Hybridoma high-throughput screening system and method

Abstract

The invention relates to a hybridoma high-throughput screening system and method.The hybridoma high-throughput screening system comprises a cell culture area, a cell analysis area and a transfer area, and the cell culture area comprises a first table board unit, a first storage unit, a first liquid working unit, a culture unit and a first transfer unit; the cell analysis area comprises a second table board unit, a second storage unit, a centrifugal unit, a cell analysis unit, a detection unit, a second liquid working unit, a cleaning unit and a second transfer unit, and the transfer area is arranged between the cell culture area and the cell analysis area. The cell plate transferring device is used for acquiring the cell plate transferred by the cell culture area / cell analysis area and transferring the cell plate to the cell analysis area / cell culture area. The intelligent hybridoma high-throughput screening system has the advantages that by means of the highly-integrated intelligent hybridoma high-throughput screening system, the steps needing manual participation are greatly reduced, the processing efficiency can be improved, and processing errors are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell screening, and in particular to a hybridoma high-throughput screening system and method. Background Art

[0002] The basic principle of hybridoma technique is to fuse mouse spleen B lymphocytes immunized with antigens with mouse myeloma cells in vitro, so that the hybrid cells have the main characteristics of both. The main characteristics of spleen B lymphocytes are that they have the ability to secrete antibodies and can grow in selective culture medium, but they cannot proliferate indefinitely; while mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have the so-called immortality. Under the action of selective culture medium, only hybrid cells fused with B lymphocytes and myeloma cells have the ability to continue to divide and proliferate, forming cells that have both the ability to secrete antibodies and maintain cell immortality, which are called hybridoma cells.

[0003] If the fused hybridoma cells are directly expanded and cultured, many problems will arise: first, if there are multiple antibody-secreting clones in the well, the final antibody may not be a monoclonal antibody; second, if the well contains a hybridoma that cannot secrete antibodies, it will eventually become a dominant cell due to its faster proliferation rate, resulting in the target hybridoma being unable to be separated or even completely disappearing; another problem is that hybridoma cells are unstable in the early stage, and as the number of cell passages increases, chromosomes may be lost and the antibody secretion ability may be lost. Therefore, after cell fusion, it is often necessary to conduct multiple rounds of identification and screening of the growing hybridoma clones and multiple cell subcloning to ensure that the target hybridoma clone is formed by the division and proliferation of a single cell, which is also the main defect of preparing monoclonal antibodies through hybridoma technology.

[0004] Under normal circumstances, it often takes about 2 months of research and development cycle from cell fusion to monoclonal antibody production. In order to obtain high-quality target antibodies for specific targets, it is usually necessary to go through multiple cell fusions and multiple rounds of throughput screening. The experimental operations involved in each cell fusion screening: changing the medium after cell fusion, sampling of cell supernatant, ELISA or FACS detection, selection and cultivation of hybridoma positive clones, antibody production and purification, etc., will consume a lot of manpower, material resources and financial resources. Defects such as long preparation cycle, cumbersome operation process and high labor cost have always been the key factors restricting the further development of hybridoma technology.

[0005] In the process of preparing monoclonal antibodies, two screenings are generally performed. The first screening is to screen hybridoma cells (i.e., selectively culture hybridoma cells), and the second screening is to further screen hybridoma cells to screen out hybridoma cells that produce specific antibodies (i.e., screen positive clones and clone them).

[0006] Hybridoma cell screening is generally done manually, including pipetting, equipment operation, coating, sampling, etc. For small batches of hybridoma cell screening, manual processing can be performed. However, for large batches of hybridoma cell screening, manual processing will result in problems such as labeling errors, pipetting errors, and data confusion.

[0007] Currently, no effective solution has been proposed for the problems existing in related technologies, such as the inability to manually handle large-scale hybridoma cell screening. Summary of the invention

[0008] The purpose of the present invention is to provide a hybridoma high-throughput screening system and method to address the deficiencies in the prior art, so as to solve the problems existing in the related art such as the inability to manually process large quantities of hybridoma cell screening.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In a first aspect, a hybridoma high-throughput screening system is provided, comprising:

[0011] A cell culture area, the cell culture area comprising a first table unit, a first storage unit, a first liquid working unit, a culture unit, and a first transport unit, wherein the first storage unit, the first liquid working unit, the culture unit, and the first transport unit are arranged on the first table unit;

[0012] A cell analysis area, the cell analysis area comprising a second table unit, a second storage unit, a centrifugal unit, a cell analysis unit, a detection unit, a second liquid working unit, a cleaning unit, and a second transport unit, wherein the second storage unit, the centrifugal unit, the cell analysis unit, the detection unit, the second liquid working unit, the cleaning unit, and the second transport unit are arranged on the second table unit;

[0013] A transfer zone is arranged between the cell culture zone and the cell analysis zone, and is used for obtaining the cell plate transferred from the cell culture zone / the cell analysis zone and transferring the cell plate to the cell analysis zone / the cell culture zone.

[0014] In some of these embodiments, the cell culture area further comprises:

[0015] A first control unit is disposed on the first table unit and is respectively connected to the first liquid working unit, the culture unit, and the first transport unit.

[0016] In some of these embodiments, the cell culture area further comprises:

[0017] The first garbage collection unit is disposed inside the first table unit.

[0018] In some of these embodiments, the cell culture area further comprises:

[0019] A first sliding unit is disposed inside the first table unit and is used to accommodate the first garbage collecting unit.

[0020] In some of these embodiments, the cell culture area further comprises:

[0021] A third storage unit is provided on the first table unit and is located on a side of the first liquid working unit.

[0022] In some of these embodiments, the cell analysis area further comprises:

[0023] A second control unit, wherein the second control unit is disposed on the second table unit and is respectively connected to the centrifugal unit, the cell analysis unit, the detection unit, the second liquid working unit, the cleaning unit, and the second transport unit.

[0024] In some of these embodiments, the cell analysis area further comprises:

[0025] A second sliding unit is disposed inside the second table unit and is used to accommodate the centrifugal unit.

[0026] In some of these embodiments, the cell analysis area further comprises:

[0027] The second garbage collecting unit is arranged inside the second table unit.

[0028] In some of these embodiments, the cell analysis area further comprises:

[0029] A third sliding unit is disposed inside the second table unit and is used to accommodate the second garbage collecting unit.

[0030] In some of these embodiments, the cell analysis area further comprises:

[0031] A fourth storage unit is provided on the second table unit and is located on a side of the centrifugal unit and a side of the detection unit.

[0032] In some of these embodiments, the transfer zone includes:

[0033] A transfer unit is disposed between the cell culture area and the cell analysis area, and is used for acquiring the cell plate transferred from the cell culture area / the cell analysis area and transferring the cell plate to the cell analysis area / the cell culture area.

[0034] In some of these embodiments, the transfer zone further comprises:

[0035] An isolation unit is disposed between the cell culture area and the cell analysis area and connected to the transfer unit, and is used to isolate the cell culture area from the cell analysis area.

[0036] In a second aspect, a method for high-throughput screening of hybridomas is provided, which is applied to the high-throughput screening system for hybridomas as described in the first aspect.

[0037] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0038] A hybridoma high-throughput screening system and method of the present invention greatly reduces the steps requiring manual participation through a highly integrated intelligent hybridoma high-throughput screening system, thereby improving processing efficiency and reducing processing errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 1 is a schematic diagram of a hybridoma high-throughput screening system according to an embodiment of the present invention (I);

[0040] Figure 2 Schematic diagram of a hybridoma high-throughput screening system according to an embodiment of the present invention (II);

[0041] Figure 3 is a schematic diagram of a cell analysis area according to an embodiment of the present invention;

[0042] Figure 4 is a top view of a cell analysis area according to an embodiment of the present invention;

[0043] Figure 5 is a schematic diagram of a cell culture area according to an embodiment of the present invention;

[0044] Figure 6 is a top view of a cell culture area according to an embodiment of the present invention;

[0045] Figure 7 is a schematic diagram of a transfer zone according to an embodiment of the present invention.

