CN112210487A - Device and method for high-throughput screening of plasma cells secreting antigen-specific antibodies - Google Patents
Device and method for high-throughput screening of plasma cells secreting antigen-specific antibodies Download PDFInfo
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Abstract
The invention discloses a device for high-throughput screening of plasma cells secreting antigen-specific antibodies, which comprises: the cross section of each micropore is not more than 500 microns, and the micropore array is used for paving an anti-IgG secondary antibody mixed solution which is coupled and labeled by the B cells and the fluorescent probes; a cover sheet for covering the microwell array, the cover sheet having an antigen embedded in a surface facing the microwell array. The invention also provides a method for high-throughput screening of secretory antigen specific antibody plasma cells. The invention can greatly shorten the development time of the monoclonal antibody, greatly improve the time efficiency of the obtained monoclonal antibody and reduce the screening cost.
Description
Technical Field
The invention relates to the technical field of cell culture and detection. More particularly, the present invention relates to a device and method for high throughput screening of antigen-specific antibody secreting plasma cells.
Background
Hybridoma screening technology is still one of the most commonly used methods for antibody development at present, (murine hybridomas) have since the invention in 1975, and the technology has become very mature through decades of development. Taking the spleen of a mouse immunized by the antigen, preparing single cell suspension, fusing with a myeloma cell line from the mouse, subcloning the fused cells for 2 to 3 times to form stable monoclonal cells, immunizing a healthy mouse by the cell lines, preparing ascites, and purifying the ascites to obtain the monoclonal antibody, wherein the whole experimental period is about 2 to 3 months. Unfortunately, in the actual monoclonal antibody preparation process, it is easy to obtain antibodies, but it is difficult to obtain antibodies with the desired properties, such as neutralizing properties (vaccine development), ELISA, western blot and immunohistochemical (clinical diagnosis) properties. In addition, the method has the limitations of long screening period, unstable gene of the generated hybridoma cell, poor screening specificity and positive rate, poor universality and the like, and is mostly applied to the preparation of the mouse monoclonal antibody at present. With the technical progress and the increasing demand of monoclonal antibodies for other species such as rabbits and alpacas, some new methods such as a cell chip method for labeling B cells, a positive and negative screening method, etc. are continuously appeared according to research and knowledge at present. However, these methods have high requirements for B cell surface markers and differentiation stages, especially there are few antibody tools for B lymphocytes containing specific surface markers in different stages, and these methods still face great challenges in specific positive rate, high throughput automation, and effective reduction of single antibody discovery cost.
Disclosure of Invention
An object of the present invention is to provide an apparatus and method for high throughput screening of plasma cells secreting antigen-specific antibodies, which can greatly shorten the time for developing monoclonal antibodies, and can greatly improve the time efficiency of the obtained monoclonal antibodies and reduce the screening cost.
To achieve these objects and other advantages in accordance with the present invention, according to one aspect of the present invention, there is provided an apparatus for high throughput screening of antigen-specific antibody-secreting plasma cells, comprising:
the cross section of each micropore is not more than 500 microns, and the micropore array is used for paving an anti-IgG secondary antibody mixed solution which is coupled and labeled by the B cells and the fluorescent probes;
a cover sheet for covering the microwell array, the cover sheet having an antigen embedded in a surface facing the microwell array.
Further, the device for high-throughput screening of plasma cells secreting antigen-specific antibodies, wherein the microwell array is formed on the bottom surface of the interior of the culture dish, and covers a part of or the whole area of the bottom surface of the interior.
Further, the device for high-throughput screening of the plasma cells secreting the antigen-specific antibody has the depth of the micropores not more than 500 microns.
Further, the device for high-throughput screening of the plasma cells secreting the antigen-specific antibody has the capacity of the micropore of not more than 150 nL.
Further, the cross-sectional shape of the micropore is a double-ear circle, a double-ear square, a double-ear hexagon, a double-ear rectangle, a double-ear rhombus, a single-ear square, a single-ear hexagon, a single-ear rectangle or a single-ear rhombus.
According to another aspect of the present invention, there is provided a method for high throughput screening of antigen-specific antibody-secreting plasma cells, comprising:
b cells and anti-IgG secondary antibody mixed solution labeled by coupling of fluorescent probes are paved on a micropore array in the device for screening and secreting antigen-specific antibody plasma cells in high flux, and a cover plate embedded with antigens is used for covering the micropore array;
putting the mixture into an incubator for incubation;
and selecting the micropores with the fluorescence signals in the micropore array to obtain the plasma cells in the micropores.
