area extends beyond the area where the through hole is located; the elastic electrode is located outside the area where the through hole is located and extends to the surface along the thickness direction of the porous soft colloid.

8. The cell culture and detection device according to claim 7, characterized in that, In the detection area, the first electrode includes an insulating layer and a metal layer. The insulating layer has a first side and a second side facing away from each other. The first side is in direct contact with the metal layer, and the second side faces the second electrode. The second electrode is planar, and the insulating layer and the metal layer form a flexible composite film with an arc protruding towards the first side, so that there is a gap between the second electrode and the second side.

9. The cell culture and detection device according to claim 3, characterized in that, The second electrode is provided with a second electrical contact for electrically connecting the second electrode to test the capacitance between the first electrode and the second electrode.

10. The cell culture and detection device according to claim 1, characterized in that, Along the direction away from the first electrode, the protein antigen layer sequentially includes a biotin sublayer, an avidin sublayer, a biotin-conjugated anti-His-tagged protein antibody sublayer, and a His-tagged target protein antigen sublayer. Alternatively, along a direction away from the first electrode, the protein antigen layer sequentially comprises a Ni-NTA sublayer and a target protein antigen sublayer with a His tag.

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