A method and device for preparing reagents for microfluidic antibody screening

Abstract

The present invention provides a method for preparing a reagent for microfluidic antibody screening, which is carried out according to the following steps: step 1, preparing corresponding reagents; step 2, pushing B cell reagents; step 3, mixing B cells with a trace amount of serum; step 4, generating screening reagents and agglutination samples; step 5, observing the agglutination state and selecting the corresponding screening reagents; step 6, extracting the screening reagents by using microfluidic technology and performing cloning culture. The preparation method and device can simultaneously mix the multi-component B cell reagents with the serum reagents on the top through the pushing mechanism at the front end, thereby providing multiple agglutination samples and screening reagents at the same time, improving the success rate of antibody production, and directly corresponding each agglutination sample to one of the screening reagents, so as to accurately judge the internal antibody production state of each screening sample, and avoid the situation where the antibody components of the agglutination sample and the screening reagent are inconsistent.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a reagent preparation method and device for microfluidic antibody screening. Background Art

[0002] During the microfluidic antibody screening process, the basic characteristics of the agglutination phenomenon will appear through the direct reaction of particulate antigens such as bacteria or cells with the corresponding antibodies. Through this phenomenon, the microfluidic technology can be used to screen and cultivate antibodies. In the prior art, after mixing serum with B cells, the agglutination phenomenon is observed in a macroscopic range. After the mixed reagent is dripped onto the slide, the slide is gently shaken and observed with the naked eye after a period of time. If a milky white agglutination block appears, it is a positive reaction; if it is still an equal emulsion, it is a negative reaction. If the result is not clear enough, the slide can be placed under a low-power microscope for observation. After confirming that agglutination has occurred, it can be judged that antibodies have been produced in this state. Therefore, the same steps are used to perform microfluidic collection of a single antibody-containing cell on the mixed reagent for subsequent cloning and cultivation. However, in this scheme, only a single reagent can be observed for agglutination at a time, and the screening efficiency is explained. At the same time, it is also impossible to avoid the difference between the screening reagent and the agglutination sample, resulting in deviations in the antibody reagent after the final screening and cultivation. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method and device for preparing reagents for microfluidic antibody screening to solve the problems raised in the above-mentioned background technology. The present invention improves the efficiency of screening, has high accuracy, and can ensure that the components of agglutination samples and screening reagents are consistent.

[0004] In order to achieve the above-mentioned purpose, the present invention is realized through the following technical scheme: a method for preparing reagents for microfluidic antibody screening, which is carried out according to the following steps: step 1, injecting B cell reagent into the pushing mechanism, injecting serum reagent into the serum reagent tube, and injecting antigen reagent into the antigen reagent tank; step 2, pushing B cell reagent into multiple mixing pipes through the front pushing mechanism; step 3, mixing B cells with a trace amount of serum through a first capillary pipe in the middle of the mixing pipe; step 4, generating screening reagents at the screening mechanism at the bottom, and generating agglutination samples in the detection component at the rear end; step 5, observing the agglutination state by naked eyes or microscope, and selecting corresponding screening reagents; step 6, extracting the screening reagents by microfluidic technology, and performing cloning culture.

[0005] Furthermore, in the step three, the B cells and a trace amount of serum are mixed and reacted inside the mixing channel by capillary action, and in the step four, the screening mechanism also extracts a small amount of screening reagent inside the screening mechanism by capillary action.

[0006] A manufacturing device used in a reagent manufacturing method for microfluidic antibody screening comprises a pushing mechanism, a screening mechanism and a detection component, wherein a plurality of mixing pipes are arranged on the top of the pushing mechanism, a serum reagent tube is installed on the top of the mixing pipe, a first injection port is arranged at one end of the serum reagent tube, the bottom end of the serum reagent tube is connected to a first capillary tube, a screening mechanism is arranged in the middle of the mixing pipe, a butt joint is arranged at the end of the mixing pipe, the butt joint is inserted into the interior of a sealing sleeve, and a detection component is installed on the side of the sealing sleeve.

[0007] Furthermore, the pushing mechanism includes a pushing tank and a B cell reagent tank, a piston rod is inserted into the top of the pushing tank, a pressure plate is connected to the top of the piston rod, an electric push rod is installed at the bottom end of the pressure plate, and a diversion pipe is connected to the bottom end of the pushing tank.

[0008] Furthermore, the interiors of the push tank and the B cell reagent tank are connected through a diversion pipe, the top of the electric push rod is fixedly connected to the bottom end of the pressure plate, the bottom end of the electric push rod is fixedly connected to the top of the push tank, and the electric push rod is connected to an external control system through wires, and each of the mixing pipes is inserted into the interior of the B cell reagent tank.