Further, the method for screening the plasma cells secreting the antigen-specific antibody in a high throughput manner uses a carbon dioxide incubator to incubate for 2-3 hours.
Further, the method for high-throughput screening of the plasma cells secreting the antigen-specific antibody scans the micropore array by using an inverted fluorescence microscope, and the micropores with fluorescence signals are determined.
Further, in the method for high-throughput screening of the plasma cells secreting the antigen-specific antibody, the fluorescent probe coupled with the anti-IgG secondary antibody comprises FITC and Cy 3.
Further, the method for high-throughput screening of plasma cells secreting antigen-specific antibodies, the method for embedding antigen on a cover slip, comprises:
coating the antigen on the surface of the cover plate, standing, sealing by bovine serum albumin, and washing by buffer solution and culture medium in sequence.
The invention at least comprises the following beneficial effects:
the invention adopts a micropore array with ultrahigh flux to separate single plasma cells, forms a closed single-cell microenvironment in the single micropore by embedding antigen through a cover plate and combining an indirect fluorescence immunoassay method, screens the antigen-specific antibody plasma secreting cells, and separates single positive plasma cells by using a single-cell microfluidic capture technology such as a glass capillary and the like, so that the time for finally obtaining the antigen-specific antibody plasma cells is shortened to one day from 2-3 weeks of the traditional hybridoma method. The invention not only can greatly shorten the development time of the monoclonal antibody, but also can greatly improve the time efficiency of the obtained monoclonal antibody and reduce the screening cost.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic structural diagram of an apparatus for high throughput screening of antigen-specific antibody secreting plasma cells according to one embodiment of the present invention;
FIG. 2 is a schematic diagram of the structure of a microwell array in one embodiment of the present invention;
FIG. 3 is a photograph of positive plasma cells taken under a fluorescence microscope using white light and fluorescence, respectively;
FIG. 4 is a dynamic diagram showing the interaction among the antigen, specific antibody and secondary antibody embedded in the patch of the present invention; 0 hour: the mixed liquid of the plasma cells is just paved and covered with an antigen embedding cover plate; 1 hour: incubating the micropore array in a cell culture box for 1 hour, gradually increasing the secreted antibody and beginning to combine with the specific antigen on the cover plate; more than 2 hours: the secreted antigen-specific antibody is fully combined with the cover plate embedded antigen and combined with the secondary antibody to trigger the increase of immunofluorescence intensity (the local fluorescence signal is enhanced, and the position of positive plasma cells in the micropore array is distinguished);
FIG. 5 is a diagram of the effect of the microscope on the bright field after B cell plating of the microwell array;
FIG. 6 is a white light/fluorescence scan under a 10X microscope and a superimposed effect plot, with the fluorescence signal showing the location of plasma cells secreting antigen-specific antibodies;
FIG. 7 is a white light/fluorescent microwell array cell scan under a 10X microscope with cover slips without embedded antigen;
FIG. 8 is a graph of the effect of fluorescence scanning of a microwell array at 30 minute intervals.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in fig. 1 to 8, embodiments of the present application provide an apparatus for high throughput screening of secretory antigen-specific antibody plasma cells, comprising: the cross section of each micropore is not more than 500 microns, and the micropore array is used for paving an anti-IgG secondary antibody mixed solution which is coupled and labeled by the B cells and the fluorescent probes; a cover sheet for covering the microwell array, the cover sheet having an antigen embedded in a surface facing the microwell array.
In this embodiment, the micro-well array 2 can be formed on the surface of any existing cell culture tool, such as a petri dish 1, which can be made of glass, polypropylene, polyethylene, or polytetrafluoroethylene. The microwell array 2 may be cut, micro-machined, embossed or stamped by light or any other standard method. The number of microwells in microwell array 2 can be adjusted as many as thousands to millions as necessary for high throughput screening. Cell culture liquid at the top of each micropore can be communicated with each other, so that cell secretion in each micropore can exchange information through the micropores around the empty upper layer space flow, and the growth of cells in a single micropore is promoted. The size of the micropores can be adjusted according to the needs, but should not be larger than 500 μm, otherwise the individual culture requirements of cell samples such as rare cell lines, single cells or monoclonal clusters cannot be met. The shape of the microwell is not limited, and may be any shape that can satisfy the culture requirements. The fluorescent probe may be any of those known in the art. After the cover plate covers the micropore array, each micropore and the cover plate form an independent single-cell microenvironment, a cell function experiment can be carried out in the independent single-cell microenvironment, and antigen-specific antibody secretory plasma cells can be rapidly analyzed and screened.