[0046] The reference numerals are: 100, cell culture area; 101, first table unit; 102, first storage unit; 103, first liquid working unit; 104, culture unit; 105, first transport unit; 106, first control unit; 107, third storage unit;

[0047] 200, cell analysis area; 201, second table unit; 202, second storage unit; 203, centrifugal unit; 204, cell analysis unit; 205, detection unit; 206, second liquid working unit; 207, cleaning unit; 208, second transport unit; 209, second control unit; 210, fourth storage unit;

[0048] 300, transfer area; 301, transfer unit; 302, isolation unit. DETAILED DESCRIPTION

[0049] 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0052] Example 1

[0053] An exemplary embodiment of the present invention. Figure 1-2As shown, a hybridoma high-throughput screening system includes a cell culture area 100, a cell analysis area 200 and a transfer area 300. The cell culture area 100 includes a first table unit 101, a first storage unit 102, a first liquid working unit 103, a culture unit 104, and a first transport unit 105, and the first storage unit 102, the first liquid working unit 103, the culture unit 104 and the first transport unit 105 are arranged on the first table unit 101; the cell analysis area 200 includes a second table unit 201, a second storage unit 202, a centrifugation unit 203, a cell analysis unit 204, a detection unit 205, a second liquid working unit 20 6. The cleaning unit 207, the second transfer unit 208, the second storage unit 202, the centrifugation unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206, the cleaning unit 207 and the second transfer unit 208 are arranged on the second table unit 201; the transfer area 300 is arranged between the cell culture area 100 and the cell analysis area 200, and is used to obtain the cell plate transferred from the cell culture area 100 / cell analysis area 200 and transfer the cell plate to the cell analysis area 200 / cell culture area 100.

[0054] like Figure 3-4 As shown, the cell culture area 100 includes a first table unit 101, a first storage unit 102, a first liquid working unit 103, a culture unit 104 and a first transfer unit 105. The first table unit 101 is arranged on a horizontal plane; the first storage unit 102 is arranged on the upper part of the first table unit 101 for storing consumables; the first liquid working unit 103 is arranged on the upper part of the first table unit 101 and is located at the side of the first storage unit 102; the culture unit 104 is arranged on the upper part of the first table unit 101 and is located at the side of the first storage unit 102 and the side of the first liquid working unit 103; the first transfer unit 105 is arranged on the upper part of the first table unit 101 and is located between the first storage unit 102, the first liquid working unit 103 and the culture unit 104, and is used to transfer the cell plate between the first storage unit 102, the first liquid working unit 103 and the culture unit 104 and transfer the cell plate to the transfer area 300.

[0055] The first table unit 101 includes a first workbench and a plurality of first sealed doors. The first workbench is arranged on a horizontal plane, and the first storage unit 102, the first liquid working unit 103, the culture unit 104 and the first transfer unit 105 are detachably mounted on the upper part of the first workbench; the plurality of first sealed doors are arranged around the side of the first workbench and are rotatably connected (such as hinged) to the first workbench respectively.

[0056] The first workbench has a hollow structure inside, which is used to install cables of the first liquid working unit 103, the culture unit 104 and the first transfer unit 105 and to place other items. The first workbench can be exposed through a plurality of first sealed doors for taking items and inspecting cables.

[0057] In some embodiments, the first table unit 101 further includes a plurality of first moving wheels. The plurality of first moving wheels are disposed at the bottom of the first workbench and are used to move the first workbench.

[0058] In some of the embodiments, the first movable wheel is a universal wheel with a braking function.

[0059] The first storage unit 102 is a light-proof structure. Specifically, the first storage unit 102 includes a first storage box and a first storage rack. The first storage box is detachably mounted on the upper part of the first workbench, and the first storage box is a light-proof structure; the first storage rack is arranged inside the first storage box for storing consumables.

[0060] In some of the embodiments, the first storage unit 102 is disposed at an end of the first table unit 101 away from the transfer area 300 .

[0061] The first liquid working unit 103 is detachably disposed on the upper portion of the first table unit 101 (ie, the first working table) and is located downstream of the first storage unit 102 .

[0062] In some of the embodiments, the first liquid working unit 103 and the first storage unit 102 are symmetrically arranged.

[0063] In some embodiments, the first liquid working unit 103 is a first liquid working station.

[0064] The culture unit 104 is detachably disposed on the upper portion of the first table unit 101 (ie, the first workbench) and is located downstream of the first storage unit 102 .

[0065] In some embodiments, the culture unit 104 and the first liquid working unit 103 are symmetrically arranged.

[0066] In some of the embodiments, the culture unit 104 is disposed at one end of the first table unit 101 close to the transfer area 300 .

[0067] In some of the embodiments, the cultivation unit 104 is an incubator.

[0068] The first transfer unit 105 is detachably disposed on the upper portion of the first table unit 101 (ie, the first workbench).

[0069] In some embodiments, the first storage unit 102 and the culture unit 104 are disposed on one side of the first transport unit 105 , and the first liquid working unit 103 is disposed on the other side of the first transport unit 105 .

[0070] The first transfer unit 105 includes a first track and a first multi-axis robot. The first track is detachably arranged on the upper part of the first workbench and is located between the first storage unit 102, the first liquid working unit 103, and the culture unit 104; the first multi-axis robot is slidably arranged on the upper part of the first track, and is used to transfer the cell plate between the first storage unit 102, the first liquid working unit 103, and the culture unit 104, and transfer the cell plate to the transfer area 300.

[0071] Furthermore, the cell culture area 100 further includes a first control unit 106. The first control unit 106 is disposed on the first table unit 101 and is connected to the first liquid working unit 103, the culture unit 104, and the first transport unit 105 respectively.

[0072] The first control unit 106 is disposed close to the first liquid working unit 103 and is located at an end of the first stage unit 101 away from the transfer area 300 .

[0073] In some embodiments, the first control unit 106 is a first control computer.

[0074] Furthermore, the cell culture area 100 further includes a first garbage collection unit (not shown in the figure). The first garbage collection unit is arranged inside the first table unit 101 .

[0075] Specifically, the first garbage collection unit is arranged inside the first workbench.

[0076] By operating the first sealing door, the first garbage collecting unit can be put into the interior of the first workbench, or the first garbage collecting unit can be taken out from the interior of the first workbench.

[0077] The first garbage collection unit includes a plurality of first garbage bins, wherein the plurality of first garbage bins are distributed inside the first workbench.

[0078] Generally, at least one first trash can is disposed near the first storage unit 102 ; at least one first trash can is disposed near the first liquid working unit 103 ; and at least one first trash can is disposed near the culture unit 104 .

[0079] Furthermore, the cell culture area 100 further includes a first sliding unit (not shown in the figure). The first sliding unit is arranged inside the first table unit 101 and is used to accommodate the first garbage collection unit.

[0080] Specifically, the first sliding unit is arranged inside the first workbench.

[0081] By providing the first sliding unit, the first garbage collecting unit can be conveniently taken out.

[0082] The first sliding unit includes a plurality of first sliding rails and a plurality of first sliding trays, wherein the plurality of first sliding rails are distributed inside the first workbench; the plurality of first sliding trays are respectively slidably connected to the corresponding first sliding rails, and at least one first trash can is disposed on the upper portion of each first sliding tray.

[0083] Furthermore, the cell culture area 100 further includes a third storage unit 107 , wherein the third storage unit 107 is disposed on the first table unit 101 and is located at a side of the first liquid working unit 103 .