[0009] Furthermore, the screening mechanism includes a docking chamber and an antibody screening slot, the antibody screening slot is arranged at the bottom of the inner side of the docking chamber, the bottom end of the mixing pipe is connected to a second capillary, and each of the screening mechanisms is connected to the detection component at the rear end.

[0010] Furthermore, the bottom end of the second capillary is inserted into the interior of the antibody screening tank, and the inner wall of the antibody screening tank is in contact with the bottom of the second capillary.

[0011] Furthermore, the detection component includes an antigen reagent tank and a bottom plate, wherein the bottom plate is arranged at the bottom of the antigen reagent tank, and an observation glass plate is embedded in the middle of the bottom plate.

[0012] Furthermore, there are multiple detection components, each detection component is connected to the corresponding mixing pipeline part, and each antigen reagent tank is installed at the same height.

[0013] Furthermore, a docking port is provided on the top of the mixing pipe, the bottom end of the first capillary is inserted into the interior of the docking port, and a delivery pipe is connected to the top of the antigen reagent tank.

[0014] Beneficial effects of the present invention: A reagent preparation method and device for microfluidic antibody screening of the present invention include a preparation method and a device body, wherein the preparation method and the device body include a pushing mechanism, a mixing pipeline, a serum reagent tube, a first injection port, a first capillary, a screening mechanism, a docking tube, a sealing sleeve, a detection component, a delivery pipeline, an antigen reagent tank, a second injection port, a bottom plate, an observation glass plate, a second capillary, a docking chamber, an antibody screening tank, a pushing tank, a B cell reagent tank, a pressure plate, an electric push rod, a piston rod, a diversion pipeline, and a docking port.

[0015] 1. The reagent preparation method for microfluidic antibody screening can simultaneously mix the multi-component B cell reagent with the serum reagent on the top through the front-end pushing mechanism, thereby providing multiple agglutination samples and screening reagents at the same time, thereby improving the success rate of antibody production.

[0016] 2. The reagent preparation method and device for microfluidic antibody screening directly corresponds each agglutination sample to one of the screening reagents. After observing each agglutination sample, the internal antibody production status of each screening sample can be accurately determined, avoiding the situation where the antibody components in the agglutination sample and the screening reagent are inconsistent.

[0017] 3. The reagent preparation method and device for microfluidic antibody screening are equipped with sealing or anti-backflow structures at the front push mechanism, the middle screening mechanism and the rear detection component to avoid external contamination affecting the reagent and improve the purity of the reagent culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A flow chart of the preparation of a reagent for microfluidic antibody screening according to the present invention;

[0019] Figure 2 This is a schematic structural diagram of the appearance of a reagent preparation device for microfluidic antibody screening according to the present invention;

[0020] Figure 3 It is a schematic structural diagram of the detection component part of a reagent preparation device for microfluidic antibody screening of the present invention;

[0021] Figure 4 It is a schematic structural diagram of the screening mechanism of a reagent preparation device for microfluidic antibody screening of the present invention;

[0022] Figure 5 It is a structural schematic diagram of a pushing mechanism part of a reagent preparation device for microfluidic antibody screening of the present invention;

[0023] In the figure: 1. pushing mechanism; 2. mixing pipeline; 3. serum reagent tube; 4. first injection port; 5. first capillary tube; 6. screening mechanism; 7. docking tube; 8. sealing sleeve; 9. detection component; 10. delivery pipeline; 11. antigen reagent tank; 12. second injection port; 13. bottom plate; 14. observation glass plate; 15. second capillary tube; 16. docking chamber; 17. antibody screening tank; 18. pushing tank; 19. B cell reagent tank; 20. pressing plate; 21. electric push rod; 22. piston rod; 23. diversion pipeline; 24. docking port. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0025] See also Figures 1 to 5 The present invention provides a technical solution: a reagent preparation method for microfluidic antibody screening, which is carried out according to the following steps: step 1, injecting B cell reagent into the push mechanism 1, injecting serum reagent into the serum reagent tube 3, and injecting antigen reagent into the antigen reagent tank 11; step 2, pushing the B cell reagent into the interior of multiple mixing pipes 2 through the front push mechanism 1; step 3, mixing B cells with a trace amount of serum through the first capillary 5 in the middle of the mixing pipe 2; step 4, generating screening reagents at the screening mechanism 6 at the bottom, and generating agglutination samples in the detection component 9 at the rear end; step 5, observing the agglutination state by naked eyes or microscope, and selecting corresponding screening reagents; step 6, extracting the screening reagents by using microfluidic technology, and performing Cloning culture is carried out. In the step three, B cells and a trace amount of serum are mixed and reacted inside the mixing pipe 2 by capillary action. In the step four, a small amount of screening reagent is extracted from the screening mechanism 6 by capillary action. Specifically, after the B cell reagent reacts with a trace amount of serum through the action of the pushing mechanism 1, antibodies can be produced under a suitable environment. While this part of the reagent flows backward along the mixing pipe 2, a small amount of mixed reagent can be flowed into the screening mechanism 6 at the bottom along the second capillary 15 as a screening reagent, and the docking pipe 7 at the end allows a large amount of mixed reagent to enter the interior of the detection component 9 for corresponding detection, and the screening reagent inside the screening mechanism 6 is judged and taken based on the mixed reagent in the detection component 9.