Alternatively, each microwell in microwell array 2 may be scanned by a microscope and then registered by software and used with its respective microwell ID number to facilitate identification and repeated viewing of the cell sample therein. Optionally, the microwell array supports single cells or monoclonal cell lines in transfer wells using glass capillaries. The cover plate is embedded and fixed with antigen, the cover plate can be made of polystyrene, the embedding mode can adopt any feasible prior art, and the cover plate embedded with the antigen is used for carrying out cell indirect immune response.
After the cover plate is covered on the micropore array, each micropore and the cover plate enclose an independent closed chamber, namely, an independent single-cell microenvironment is provided, non-contact plasma cell antibody secretion amount is measured, the antigen is embedded and fixed on the cover plate, exogenous antigen is not contacted with the cell surface, cell surface antibody reaction cannot be caused, the secreted antibody can freely float to the vicinity of the upper cover plate to be combined with the antigen and trigger the immunofluorescence reaction (the antibody is fully combined with the cover plate embedded antigen and combined with a second antibody to trigger the immunofluorescence intensity to be increased), namely, an indirect fluorescence immunoassay method, and then accurate antigen-specific antibody detection is carried out through fluorescence signals, and plasma cells are screened. When the device is used, the anti-IgG secondary antibody mixed solution labeled by coupling B cells and fluorescent probes is paved on a micropore array in the device for screening the plasma cells secreting the antigen-specific antibodies in high flux, a cover plate embedded with the antigens is used for covering and closing the micropore array, the micropore array is placed into an incubator for incubation, micropores with fluorescent signals in the micropore array are selected, the plasma cells in the micropores are obtained, screening is completed, and then downstream analysis such as nucleic acid extraction, PCR amplification, RNA sequencing and the like is carried out, or culture and the like are carried out. In the embodiment, a micropore array with ultrahigh flux is adopted to separate single plasma cells, a closed single-cell microenvironment is formed in the single micropore by the establishment of a cover plate embedding antigen and an indirect fluorescence immunoassay, the antigen-specific antibody secreting plasma cell screening is carried out, single positive plasma cells are separated by using single-cell microfluidic capture technologies such as a glass capillary and the like, and the time for finally obtaining the antigen-specific antibody plasma cells is shortened to one day from 2-3 weeks of the traditional hybridoma method. Therefore, the embodiment not only can greatly shorten the development time of the monoclonal antibody, but also can greatly improve the time efficiency of the obtained monoclonal antibody and reduce the screening cost.
In other embodiments, the microwell array 2 is formed on the inner bottom surface of the culture dish 1, covering a part of or the whole area of the inner bottom surface. In the present embodiment, the above description is referred to with respect to the microwell array 2, and the culture dish 1 in the present embodiment may be any existing culture dish 1. Table 1 shows some examples of culture dishes and microwells.
TABLE 1 number and Specifications of micropores in Petri dish
| Diameter or length | High or deep | Volume of micro pores | Cell culture dish well number | Number of micropores per dish hole | Total number of micropores | |
| 350μm | 350μm | 60nl | 6/24 | 4000/750 | 2.4 ten thousand/1.8 ten thousand | |
| 200μm | 100μm | 4nl | 6/24 | 2.2 Wan/4300 | 13 ten thousand/10 ten thousand | |
| 100μm | 50μm | 500pl | 6/24 | 8 Wan/15700 | 48 ten thousand/38 ten thousand | |
| 100μm | 100μm | 800pl | 6 | 6 ten thousand | 36 ten thousand | |
| 40μm | 40μm | 50pl | 6 | 15 ten thousand | 90 ten thousand | |
| | 25μm | 12pl | 2 | 20 ten thousand | 40 ten thousand | |
| 25μm | 100μm | 48pl | 6 | 20 ten thousand | 120 ten thousand | |
| 12μm | 12μm | 1.5pl | 6 | 90 ten thousand | 500 ten thousand |
In other embodiments, the microwells are no more than 500 μm deep and should be of sufficient depth to achieve separation of cells, typically no more than 500 μm.
In other embodiments, the volume of the microwells is no greater than 150nL, as long as the volume of the microwells is sufficient for cell culture, and is typically no greater than 150 nL. .
In other embodiments, the cross-sectional shape of the microwell is binaural round, binaural square, binaural hexagon, binaural rectangle, binaural rhombus, monaural square, monaural hexagon, monaural rectangle, or monaural rhombus, which are more convenient for capturing cells with a glass capillary.