[0084] In some of the embodiments, the third storage unit 107 and the first liquid working unit 103 are located on the same side of the first transfer unit 105 .

[0085] The third storage unit 107 includes at least one first material bin, wherein the first material bin is disposed on the upper part of the first workbench and located on the side of the first liquid working unit 103, and is used to place the cell plate.

[0086] In some embodiments, there are multiple first material bins, which are distributed on the side of the first liquid working unit 103 .

[0087] Generally, at least one first material bin is disposed on one side of the first liquid working unit 103 , and at least one first material bin is disposed on the other side of the first liquid working unit 103 .

[0088] Furthermore, the cell culture area 100 further includes a first constant temperature unit, wherein the first constant temperature unit is arranged inside and / or outside the first liquid working unit 103, and is used to perform a constant temperature treatment (generally 4°C) on the cell plate.

[0089] like Figure 5-6As shown, the cell analysis area 200 includes a second table unit 201, a second storage unit 202, a centrifugal unit 203, a cell analysis unit 204, a detection unit 205, a second liquid working unit 206, a cleaning unit 207, and a second transport unit 208. The second table unit 201 is arranged on a horizontal plane; the second storage unit 202 is arranged on the upper part of the second table unit 201 for storing consumables; the centrifugal unit 203 is arranged on the upper part of the second table unit 201 and is located at the side of the second storage unit 202; the cell analysis unit 204 is arranged on the upper part of the second table unit 201 and is located at the side of the second storage unit 202; the detection unit 205 is arranged on the upper part of the second table unit 201 and is located at the side of the cell analysis unit 204; the second liquid working unit 206 is arranged on the upper part of the second table unit 201 and is located at the detection unit 208. The cleaning unit 207 is arranged on the upper part of the second table unit 201 and is located on the side of the second liquid working unit 206; the second transfer unit 208 is arranged on the upper part of the second table unit 201 and is located between the second storage unit 202, the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206 and the cleaning unit 207, and is used for transferring between the second storage unit 202, the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206 and the cleaning unit 207.

[0090] The second table unit 201 includes a second workbench and a plurality of second sealed doors. The second workbench is arranged on a horizontal plane, and the second storage unit 202, the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206, the cleaning unit 207 and the second transfer unit 208 are detachably mounted on the upper part of the second workbench; the plurality of second sealed doors are arranged around the side of the second workbench and are rotatably connected (such as hinged) to the second workbench respectively.

[0091] The interior of the second workbench is a hollow structure, which is used to install the cables of the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206, the cleaning unit 207 and the second transfer unit 208, and to place other items. Through a plurality of second sealed doors, the interior of the second workbench can be exposed for taking out items and inspecting cables.

[0092] In some embodiments, the second table unit 201 further includes a plurality of second moving wheels. The plurality of second moving wheels are disposed at the bottom of the second workbench and are used to move the second workbench.

[0093] In some of the embodiments, the second movable wheel is a universal wheel with a braking function.

[0094] The second storage unit 202 is a light-proof structure. Specifically, the second storage unit 202 includes a second storage box and a second storage rack. The second storage box is detachably mounted on the upper part of the second workbench, and the second storage box is a light-proof structure; the second storage rack is arranged inside the second storage box for storing consumables.

[0095] In some of the embodiments, the second storage unit 202 is disposed at one end of the second table unit 201 close to the transfer area 300 .

[0096] The centrifugal unit 203 is detachably disposed inside the second table unit 201 (ie, the second workbench).

[0097] In some of the embodiments, the centrifugal unit 203 is disposed at one end of the second table unit 201 close to the transfer area 300 .

[0098] In some of the embodiments, the centrifugal unit 203 is a well plate centrifuge.

[0099] The cell analysis unit 204 is detachably disposed on the upper portion of the second table unit 201 (ie, the second workbench).

[0100] In some embodiments, there are multiple stem cell analysis units 204. If the stem cell analysis units 204 are arranged at intervals along the length direction of the second table unit 201.

[0101] In some embodiments, the cell analysis unit 204 is a flow cytometer.

[0102] The detection unit 205 is detachably disposed on the upper portion of the second table unit 201 (second workbench).

[0103] In some of the embodiments, the detection unit 205 is disposed at an end of the second table unit 201 away from the transfer area 300 .

[0104] In some of the embodiments, the detection unit 205 is an ELISA reader.

[0105] The second liquid working unit 206 is detachably disposed on the upper portion of the second stage unit 201 (second work stage).

[0106] In some of the embodiments, the second liquid working unit 206 and the cell analysis unit 204 are symmetrically arranged.

[0107] In some embodiments, the second liquid working unit 206 is a second liquid working station.

[0108] The cleaning unit 207 is detachably disposed on the upper portion of the second table unit 201 (second workbench).

[0109] In some of the embodiments, the cleaning unit 207 is disposed between the detection unit 205 and the second liquid working unit 206 .

[0110] In some of the embodiments, the cleaning unit 207 is disposed at an end of the second table unit 201 away from the transfer area 300 .

[0111] In some of the embodiments, the cleaning unit 207 is a plate washer.

[0112] The second transfer unit 208 is detachably disposed on the upper portion of the second table unit 201 (ie, the second workbench).

[0113] The second transfer unit 208 includes a second track and a second multi-axis robot. The second track is detachably arranged on the upper part of the second workbench and is located between the second storage unit 202, the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206 and the cleaning unit 207; the second multi-axis robot is slidably arranged on the upper part of the second track and is used for transferring between the second storage unit 202, the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206 and the cleaning unit 207.

[0114] In some of the embodiments, the axis of the second transfer unit 208 is coplanar with the axis of the first transfer unit 105 .

[0115] In some of the embodiments, the second storage unit 202 and the cell analysis unit 204 are arranged on one side of the second transfer unit 208, the centrifugation unit 203 and the second liquid working unit 206 are arranged on the other side of the second transfer unit 208, and the detection unit 205 and the cleaning unit 207 are arranged at one end of the second transfer unit 208 away from the transfer area 300.

[0116] Furthermore, the cell analysis area 200 further includes a second control unit 209. The second control unit 209 is disposed on the second table unit 201 and is connected to the centrifugal unit 203, the cell analysis unit 204, the detection unit 205, the second liquid working unit 206, the cleaning unit 207, and the second transport unit 208 respectively.

[0117] In some embodiments, there are multiple second control units 209 , wherein at least one second control unit 209 is disposed close to the cell analysis unit 204 , and at least one second control unit 209 is disposed close to the second liquid working unit 206 .

[0118] In some embodiments, the second control unit 209 is a second control computer.

[0119] Furthermore, the cell analysis area 200 further includes a second sliding unit (not shown in the figure). The second sliding unit is disposed inside the second table unit 201 and is used to accommodate the centrifugal unit 203 .

[0120] Specifically, the second sliding unit is arranged inside the second workbench.

[0121] By providing the second sliding unit, the centrifugal unit 203 can be replaced conveniently.

[0122] The second sliding unit includes a second slide rail and a second slide tray, wherein the second slide rail is arranged inside the second workbench; the second slide tray is slidably connected to the second slide rail, and a centrifugal unit 203 is arranged on the upper part of the second slide tray.

[0123] Furthermore, the cell analysis area 200 further includes a second garbage collection unit (not shown in the figure). The second garbage collection unit is disposed inside the second table unit 201 .

[0124] Specifically, the second garbage collection unit is arranged inside the second workbench.

[0125] By operating the second sealing door, the second garbage collecting unit can be put into the interior of the second workbench, or the second garbage collecting unit can be taken out from the interior of the second workbench.

[0126] The second garbage collection unit includes a plurality of second garbage cans, wherein the plurality of second garbage cans are distributed inside the second workbench.