[0026] The present embodiment also provides a production device used in a reagent production method for microfluidic antibody screening, comprising a pushing mechanism 1, a screening mechanism 6 and a detection component 9, wherein a plurality of mixing pipes 2 are arranged on the top of the pushing mechanism 1, a serum reagent tube 3 is installed on the top of the mixing pipe 2, a first injection port 4 is arranged at one end of the serum reagent tube 3, and the bottom end of the serum reagent tube 3 is connected to a first capillary 5, a screening mechanism 6 is arranged in the middle position of the mixing pipe 2, a butt joint pipe 7 is arranged at the end of the mixing pipe 2, the butt joint pipe 7 is inserted into the interior of a sealing sleeve 8, and a detection component 9 is installed on the side of the sealing sleeve 8, the pushing mechanism 1 comprises a pushing tank 18 and a B cell reagent tank 19, a piston rod 22 is inserted into the top of the pushing tank 18, a pressing plate 20 is connected to the top of the piston rod 22, an electric push rod 21 is installed at the bottom end of the pressing plate 20, and a diversion pipe is connected to the bottom end of the pushing tank 18 23, the interior of the push tank 18 and the B cell reagent tank 19 are kept in communication through the guide pipe 23, the top of the electric push rod 21 is fixedly connected to the bottom end of the pressure plate 20, the bottom end of the electric push rod 21 is fixedly connected to the top of the push tank 18, and the electric push rod 21 is connected to the external control system through wires, each of the mixing pipes 2 is inserted into the interior of the B cell reagent tank 19, and the multi-component B cell reagent can be mixed with the serum reagent on the top at the same time through the front-end pushing mechanism 1, thereby providing multiple agglutination samples and screening reagents at the same time, thereby improving the success rate of antibody production. Specifically, by connecting to each electric push rod 21 through an external control system, the electric push rod 21 can be controlled to pull the top pressure plate 20 downward, and the B cell reagent inside the push tank 18 and the B cell reagent tank 19 can be pushed to the top of the mixing pipe 2 through the piston action, so as to carry out the subsequent mixing process.

[0027] In this embodiment, the screening mechanism 6 includes a docking chamber 16 and an antibody screening groove 17, wherein the antibody screening groove 17 is arranged at the bottom of the inner side of the docking chamber 16, and the bottom end of the mixing pipe 2 is connected with a second capillary 15, and each of the screening mechanisms 6 is connected with the detection component 9 at the rear end, and the bottom end of the second capillary 15 is inserted into the interior of the antibody screening groove 17, and the inner wall of the antibody screening groove 17 is in contact with the bottom of the second capillary 15. In the screening mechanism 6, the solution obtained by mixing the B cell reagent and the serum reagent flows by the capillary action through the second capillary 15 at the bottom until it flows into the interior of the screening mechanism 6 at the bottom, and a small amount is collected by the antibody screening groove 17. After the detection process is completed by the detection component 9 at the rear end, the subsequent antibody cloning and cultivation process can be carried out by directly removing the docking chamber 16.