Embodiments of the present application also provide a method for high throughput screening of secretory antigen specific antibody plasma cells, comprising: b cells and anti-IgG secondary antibody mixed solution labeled by coupling of fluorescent probes are paved on a micropore array in the device for screening and secreting antigen-specific antibody plasma cells in high flux, and a cover plate embedded with antigens is used for covering the micropore array; putting the mixture into an incubator for incubation; and selecting the micropores with the fluorescence signals in the micropore array to obtain the plasma cells in the micropores.
In the above examples, reference is made to the above description for a device for high throughput screening of antigen-specific antibody-secreting plasma cells. The antigen is bound to the cover sheet by physical adsorption, the preferable material of the cover sheet is polystyrene, the source of the B cell can be found in the prior art, the spleen cell derived from mammal such as mouse, rat, rabbit, etc. is preferable, and any fluorescent probe in the prior art can be used as the fluorescent probe. After the cover plate is covered on the micropore array, each micropore forms an independent closed chamber, namely, an independent single-cell microenvironment is provided, non-contact plasma cell antibody secretion amount is measured, the antigen is embedded and fixed on the cover plate, exogenous antigen is not contacted with the cell surface, cell surface antibody reaction is not caused, the secreted antibody can freely float to the position near the upper cover plate to be combined with the antigen, and immunofluorescence reaction of the antibody is triggered (the antibody is fully combined with the cover plate embedded antigen and combined with a second antibody to trigger immunofluorescence intensity to be increased), and then accurate antigen-specific antibody detection is carried out through fluorescence signals, and plasma cells are screened. A cell suspension liquid mixed by B cells and an anti-IgG antibody fluorescent secondary antibody is used, and uniform Z-shaped liquid is required to be added when the plate is paved, and a small amount of liquid is added for many times.
It can be seen that this example enables high throughput screening, using a large number of wells, but concentrated in a single plate; a single plasma cell secretes the antibody, and each micropore can realize single cell separation, observation and test through the antibody secretion process for 2-3 hours; an indirect fluorescence immunoassay method, which does not depend on direct marker marking on the surface of B cells, but indirectly screens antibodies secreted by plasma cells with antigen specificity by using a fluorescence-labeled secondary antibody; the cover plate embedded with the antigen is combined with a fluorescent secondary antibody, and the indirect fluorescence immune screening of positive plasma cells is realized through microscopic scanning); accurate localization tracking, by microwell ID number, accurately identifies positive plasma cells and captures them for downstream analysis.
In other embodiments, the incubation is performed using a carbon dioxide incubator for 2 to 3 hours, preferably 37 ℃.
In other embodiments, the microwell array is scanned with an inverted fluorescence microscope to determine microwells for which a fluorescent signal is present. After incubation, the microporous array plate with cells and a cover is placed on an inverted fluorescence microscope, and a 10-fold objective bright field and fluorescence scanning are used to set the fluorescence signal threshold and other detection parameters. Selecting a hole with a fluorescent signal (identifying target plasma cells expressed by specific antibodies), picking single plasma cells in each micropore by a glass capillary or other single-cell microfluidic capture technology, transferring the single plasma cells to a PCR (polymerase chain reaction) tube, and then carrying out downstream analysis such as nucleic acid extraction, PCR amplification and RNA sequencing to find a target antibody sequence or carrying out re-culture.
In other embodiments, the fluorescent probe coupled to the anti-IgG secondary antibody comprises FITC, Cy 3. These examples provide alternative fluorescent probes, which may be selected according to actual needs and do not represent specific limitations on fluorescent probes.
In other embodiments, the method of embedding an antigen on a coverslip comprises: coating the antigen on the surface of the cover plate, standing, sealing by bovine serum albumin, washing by buffer solution and culture medium in sequence, wherein the type, concentration, standing time, sealing time, buffer solution type and the like of the antigen can be selected according to actual requirements.
The following is further illustrated by a specific example:
screening rabbit antigen-specific antibody secreting plasma cells:
1. the rabbit was taken, weighed four jin above, with obvious auricless, arterial and healthy, and was injected intravenously one week earlier with 0.4g of the purified emulsified antigen, in this example Human alpha-fetoprotein (Human AFP) (CUSABIO), and the back multipoint injection method was used.
2. One week later, the rabbits were sacrificed, spleens were separated according to standard procedures required by the animal center laboratories, and splenocytes were obtained.