[0127] Generally, at least one second trash can is set near the second storage unit 202; at least one second trash can is set near the centrifugation unit 203; at least one second trash can is set near the cell analysis unit 204; at least one second trash can is set near the detection unit 205; at least one second trash can is set near the second liquid working unit 206; and at least one second trash can is set near the cleaning unit 207.

[0128] Furthermore, the cell analysis area 200 further includes a third sliding unit (not shown in the figure). The third sliding unit is arranged inside the second table unit 201 to accommodate the second garbage collection unit.

[0129] Specifically, the third sliding unit is arranged inside the second workbench.

[0130] By providing the third sliding unit, the second garbage collecting unit can be conveniently taken out.

[0131] The third sliding unit includes a plurality of third slide rails and a plurality of third slide trays, wherein the plurality of third slide rails are distributed inside the third workbench; the plurality of third slide trays are respectively slidably connected to the corresponding third slide rails, and at least one third trash can is arranged on the upper part of each third slide tray.

[0132] Furthermore, the cell analysis area 200 further includes a fourth storage unit 210 , wherein the fourth storage unit 210 is disposed on the second table unit 201 and is located on the side of the centrifugal unit 203 and the side of the detection unit 205 .

[0133] The fourth storage unit 210 includes at least two fourth material bins, wherein at least one fourth material bin is disposed on the side of the centrifugal unit 203 , and at least one fourth material bin is disposed on the side of the detection unit 205 .

[0134] Furthermore, the cell analysis area 200 further includes a second constant temperature unit, which is disposed inside and / or outside the second liquid working unit 206 and is used to perform a constant temperature treatment (generally 4° C.) on the cell plate.

[0135] like Figure 7 As shown, the transfer area 300 includes a transfer unit 301. The transfer unit 301 is disposed between the cell culture area 100 and the cell analysis area 200, and is used to obtain the cell plate transferred from the cell culture area 100 / cell analysis area 200 and transfer the cell plate to the cell analysis area 200 / cell culture area 100.

[0136] Specifically, the transfer unit 301 is disposed between the first stage unit 101 and the second stage unit 201 .

[0137] Generally, the transfer unit 301 is located between the first transfer unit 105 and the second transfer unit 208, and the axis of the transfer unit 301, the axis of the first transfer unit 105, and the axis of the second transfer unit 208 are collinear.

[0138] In some of the embodiments, the transfer unit 301 is an automatic transfer window.

[0139] Furthermore, the transfer area 300 further includes an isolation unit 302 , which is disposed between the cell culture area 100 and the cell analysis area 200 and connected to the transfer unit 301 , and is used to isolate the cell culture area 100 from the cell analysis area 200 .

[0140] Specifically, the isolation unit 302 is disposed between the first mesa unit 101 and the second mesa unit 201 .

[0141] In some embodiments, the isolation unit 302 is a transparent isolation window.

[0142] The hybridoma high-throughput screening method of the present invention is applied to the hybridoma high-throughput screening system as described above, comprising:

[0143] Step 1: Plating of fused cells

[0144] The first transport unit 105 transports the first cell plate from the first storage unit 102 to the first liquid working unit 103;

[0145] The first liquid working unit 103 performs plating;

[0146] The first transport unit 105 transports the laid first cell plate to the culture unit 104 for culture;

[0147] Repeat the above steps until all first cell plates are plated;

[0148] Step 2: Replace the medium of fused cells

[0149] After the liquid replacement time is reached, the first transport unit 105 transports the first cell plate from the culture unit 104 to the first liquid working unit 103;

[0150] The first liquid working unit 103 performs liquid replacement;

[0151] The first transport unit 105 transports the first cell plate after the medium replacement to the culture unit 104 for culture;

[0152] Step (III) ELISA coating

[0153] The second transport unit 208 transports the first ELISA plate from the second storage unit 202 to the second liquid working unit 206;

[0154] The second liquid working unit 206 performs ELISA coating;

[0155] The second transport unit 208 transports the coated first ELISA plate to the cleaning unit 207;

[0156] The cleaning unit 207 cleans the first ELISA plate to remove the coating solution;

[0157] The second transport unit 208 transports the cleaned first ELISA plate to the second liquid working unit 206;

[0158] The second liquid working unit 206 adds a blocking solution to the first ELISA plate;

[0159] The second transport unit 208 transports the sealed first ELISA plate to the cleaning unit 207;

[0160] The cleaning unit 207 cleans the first ELISA plate to remove the blocking solution;

[0161] The second transport unit 208 transports the cleaned first ELISA plate to the transfer unit 301;

[0162] Step 4: ELISA sampling

[0163] The first transport unit 105 transports the first ELISA plate to the first liquid working unit 103;

[0164] The first liquid working unit 103 adds the supernatant of the first cell plate to the corresponding wells of the first ELISA plate, and reserves negative control wells and positive control wells;

[0165] The first transport unit 105 transports the first ELISA plate processed by the first liquid working unit 103 to the third storage unit 107 for room temperature binding;

[0166] The first transport unit 105 transports the first ELISA plate after room temperature binding to the transfer unit 301;

[0167] Step 5: ELISA detection of hybridoma supernatant

[0168] The second transport unit 208 transports the first ELISA plate to the cleaning unit 207;

[0169] The cleaning unit 207 cleans the first ELISA plate to remove the supernatant;

[0170] The second transport unit 208 transports the cleaned first ELISA plate to the second liquid working unit 206;

[0171] The second liquid working unit 206 adds a secondary antibody to the first ELISA plate;

[0172] The second transport unit 208 transports the first ELISA plate with the secondary antibody added to the cleaning unit 207;

[0173] The cleaning unit 207 cleans the first ELISA plate to remove the secondary antibody;

[0174] The second transport unit 208 transports the cleaned first ELISA plate to the second liquid working unit 206;

[0175] The second liquid working unit 206 sequentially adds a color developing reagent and a stop reagent to the first ELISA plate;

[0176] The second transport unit 208 transports the first ELISA plate to the detection unit 205;

[0177] The detection unit 205 performs ELISA detection on the first enzyme label plate;

[0178] Step 6: Hybridoma FACS sampling

[0179] The first transfer unit 105 transfers the first V-bottom plate from the first storage unit 102 to the first liquid working unit 103;

[0180] The first transport unit 105 transports the first cell plate from the culture unit 104 to the first liquid working unit 103;

[0181] According to the ELISA test results, the first liquid working unit 103 selects positive wells and sequentially adds the supernatant of the first cell plate to the first V bottom plate;

[0182] The first transport unit 105 transports the first cell plate from which the supernatant has been taken to the culture unit 104;

[0183] The first transport unit 105 transports the first V-bottom plate containing the supernatant to the transfer unit 301;

[0184] Step (VII) FACS detection of hybridoma supernatant

[0185] The second transfer unit 208 transfers the second V-bottom plate from the second storage unit 202 to the second liquid working unit 206;

[0186] The second transfer unit 208 transfers the first V-bottom plate to the second liquid working unit 206;

[0187] The second liquid working unit 206 sequentially adds cells and hybridoma supernatant stock solution to the second V-bottom plate and incubates them;

[0188] The second transport unit 208 transports the incubated second V-bottom plate to the centrifugal unit 203;

[0189] The centrifugal unit 203 centrifuges the second V-bottom plate;

[0190] The second transfer unit 208 transfers the second V-bottom plate after centrifugation to the second liquid working unit 206;

[0191] The second liquid working unit 206 adds buffer to the second V bottom plate;

[0192] The second transport unit 208 transports the second V-bottom plate to the cell analysis unit 204;

[0193] The cell analysis unit 204 performs FACS detection on the second V bottom plate;