[0028] In this embodiment, the detection component 9 includes an antigen reagent tank 11 and a bottom plate 13, the bottom plate 13 is arranged at the bottom of the antigen reagent tank 11, and an observation glass plate 14 is embedded in the middle of the bottom plate 13. The detection component 9 is provided with multiple detection components, each detection component 9 is connected to the corresponding mixing pipeline 2 part, and each antigen reagent tank 11 is installed at the same height, and a docking port 24 is opened on the top of the mixing pipeline 2, and the bottom end of the first capillary 5 is inserted into the interior of the docking port 24. The top of the antigen reagent tank 11 is connected to the delivery pipeline 10, and the pushing mechanism 1 at the front end, the screening mechanism 6 in the middle and the detection component 9 at the rear end are all provided. There is a sealing or anti-backflow structure to prevent external pollution from affecting the reagents, thereby improving the purity of the reagent culture. In the detection component 9, the mixed reagent solution inside the mixing pipe 2 is collected and accumulated through the connecting pipe 7 and the sealing sleeve 8, and this part of the reagent is transported to the antigen reagent tank 11 through the top conveying pipe 10. After being injected into the interior, it directly contacts the antigen reagent. If antibodies have been produced in this part of the mixed reagent, agglutination blocks are produced after the antibodies and antigens are contacted and mixed, and it can be judged that part of the reagent inside the mixing pipe 2 has produced antibodies. In this state, the screening reagent inside the corresponding screening mechanism 6 can be extracted for later cloning and cultivation.

[0029] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0030] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A device for microfluidic antibody screening, characterized in that: The invention comprises a pushing mechanism (1), a screening mechanism (6) and a detection assembly (9), wherein a plurality of mixing pipes (2) are arranged on the top of the pushing mechanism (1), a serum reagent tube (3) is installed on the top of the mixing pipe (2), a first injection port (4) is arranged at one end of the serum reagent tube (3), the bottom end of the serum reagent tube (3) is connected to a first capillary tube (5), a screening mechanism (6) is arranged in the middle of the mixing pipe (2), a butt joint pipe (7) is arranged at the end of the mixing pipe (2), the butt joint pipe (7) is inserted into the interior of a sealing sleeve (8), a detection assembly (9) is installed on the side of the sealing sleeve (8), and the like. The pushing mechanism (1) comprises a pushing tank (18) and a B cell reagent tank (19), a piston rod (22) is inserted into the top of the pushing tank (18), a pressing plate (20) is connected to the top of the piston rod (22), an electric push rod (21) is installed at the bottom of the pressing plate (20), a diversion pipe (23) is connected to the bottom of the pushing tank (18), the inside of the pushing tank (18) and the B cell reagent tank (19) are connected through the diversion pipe (23), the top of the electric push rod (21) is fixedly connected to the bottom of the pressing plate (20), and the bottom of the electric push rod (21) is fixedly connected to the top of the pushing tank (18), and The electric push rod (21) is connected to an external control system through an electric wire. Each of the mixing pipes (2) is inserted into the interior of the B cell reagent tank (19). The screening mechanism (6) includes a docking chamber (16) and an antibody screening tank (17). The antibody screening tank (17) is arranged at the bottom of the inner side of the docking chamber (16). The bottom end of the mixing pipe (2) is connected to a second capillary (15). The bottom end of the second capillary (15) is inserted into the interior of the antibody screening tank (17). The inner wall of the antibody screening tank (17) is in contact with the bottom of the second capillary (15). The detection component (9) includes an antigen reagent tank (11) and a bottom plate (11). 3), the bottom plate (13) is arranged at the bottom of the antigen reagent tank (11), an observation glass plate (14) is embedded in the middle of the bottom plate (13), a second injection port (12) is arranged at the top of the antigen reagent tank (11), a plurality of detection components (9) are arranged, each detection component (9) is connected to a corresponding mixing pipe (2) part, and each antigen reagent tank (11) is installed at the same height, a docking port (24) is opened at the top of the mixing pipe (2), the bottom end of the first capillary (5) is inserted into the interior of the docking port (24), and the top of the antigen reagent tank (11) is connected to a conveying pipe (10).

2. The method for using the device for microfluidic antibody screening according to claim 1, characterized in that: The process is carried out according to the following steps: Step 1, injecting the B cell reagent into the pushing mechanism (1), injecting the serum reagent into the serum reagent tube (3), and injecting the antigen reagent into the antigen reagent tank (11); Step 2, pushing the B cell reagent into the plurality of mixing pipes (2) through the front pushing mechanism (1); Step 3, mixing the B cells with a trace amount of serum through the first capillary (5) in the middle of the mixing pipe (2); Step 4, generating a screening reagent at the screening mechanism (6) at the bottom, and generating an agglutination sample in the detection component (9) at the rear end; Step 5, observing the agglutination state with the naked eye or a microscope, and selecting a corresponding screening reagent; Step 6, extracting the screening reagent using microfluidics technology, and performing cloning culture.

3. The method of use according to claim 2, characterized in that: In the step three, the B cells and a trace amount of serum are mixed and reacted inside the mixing pipe (2) by capillary action. In the step four, a small amount of screening reagent is also generated inside the screening mechanism (6) by capillary action.

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