Screening the cells, centrifuging, removing supernatant, cracking red blood cells, centrifuging, filtering and the like to remove impurities, and counting 100 microliters of cells;
3. using a microwell array (microwell size: 25 μm ID, 100 μm deep, 48pl volume) with a total of 20 ten thousand microwells, 1ml of absolute ethanol was injected into the whole array patch to fill the whole patch, and centrifuged (2000g, 2min) at room temperature to remove a small amount of air bubbles on the surface of the patch.
4. And (3) washing the cover plate: 0.5ml of absolute ethanol was aspirated, and 2ml of CBS buffer (pH 9.6) was added: na (Na)2CO3 1.59g+NaHCO32.94g, then siphoned off again and CBS washed 5 times in total. The surface of the cover slip cannot be kept free of liquid (at least 0.5ml) and does not need to be tilted during movement.
5. Cover sheet coating
0.5ml of antigenic Human alpha-fetoprotein (Human AFP) was added, at a concentration of 80-100. mu.g/ml being recommended, and the whole patch was covered. Left overnight at 4 ℃. The next day, the coating solution was discarded, washed 3 times with CBS buffer, and blocked by adding 0.5ml of 2% bovine serum albumin BSA at 37 ℃ for 1 hour. After blocking, the cells were washed 3 times with CBS and 3 times with medium.
6. B cell plating
0.5ml of a cell suspension (density 106 cells/ml) and a mixture of fluorescent probe Cy 3-labeled anti-rabbit IgG antibody (concentration 20. mu.g/ml, ThermoFisher) were added to each dish, and a uniform "Z" -shaped gentle solution was added as many times as possible during plating. Standing for 10 minutes at room temperature; centrifuge for 3 minutes at 300 g.
7. Incubation and scanning
The embedded cover plate is covered on the micropore array and put into a carbon dioxide incubator for incubation for 2-3 hours at 37 ℃. The 0 hour can be scanned with microscope white light as in fig. 5. If necessary, the culture time may be appropriately prolonged, but it is not recommended to exceed 4 hours.
And (3) screening positive plasma cells by using an automatic fluorescence inverted microscope and a 10-fold objective lens to scan a bright field and a fluorescence visual field, setting a fluorescence signal threshold value and other detection parameters, wherein the screened cells are Cy3-IgG positive single-cell micropores (single plasma cells with specific antibody expression). Other differentiation stage B cells such as mature B lymphocytes and plasma cells can be distinguished under a white light field of view by a 40x objective lens.
8. Taking down the cover plate, separating the single plasma cells in the positive micropores into a PCR tube by a glass capillary or other single cell microfluidic capture technologies, and then performing downstream analysis such as nucleic acid extraction, PCR amplification, RNA sequencing and the like, or performing culture and the like.
After continuous scanning of the microplate array culture dishes without removing the cover plate, the antibody secretion of plasma cells was observed to increase from 1 hour of culture and gradually stabilize at about 2-3 hours after observation for 5 hours, as shown in FIG. 8, which shows that the antibody secretion decreased or gradually stopped, and the activity of plasma cells may be decreased with the increase of culture time. The method can clearly and visually see the change of the antibody secretion of each plasma cell along with time, and can carry out parallel quantitative tracking; the optimal culture scanning time can be set through the experiment, the plasma cells with the best secretory antibody positive rate are collected one by one through a single-cell microfluidic capture technology and transferred to a PCR tube for reverse transcription, and other applications are carried out. Because the number of target antibodies in the specific plasma cells is the largest, the probability of obtaining effective sequences of the antibodies through single-cell reverse transcription, gene amplification and sequencing is greatly improved, the workload of expression verification of downstream fusion proteins is greatly reduced, and the screening efficiency of the antibody sequences is greatly improved.
To verify the specificity of the positive fluorescence signal in this application, we designed control experiments: the microwell array coverslips were free of embedded antigen and the B cells (containing plasma cells) and CY 3-anti-rabbit IgG secondary antibody mixture were cultured directly. The experimental result can verify that the added fluorescent secondary antibody is combined with the antibody secreted by the plasma cells in the absence of the antigen, but any biological or chemical reaction is a dynamic process, the secreted antigen-specific antibody is distributed and dispersed in the pores under the condition of lacking the antigen for fixing, and cannot generate aggregation effect within a certain time, so that the experimental result is reflected, the fluorescent signal depends on the distribution characteristics of the antibody secreted by the plasma cells, the aggregation effect cannot be formed, and the fluorescent signal tested in the system is close to the background light and has no detectable fluorescent signal.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. An apparatus for high throughput screening of plasma cells secreting antigen-specific antibodies, comprising:
the cross section of each micropore is not more than 500 microns, and the micropore array is used for paving an anti-IgG secondary antibody mixed solution which is coupled and labeled by the B cells and the fluorescent probes;
a cover sheet for covering the microwell array, the cover sheet having an antigen embedded in a surface facing the microwell array.