[0194] Step 8: First cell subcloning

[0195] The first transport unit 105 transports the second cell plate from the first storage unit 102 to the first liquid working unit 103;

[0196] The first transport unit 105 transports the first cell plate from the culture unit 104 to the first liquid working unit 103;

[0197] The first liquid working unit 103 adds complete culture medium to the second cell plate;

[0198] According to the FACS test results, the first liquid working unit 103 selects the positive wells that need to be subcloned, and transfers the positive wells that need to be subcloned to the corresponding second cell plate in order (Mix before transfer), and blows the wells to be subcloned in the second cell plate several times to resuspend and mix the cells;

[0199] The first transport unit 105 transports the second cell plate from the first liquid working unit 103 to the culture unit 104;

[0200] Conduct subcloning experiments to obtain the first subclone cell plate;

[0201] Step 9: ELISA coating

[0202] The second transport unit 208 transports the second ELISA plate from the second storage unit 202 to the second liquid working unit 206;

[0203] The second liquid working unit 206 performs ELISA coating;

[0204] The second transport unit 208 transports the coated second ELISA plate to the cleaning unit 207;

[0205] The cleaning unit 207 cleans the second ELISA plate to remove the coating solution;

[0206] The second transport unit 208 transports the cleaned second ELISA plate to the second liquid working unit 206;

[0207] The second liquid working unit 206 adds a blocking solution to the second ELISA plate;

[0208] The second transport unit 208 transports the sealed second ELISA plate to the cleaning unit 207;

[0209] The cleaning unit 207 cleans the second ELISA plate to remove the blocking solution;

[0210] The second transport unit 208 transports the cleaned second ELISA plate to the transfer unit 301;

[0211] Step 10: Subclone cell ELISA sampling

[0212] The first transport unit 105 transports the second ELISA plate to the first liquid working unit 103;

[0213] The first liquid working unit 103 adds the supernatant of the first subclone cell plate to the corresponding wells of the second ELISA plate, and reserves negative control wells and positive control wells;

[0214] The first transport unit 105 transports the second ELISA plate processed by the first liquid working unit 103 to the third storage unit 107 for room temperature binding;

[0215] The first transport unit 105 transports the second ELISA plate after room temperature binding to the transfer unit 301;

[0216] Step (XI) ELISA detection of subcloned cell supernatant

[0217] The second transport unit 208 transports the second ELISA plate to the cleaning unit 207;

[0218] The cleaning unit 207 cleans the second ELISA plate to remove the supernatant;

[0219] The second transport unit 208 transports the cleaned second ELISA plate to the second liquid working unit 206;

[0220] The second liquid working unit 206 adds a secondary antibody to the second ELISA plate;

[0221] The second transport unit 208 transports the second ELISA plate with the secondary antibody to the cleaning unit 207;

[0222] The cleaning unit 207 cleans the second ELISA plate to remove the secondary antibody;

[0223] The second transport unit 208 transports the cleaned second ELISA plate to the second liquid working unit 206;

[0224] The second liquid working unit 206 sequentially adds a color developing reagent and a stop reagent to the second ELISA plate;

[0225] The second transport unit 208 transports the second ELISA plate to the detection unit 205;

[0226] The detection unit 205 performs ELISA detection on the second enzyme label plate;

[0227] Step 12: FACS sampling of subcloned cells

[0228] The first transfer unit 105 transfers the third V-bottom plate from the first storage unit 102 to the first liquid working unit 103;

[0229] The first transport unit 105 transports the first subclone cell plate from the culture unit 104 to the first liquid working unit 103;

[0230] According to the ELISA test results, the first liquid working unit 103 selects positive wells and sequentially adds the supernatant of the first subclone cell plate to the third V bottom plate;

[0231] The first transport unit 105 transports the first cell plate from which the supernatant has been taken to the culture unit 104;

[0232] The first transport unit 105 transports the third V-bottom plate containing the supernatant to the transfer unit 301;

[0233] Step (13) FACS detection of subcloned cell supernatant

[0234] The second transfer unit 208 transfers the fourth V-bottom plate from the second storage unit 202 to the second liquid working unit 206;

[0235] The second transfer unit 208 transfers the third V-bottom plate to the second liquid working unit 206;

[0236] The second liquid working unit 206 sequentially adds cells and hybridoma supernatant stock solution to the fourth V bottom plate and incubates them;

[0237] The second transport unit 208 transports the incubated fourth V-bottom plate to the centrifugal unit 203;

[0238] The centrifugal unit 203 centrifuges the fourth V bottom plate;

[0239] The second transfer unit 208 transfers the fourth V-bottom plate after centrifugation to the second liquid working unit 206;

[0240] The second liquid working unit 206 adds buffer to the fourth V bottom plate;

[0241] The second transport unit 208 transports the fourth V-bottom plate to the cell analysis unit 204;

[0242] The cell analysis unit 204 performs FACS detection on the fourth V bottom plate;

[0243] Step 14: Second cell subcloning

[0244] The first transport unit 105 transports the third cell plate from the first storage unit 102 to the first liquid working unit 103;

[0245] The first transport unit 105 transports the first subclone cell plate from the culture unit 104 to the first liquid working unit 103;

[0246] The first liquid working unit 103 adds complete culture medium to the third cell plate;

[0247] According to the FACS test results, the first liquid working unit 103 selects the positive wells that need to be subcloned, and transfers the positive wells that need to be subcloned to the corresponding third cell plate in order (Mix before transfer), and blows the wells to be subcloned in the third cell plate several times to resuspend and mix the cells;

[0248] The first transport unit 105 transports the third cell plate from the first liquid working unit 103 to the culture unit 104;

[0249] Perform subcloning experiments to obtain a second subclone cell plate;

[0250] Step 15: Planting and expansion

[0251] The first transport unit 105 transports the fourth cell plate from the first storage unit 102 to the first liquid working unit 103;

[0252] The first liquid working unit 103 adds complete culture medium to the fourth cell plate;

[0253] The first transport unit 105 transports the second subclone cell plate to the first liquid working unit 103;

[0254] The first liquid working unit 103 suspends the cells in the fixed-line wells of the second subclone cell plate and transfers them to the corresponding fourth cell plate, and mixes them evenly by pipetting;

[0255] The first transport unit 105 transfers the fourth cell plate to the culture unit 104 for culture.

[0256] The hybridoma high-throughput screening system and method of the present invention greatly reduces the steps requiring manual participation through a highly integrated intelligent hybridoma high-throughput screening system, thereby improving processing efficiency and reducing processing errors.

[0257] Example 2

[0258] A specific embodiment of the present invention.

[0259] A method for high-throughput screening of hybridomas, comprising:

[0260] (I) Cell area: Plating of fused cells

[0261] Throughput: can meet the needs of 40 96-well cell plates (4 plates per batch, completed in 10 batches);

[0262] Consumables enter the workstation: 1-4 pieces of 96WP enter the workstation from the consumables stack;

[0263] Manually place 3 reagent tanks (containing 300mL of fused cells) on the workstation table

[0264] Manually place the 300uLTip head on the workstation table (this step requires a total of 10 boxes for 96 heads of the liquid workstation);

[0265] 1) Run the workstation method: 1_1_CellPlanking: 150uL / well. After all the plates are in, open the lid, reset the tip, inoculate cells, replace the tip box, and close the lid;

[0266] 2) Consumables leave the liquid workstation: 1-4 96WPs leave the liquid workstation and enter the cell culture incubator, and the timing starts at this time;

[0267] 3) Repeat steps 1-3 until all 40 boards are laid;

[0268] 4) After all 40 tiles are laid: manually clean the countertop;

[0269] (II) Cell area: cell medium replacement

[0270] Throughput: can meet the needs of 40 96-well cell plates for medium replacement (4 plates per batch, completed in 10 batches);