2. The apparatus for high throughput screening of antigen-specific antibody secreting plasma cells according to claim 1, wherein said microwell array is formed on the bottom surface of the interior of the culture dish covering a portion or the entire area of said bottom surface.
3. The device for high throughput screening of antigen-specific antibody secreting plasma cells of claim 1, wherein said microwells are no deeper than 500 μm.
4. The device for high throughput screening of antigen-specific antibody secreting plasma cells of claim 1 wherein said microwell has a volume of no more than 150 nL.
5. The device for high throughput screening of antigen-specific antibody secreting plasma cells according to claim 1, wherein said microwell has a cross sectional shape of a binaural circle, a binaural square, a binaural hexagon, a binaural rectangle, a binaural rhombus, a monaural square, a monaural hexagon, a monaural rectangle, a monaural rhombus, or an irregular shape having one or both ears.
6. A method for high throughput screening of plasma cells secreting antigen-specific antibodies comprising:
spreading a mixed solution of B cells and an anti-IgG secondary antibody which is coupled and labeled by a fluorescent probe to a micropore array in the device for screening the plasma cells secreting the antigen-specific antibody in high throughput according to claim 1, and covering the micropore array with a cover plate embedded with the antigen;
putting the mixture into an incubator for incubation;
and selecting the micropores with the fluorescence signals in the micropore array to obtain the plasma cells in the micropores.
7. The method for high-throughput screening of plasma cells secreting antigen-specific antibody according to claim 6, wherein the incubation is performed for 2 to 3 hours using a carbon dioxide incubator.
8. The method for high throughput screening of antigen-specific antibody secreting plasma cells of claim 6 wherein said microwell array is scanned with an inverted fluorescence microscope to determine microwells that have a fluorescent signal present.
9. The method for high throughput screening of antigen-specific antibody secreting plasma cells of claim 6 wherein the fluorescent probe conjugated to the anti-IgG secondary antibody comprises FITC, Cy 3.
10. The method for high throughput screening of antigen-specific antibody secreting plasma cells of claim 6 wherein the method of embedding antigen on a patch comprises:
coating the antigen on the surface of the cover plate, standing, sealing by bovine serum albumin, and washing by buffer solution and culture medium in sequence.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100152054A1 (en) * | 2005-09-16 | 2010-06-17 | Love J Christopher | Screening assays and methods |
| US20140287402A1 (en) * | 2011-06-27 | 2014-09-25 | Valneva | Method for screening cells |
| US20170074872A1 (en) * | 2015-07-13 | 2017-03-16 | Nidus Biosciences, LLC | High-throughput single cell sorting using microbubble well arrays |
| CN109154017A (en) * | 2016-03-17 | 2019-01-04 | 不列颠哥伦比亚大学 | The device and method of cell secretion analysis |
| US20190056398A1 (en) * | 2016-02-26 | 2019-02-21 | Sri International | Identification and isolation of antibodies from white blood cells |
-
2020
- 2020-09-30 CN CN202011064202.4A patent/CN112210487A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100152054A1 (en) * | 2005-09-16 | 2010-06-17 | Love J Christopher | Screening assays and methods |
| US20140287402A1 (en) * | 2011-06-27 | 2014-09-25 | Valneva | Method for screening cells |
| US20170074872A1 (en) * | 2015-07-13 | 2017-03-16 | Nidus Biosciences, LLC | High-throughput single cell sorting using microbubble well arrays |
| US20190056398A1 (en) * | 2016-02-26 | 2019-02-21 | Sri International | Identification and isolation of antibodies from white blood cells |
| CN109154017A (en) * | 2016-03-17 | 2019-01-04 | 不列颠哥伦比亚大学 | The device and method of cell secretion analysis |
Non-Patent Citations (2)
| Title |
|---|
| NAVIN VARADARAJAN ET AL.: ""Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving"", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE OF THE UNITED STATES OF AMERICA》 * |
| 李燕主编: "《精编分子生物学实验技术》", 30 September 2017 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115166241A (en) * | 2022-08-22 | 2022-10-11 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
| CN115166241B (en) * | 2022-08-22 | 2023-03-24 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
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