[0271] Manually place the 300uLTip head on the workstation table;

[0272] Manually place an empty 290mL reagent tank to the peristaltic filling position of the liquid workstation (for adding fresh culture medium);

[0273] Manually place the bottle of fresh culture medium to the peristaltic pump (a total of 576 mL is required, and the dead volume is to be observed);

[0274] When the cell medium replacement time is reached (about 4 days, the time can be set by the user), an empty 290mL reagent tank is manually placed into the workstation to hold the discarded culture medium;

[0275] 1) Use a peristaltic pump to add fresh culture medium (pump once for every 20 pieces, 300 mL each time);

[0276] 2) 1-4 96WPs enter the workstation from the incubator;

[0277] 3) Run the workstation method: 2_1_Medium Exchange: discard 70uL / well of old medium, add 150uL / well of new medium, and change the pipette tip between aspirating old medium and new medium;

[0278] 4) Consumables leave the liquid workstation: 1-4 96WPs leave the liquid workstation and enter the cell culture box, and the timing starts at this time;

[0279] 5) Repeat steps 2-5 until all 40 cell plates have been completely replaced;

[0280] 6) Manually discard the waste liquid tank on the workstation table (containing 270mL of old culture medium);

[0281] 7) Manually discard the reagent tank at the peristaltic pump filling position (containing a certain dead volume of fresh culture medium);

[0282] 8) Pump tube flushing;

[0283] (III) Analysis area: ELISA plate coating_1

[0284] Throughput: 10 ELISA plates, the number of input ELISA plates can be controlled and executed in batches, 4 plates / batch;

[0285] 1) Consumables enter the workstation: 1-4 384WP ELISA plates (with covers) are scanned from the consumables stack and transferred to the liquid workstation table in the analysis area;

[0286] 2) Run the analysis area liquid workstation method: 3_1_ELISA_PlatePlaning: Open the cover, then add 25ul / well of 1ug / ml antigen solution in PBS to the 384 ELISA plate (without changing the pipette tip), and close the cover;

[0287] 3) Transport the 384 ELISA plates with added antigens to a temporary storage location at 4°C and coat overnight (usually 12 hours, and the next step of plate washing can be done at regular intervals); (repeat steps 1-2 until all ELISA plates have completed the addition of antigens);

[0288] 4) A 384 ELISA plate is transferred from the 4°C temporary storage location to the workstation table and opened;

[0289] 5) Transfer the opened ELISA plate to the plate washer, discard the coating solution, and wash the plate once;

[0290] 6) Use ISWAP to transfer the washed 384-ELISA plate from the plate washer to the workstation and cover the lid;

[0291] 7) Transfer out of the workstation and transfer the washed 384 ELISA plates from the workstation to the temporary storage rack next to the plate washer;

[0292] 8) Repeat the above steps until all 10 plates are cleaned;

[0293] 9) Use a peristaltic pump to add 160 mL of 3% milk / 1X PBS to the reagent tank (for batches 4+4+2, add 160 mL each time for the first two batches, and no addition is required for the last two plates);

[0294] 10) 1-4 ELISA plates are transferred from the temporary storage rack next to the plate washer to the liquid workstation in the analysis area. Run the workstation method: 3_2_ELISA_PlatePlaning: open the cover, add 80ul / well, 3% milk / 1XPBS from the peristaltic pump filling position, and close the cover. (4 plates require at least 123+25mL, 10 plates require 308mL). The peristaltic pump can be controlled to add liquid before the experiment, and the filling volume can be controlled;

[0295] 11) The consumables are transported out of the workstation to the temporary storage location next to the plate washer and sealed at room temperature for 2 hours;

[0296] 12) Repeat steps 7)-8) until 10 plates are filled with liquid;

[0297] 13) Manually throw the reagent tank on the liquid workstation table in the analysis area into the trash can (containing a 1ug / ml antigen solution reagent tank with a certain dead volume of PBS);

[0298] 14) Manually throw the reagent tank filled with liquid by the peristaltic pump on the liquid workstation table in the analysis area into the trash can (the 3% milk / 1XPBS containing a certain dead volume must be poured into the corresponding waste liquid bucket first);

[0299] 15) After blocking for 2 hours at room temperature, transfer 1-10 ELISA plates from the temporary storage position next to the plate washer to the workstation in turn, then open the cover of the workstation, grab the plate washer with iswap, discard the blocking solution, and wash the plate washer three times;

[0300] 16) The washed plates are returned to the temporary storage rack next to the plate washer (steps 1-12 are expected to be completed before work the next day);

[0301] (IV) Cell area: ELISA test sampling

[0302] Throughput requirement: supernatant from 40 96WP cell culture plates can be taken;

[0303] 1) Consumables enter the workstation: When the cell culture time is reached, a 384-well ELISA plate is transferred from the temporary storage rack next to the plate washer in the analysis area to the liquid workstation in the cell area;

[0304] 2) Four 96WP cell culture plates are transferred out of the incubator and into the liquid workstation in the cell area;

[0305] 3) Operation workstation method: 4_1_ELISASampling: Open the cell culture plate and the ELISA plate, and the liquid workstation aspirates the supernatant from the fused cell plate and adds it to the corresponding wells of the ELISA plate, and reserves negative and positive control wells. Sample volume: 25ul / well, close the cell culture plate and the ELISA plate; need to change the gun tip; one 384 ELISA plate requires 4 boxes of gun tips, 10 384 plates require 40 boxes of gun tips; gun tips need to be manually replenished in the middle;

[0306] 4) Consumables out of the workstation: 4 96WP cell culture plates are out of the liquid workstation and into the incubator;

[0307] 5) Consumables out of the workstation: 1 384-well ELISA plate is taken out of the liquid workstation and put into the transfer plate rack in the analysis area for binding at room temperature for 1 hour;

[0308] 6) Repeat steps 1) to 5) until all supernatants from 40 cell plates are collected;

[0309] (V) Analysis area: Hybridoma supernatant ELISA test

[0310] 1) After 1 hour of binding, transfer the 384-well ELISA plate to the workstation, open the cover of the workstation, grab the plate washer with iswap, discard the supernatant, and wash the plate 3 times with PBST (1‰ T20); (do it in batches of 10 plates);

[0311] 2) Place the 384-well ELISA plate after washing on the temporary storage rack next to the plate washer;

[0312] 3) Transfer 4 384-well ELISA plates to the liquid workstation in the analysis area and run the workstation method 5_1_ELISA_AddIgG: open the cover, add Goat-anti-mouse IgG (1:10000 in 3% milk-PBS), 25ul / well; close the cover (do 10 plates in batches);

[0313] 4) Transfer the 384-well ELISA plate to the transfer plate rack and bind at room temperature for 1 hour;

[0314] 5) Transfer the 384-well ELISA plate from the transfer plate rack to the workstation, open the cover of the workstation, use iSwap to grab the plate washer, discard the secondary antibody, and wash the plate 9 times with PBST (1‰T20);

[0315] 6) Place the 384-well ELISA plate after washing on the temporary storage rack next to the plate washer;

[0316] 7) Transfer 4 384-well ELISA plates to the liquid workstation in the analysis area and run the workstation method: 5_2_ELISA_AddTMBand HCl: Open the cover, add HRP substrate TMB, 25ul / well, color development for 12min at room temperature in the dark, the positive wells will turn blue; (do 10 plates in batches); add stop solution 2MHCl, 25ul / well to stop color development, the system will turn yellow;

[0317] 8) Transfer the 384-well ELISA plate to an ELISA reader for detection at 450 nm absorbance;

[0318] 9) Transfer the tested ELISA plate from the ELISA instrument to the trash can;

[0319] (VI) Cell Area: FACS Sampling of Hybridoma Cells

[0320] 1) Consumables enter the workstation: 1-2 96-well V-bottom plates are placed from the consumables stack into the liquid workstation;

[0321] 2) 1-2 96WP cell culture plates (with lids) are transferred from the incubator (cell culture plates with positive ELISA test results) to the liquid workstation table in the cell area;

[0322] 3) Run the cell area liquid workstation method: 6_1_FACS Sampling: According to the ELISA test results, select the positive wells, carefully pipette the supernatant of the corresponding plate number and well number into a 96-well V-bottom plate in order, and the sample volume: 60ul / well for one FACS experiment, 110ul / well for two FACS experiments, and 160ul / well for three FACS experiments;

[0323] 4) Remove consumables from the workstation: transfer 1-2 96WP cell plates from the workstation table to the incubator;

[0324] 5) Consumables out of the workstation: 1-2 96-well V-bottom plates containing hybridoma supernatants are taken out of the liquid workstation and temporarily stored at 4°C in the liquid workstation in the analysis area;

[0325] (VII) Analysis area: FACS detection of hybridoma supernatant

[0326] Manually place the thawed PBS-resuspended cell suspension on the liquid workstation table in the analysis area;

[0327] Manually place PBS at the peristaltic pump position;

[0328] Manually place the 300uLTip head on the workstation table;

[0329] Manual placement of secondary antibody reagent tank;

[0330] Manual placement of waste tanks;

[0331] Each plate produced 115 mL of waste;

[0332] 1) Consumables enter the workstation: 1-2 new 96-well V-shaped plates are transferred from the consumables stack to the liquid workstation in the analysis area;

[0333] 2) 1-2 96-well V-bottom plates containing hybridoma supernatants are transferred from the 4°C temporary storage position of the analysis area liquid workstation to the analysis area liquid workstation;

[0334] 3) Run the workstation method: 7_1_FACS_Add Cell and Supernate: Add 5E5 cells to a new 96-well V-bottom plate. The wells where the cells are added should be consistent with the wells with hybridoma supernatant stock solution. The cell density should be manually adjusted in advance. Then add 50ul / well of hybridoma supernatant stock solution (in the experiment, a no stain group, a secondary antibody single staining group, and a positive control group must be designed). Resuspend the cells by pipetting in a small volume (try to avoid generating bubbles);

[0335] 4) Transfer 1-2 96-well V-bottom plates with cells and supernatant from the liquid workstation in the analysis area to the 4°C temporary storage position on the work surface and incubate for 30 minutes;

[0336] 5) Manually transfer the plate containing hybridoma supernatant from the liquid workstation table in the analysis area to the trash can;

[0337] 6) Manually transfer the cell suspension reagent tank from the liquid workstation table in the analysis area to the trash can;

[0338] 7) Use a peristaltic pump to add PBS to the reagent tank;

[0339] 8) Consumables enter the workstation: 290mL reagent tank enters the liquid workstation in the analysis area;

[0340] 9) Centrifuge the incubated 96-well V-bottom plate and transfer it to the liquid workstation table in the analysis area. Run the workstation method: 7_2_FACS_Wash1: discard the supernatant and wash once with 150ul / well PBS;

[0341] 10) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0342] 11) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_3_FACS_Wash2: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0343] 12) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0344] 13) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_4_FACS_Wash3: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0345] 14) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0346] 15) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_5_FACS_AddAntibody: discard the supernatant and add the secondary antibody to the corresponding wells, 50ul / well;

[0347] 16) Transfer the 96-well V-bottom plate from the liquid workstation to the 4°C chamber and incubate in the dark for 30 minutes.

[0348] 17) Transfer the incubated 96-well V-bottom plate to the liquid workstation table and run the workstation method 7_2_FACS_Wash1: discard the supernatant and wash once with 150ul / well PBS;

[0349] 18) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0350] 19) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_3_FACS_Wash2: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0351] 20) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0352] 21) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_4_FACS_Wash3: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0353] 22) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0354] 23) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 7_6_FACS_AddPBS: discard the supernatant and add 50ul / well PBS to the corresponding wells to resuspend the cells;

[0355] 24) Transfer the 96-well V-bottom plate from the liquid workstation to the flow cytometer for testing (there are 3 flow cytometers);

[0356] (VIII) Cell area: Cell subclone 1 (subclone culture for about 7 days)

[0357] 1) Consumables enter the workstation: 1-4 24WP cell culture plates are transferred from the consumables stack to the cell area liquid workstation;

[0358] 2) A 96WP cell culture plate was transferred from the incubator (positive by FACS) to the liquid workstation;

[0359] 3) Run the workstation method: 5_1_Subclone Sampling: According to the FACS test results, select the positive wells that need to be subcloned, and transfer the wells that need to be subcloned to the corresponding 24-well plate in the order of (transfer volume to be determined) (Mix before transfer), and pipette the wells to be subcloned in the 24-well plate several times with a 1ml pipette tip to resuspend and mix the cells;

[0360] 4) Consumables out of the workstation: 24-well plate out of the workstation to the incubator;

[0361] 5) Consumables out of the workstation: 96WP cell culture plate out of the workstation;

[0362] 6) Manual intervention, starting subcloning experiments, and finally obtaining 96WP containing the first subclone cells (named "96WP subclone cell plate 1");

[0363] (IX) Analysis area: ELISA plate coating_2

[0364] All the processes are the same as in “(III) Analysis Zone: ELISA Plate Coating_1”. The number of coated plates can be selected or determined based on the number of positive cells in the first FACS result.

[0365] Throughput: at least 10 ELISA plates, the number of ELISA plates to be coated can be controlled, and the process is completed in batches of 4;

[0366] Manually place 1ug / ml antigen solution prepared in PBS on the work surface 5 (100mL is required for 10 ELISA plates);

[0367] (X) Cell Area: Subclone Cells_ELSA Sampling

[0368] Note: Manually prepare the "96WP Subcloning Cell Plate 1" for the first subcloning to start the experiment;

[0369] Import the information of the first subcloning cell plate;

[0370] 1) Consumables enter the workstation: A 384-well ELISA plate is transferred from the consumables stack in the analysis area (the temporary storage rack next to the plate washer) to the liquid workstation in the cell area;

[0371] 2) 1-4 96WP subclone cell plates are transferred out of the incubator and put into the cell area liquid workstation to run the workstation method: 10_1_Subclone ELISA Sampling;

[0372] 3) Consumables out of the workstation: 4 96WP subcloning cell plates are taken out of the liquid workstation and put into the incubator;

[0373] 4) Consumables out of the workstation: 1 384-well ELISA plate is taken out of the liquid workstation and put into the transfer plate rack in the analysis area for binding at room temperature for 1 hour;

[0374] (XI) Analysis area: ELISA detection of subclone cell supernatant

[0375] Note: The actions are the same as those in “(V) Analysis Area: Hybridoma Supernatant ELISA Test”, except that the number of ELISA plates is a variable and may not be 10;

[0376] (XII) Cell Area: Subclone Cells_FACS Sampling

[0377] 1) Consumables enter the workstation: 1-2 96-well V-bottom plates are placed from the consumables stack into the liquid workstation;

[0378] 2) 1-2 96WP subclone cell plates are transferred from the incubator (cell culture plates with positive ELISA test results) to the liquid workstation table in the cell area;

[0379] 3) Run the cell area liquid workstation method: 12_1_Subclone FACS Sampling: According to the ELISA test results, select the positive wells and carefully pipette the supernatant of the corresponding plate number and well number into a 96-well V-bottom plate in order;

[0380] 4) Remove consumables from the workstation: transfer 1-2 96WP subcloning cell plates from the workstation table to the incubator;

[0381] 5) Consumables out of the workstation: 1-2 96-well V-bottom plates are taken out of the liquid workstation and temporarily stored at 4°C in the liquid workstation in the analysis area;

[0382] (XIII) Analysis area: Subclone cells_FACS detection

[0383] Thaw the cell suspension resuspended in PBS and place it on the liquid workstation table in the analysis area;

[0384] 1) Consumables enter the workstation: 1-2 new 96-well V-shaped plates are transferred from the consumables stack to the liquid workstation in the analysis area;

[0385] 2) 1-2 96-well V-bottom plates containing hybridoma supernatants are transferred from the 4°C temporary storage position of the analysis area liquid workstation to the analysis area liquid workstation;

[0386] 3) Run the workstation method: 13_1_FACS_Add Cell and Supernate: Add 5E5 cells to a new 96-well V-bottom plate. The wells where the cells are added should be consistent with the wells with hybridoma supernatant stock solution. Then add 50ul / well of hybridoma supernatant stock solution (in the experiment, a nostain group, a secondary antibody single staining group, and a positive control group must be designed). Resuspend the cells by pipetting in a small volume (try to avoid generating bubbles);

[0387] 4) Transfer 1-2 96-well V-bottom plates with cells and supernatant from the liquid workstation in the analysis area to the 4°C temporary storage position on the work surface and incubate for 30 minutes;

[0388] 5) The incubated 96-well V-bottom plate is transferred to the liquid workstation table in the analysis area, and the workstation method is run: 13_2_FACS Wash1: discard the supernatant and wash once with 150ul / well PBS;

[0389] 6) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0390] 7) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_3_FACSWash2: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0391] 8) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0392] 9) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_4_FACSWash3: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0393] 10) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0394] 11) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_5_FACS_AddAntibody: discard the supernatant and add secondary antibody to the corresponding wells, 50ul / well;

[0395] 12) Transfer the 96-well V-bottom plate from the liquid workstation to a 4°C chamber and incubate in the dark for 30 minutes;

[0396] 13) Transfer the incubated 96-well V-bottom plate to the liquid workstation table and run the workstation method 13_2_FACSWash1: discard the supernatant and wash once with 150ul / well PBS;

[0397] 14) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0398] 15) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_3_FACSWash2: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0399] 16) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0400] 17) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_4_FACSWash3: discard the supernatant (aspirate as clean as possible), wash once with 200ul / well PBS;

[0401] 18) Transfer the 96-well V-bottom plate from the liquid handling station to the centrifuge and centrifuge at 1500 rpm for 1 min;

[0402] 19) Transfer the 96-well V-bottom plate from the centrifuge to the liquid workstation table and run the workstation method: 13_6_FACS_AddPBS: discard the supernatant and add 50ul / well PBS to the corresponding wells to resuspend the cells;

[0403] 20) Transfer the 96-well V-bottom plate from the liquid workstation to the flow cytometer for testing (there are 3 flow cytometers);

[0404] (XV) Cell area: Cell subclone 2 (subclone culture for about 7 days)

[0405] 1) Consumables enter the workstation: 1-4 24WP cell culture plates are transferred from the consumables stack to the cell area liquid workstation;

[0406] 2) One 96WP subclone cell plate was transferred from the incubator (positive for the second FACS test) to the liquid workstation;

[0407] 3) Run the workstation method: 8_1_Subclone2Sampling: According to the FACS test results, select the positive wells that need to be subcloned, transfer the wells that need to be subcloned to the corresponding 24-well plate in order (Mix before transfer), and pipette the wells to be subcloned in the 24-well plate several times with a 1 ml pipette tip to resuspend and mix the cells;

[0408] 4) Consumables out of the workstation: 24-well plate out of the workstation to the incubator;

[0409] 5) Consumables out of the workstation: 96WP subcloning cell plate out of the workstation;

[0410] 6) Manual intervention, starting subcloning experiments, and finally obtaining 96WP containing the second subclone cells (named "96WP subclone cell plate 2");

[0411] (XV) Cell Area: Identification and Expansion

[0412] According to the results of ELISA and FACS, two positive monoclonal wells in good condition were selected for each hybridoma cell line for cell identification (manual operation);

[0413] 1) Consumables enter the workstation: 1-4 24WP cell culture plates (with lids) are scanned from the consumables stack and transferred to the liquid workstation table in the cell area; (the number of plates transferred is determined by the second FACS results);

[0414] 2) A 96WP subcloning cell plate II is transferred from the incubator to the liquid workstation;

[0415] 3) Run the workstation method: 9_1_Subclone2Sampling: Add complete medium to a new 24-well cell culture plate, 1 mL / well, suspend the cells that have undergone two subclones by pipetting and transfer them to the corresponding 24WP cell culture plate, and mix them evenly by pipetting;

[0416] 4) Consumables out of the workstation: 1-4 24WP cell culture plates (with lids) are transferred from the workstation to the incubator.

[0417] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma high-throughput screening system, characterized in that: include: A cell culture area, the cell culture area comprising a first table unit, a first storage unit, a first liquid working unit, a culture unit, and a first transport unit, wherein the first storage unit, the first liquid working unit, the culture unit, and the first transport unit are arranged on the first table unit; A cell analysis area, the cell analysis area comprising a second table unit, a second storage unit, a centrifugal unit, a cell analysis unit, a detection unit, a second liquid working unit, a cleaning unit, and a second transport unit, wherein the second storage unit, the centrifugal unit, the cell analysis unit, the detection unit, the second liquid working unit, the cleaning unit, and the second transport unit are arranged on the second table unit; A transfer zone is arranged between the cell culture zone and the cell analysis zone, and is used for obtaining the cell plate transferred from the cell culture zone / the cell analysis zone and transferring the cell plate to the cell analysis zone / the cell culture zone.

2. The hybridoma high-throughput screening system according to claim 1, characterized in that: The cell culture area also includes: A first control unit is disposed on the first table unit and is respectively connected to the first liquid working unit, the culture unit, and the first transport unit.

3. The hybridoma high-throughput screening system according to claim 1 or 2, characterized in that: The cell culture area also includes: The first garbage collection unit is disposed inside the first table unit.

4. The hybridoma high-throughput screening system according to claim 3, characterized in that: The cell culture area also includes: A first sliding unit is disposed inside the first table unit and is used to accommodate the first garbage collecting unit.

5. The hybridoma high-throughput screening system according to claim 1, characterized in that: The cell analysis area also includes: a second control unit, which is disposed on the second table unit and is respectively connected to the centrifugal unit, the cell analysis unit, the detection unit, the second liquid working unit, the cleaning unit, and the second transport unit; and / or A second sliding unit is disposed inside the second table unit and is used to accommodate the centrifugal unit.

6. The hybridoma high-throughput screening system according to claim 1 or 5, characterized in that: The cell analysis area also includes: The second garbage collecting unit is arranged inside the second table unit.

7. The hybridoma high-throughput screening system according to claim 6, characterized in that: The cell analysis area also includes: A third sliding unit is disposed inside the second table unit and is used to accommodate the second garbage collecting unit.

8. The hybridoma high-throughput screening system according to claim 1, characterized in that: The transfer area includes: A transfer unit is disposed between the cell culture area and the cell analysis area, and is used for acquiring the cell plate transferred from the cell culture area / the cell analysis area and transferring the cell plate to the cell analysis area / the cell culture area.

9. The hybridoma high-throughput screening system according to claim 1 or 8, characterized in that: The transfer area also includes: An isolation unit is disposed between the cell culture area and the cell analysis area and connected to the transfer unit, and is used to isolate the cell culture area from the cell analysis area.

10. A method for high-throughput screening of hybridomas, applied to the high-throughput screening system for hybridomas as claimed in any one of claims 1 to 9.

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