CN118006552A - Screening method of antigen-specific plasma cells and application thereof - Google Patents
Screening method of antigen-specific plasma cells and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of antibody preparation, and provides a screening method of antigen-specific plasma cells and application thereof. The screening method of the antigen specific plasma cells sequentially comprises the steps of obtaining the bispecific antibody, obtaining the fluorescent antigen, obtaining the cells with fluorescence and separating out the single antigen specific plasma cells, so that the single antigen specific plasma cells can be obtained, the preparation cost is low, the operation is simple, and the screening time is short.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a screening method of antigen-specific plasma cells and application thereof.
Background
Monoclonal antibodies (monoclonal antibodies) have high specificity and affinity and are now widely used in the diagnosis and treatment of viral infections, tumors, autoimmune diseases, neurological diseases, etc., and high quality monoclonal antibodies are important for disease treatment and development of high performance immunodiagnostic reagents. Therefore, the rapid and efficient antibody discovery technology has important significance for developing antibody medicaments and diagnosing diseases.
Plasma cells (PLASMA CELL, PC), also known as Antibody secreting cells (ASC, SECRETING CELL), are the most mature B cells in the immune system that release large amounts of antibodies, monoclonal antibodies are highly homogeneous antibodies raised by single B cell clones and directed against only one specific epitope, whereas antibodies are raised by plasma cells by the body due to stimulation of antigens, so direct screening of single antigen-specific plasma cells is the most ideal, most direct and most efficient way of monoclonal Antibody discovery, and is also the heart of monoclonal Antibody discovery technology.
In addition, the number of plasma cells in the peripheral blood of the organism is not large, and the number of the plasma cells is usually about 50 antigen-specific plasma cells/10000 plasma cells after immunization, so that the screening accuracy is higher, and the subsequent sequencing and expression costs are lower. However, the membrane surface of plasma cells has no membrane-bound antibody, the antibody is secreted once it is mature, and plasma cells have no means to culture them in vitro for a long period of time, the amount of antibody secreted by single cells is very small, and the secreted antibody is difficult to detect, so it is very difficult to directly screen single antigen-specific plasma cells.
In order to solve the problem that screening single antigen specific plasma cells is very difficult, related technicians develop a microwell array for capturing secondary antibodies, each microwell can just hold single plasma cells, plasma cells are distributed into each microwell to be cultured for 3.5 hours, antibodies secreted by the plasma cells are captured by the secondary antibodies, then antigens coupled with fluorescent dyes are added to dye, the antibodies secreted by the plasma cells in the holes with strong fluorescence have better performance, and a micromanipulator can be used for picking the plasma cells to sequence to obtain an antibody sequence.
In addition, related technicians also developed a Beacon system for screening plasma cells based on an optical tweezers technology, which comprises a microfluidic chip and a set of optical tweezers equipment, wherein the microfluidic chip is divided into a plurality of small chambers, each plasma cell is separated into each small chamber to be cultured for a period of time, then the culture supernatant is developed through microspheres coupled with antigens and secondary antibodies coupled with fluorescence, and the plasma cells in the chambers with strong fluorescence are separated by using the optical tweezers system to sequence to obtain an antibody sequence.
Although both of the above techniques can achieve single antigen-specific plasma cell screening, the cost per set of equipment is high, and the operation is complicated and the screening time is long.
Disclosure of Invention
The invention provides a screening method of antigen-specific plasma cells and application thereof, and aims to solve the problems that the cost of screening single antigen-specific plasma cells is high, the operation is complex and the screening time is long in the prior art.
In a first aspect, the present invention provides a method of screening antigen-specific plasma cells comprising the steps of:
S1, carrying out a first reaction on a specific antibody of an anti-species and a 2-IT solution, filtering and desalting the specific antibody after the reaction, and redissolving the specific antibody by using a buffer solution to filter and desalt the specific antibody;
Carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution;
Uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring uniformly at a low temperature to obtain a bispecific antibody;
s2, diluting an antigen to be marked into an antigen solution of 10mg/ml by using a CB solution, and filling the antigen solution into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; absorbing the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain fluorescent antigen;
S3, extracting PBMC, adding an anti-CD 16/32 antibody to block Fc receptor of the PBMC, and performing first incubation; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells;
S4, separating out the single antigen specific plasma cells by a single cell separation technology to obtain the single antigen specific plasma cells.
Preferably, in the step S1, the first reaction and the second reaction are performed by using a rotary incubator, respectively; the mass of the specific antibody of the species to be anti-and the antibody of the surface marker molecule specific to the plasma cell to be marked is 0.1 mg; the volume of the 2-IT solution is 2 mu L, and the concentration is 6 mg/mL; the temperature of the rotary incubator is 25 ℃ during the first reaction, and the reaction time is 60min; the buffer was 0.05M PBS buffer at pH 7.0.
Preferably, in the step S1, the volume of the SMCC solution is 6 mu L, and the concentration is 7 mg/mL; the temperature of the rotary incubator is 25 ℃ during the second reaction, and the reaction time is 30min; the mixing is carried out by a refrigerator at the temperature of 4 ℃ for 18 hours.
Preferably, in the step S2, the concentration of the CB solution is 0.025mol/L and the pH value is 9.0; the stirring is performed by an electromagnetic stirrer.
Preferably, in the step S2, the volume of the fluorescein solution is 10 times that of the antigen solution when immersed in the fluorescein solution; after immersing the dialysis bag in the fluorescein solution, stirring the fluorescein solution in an electromagnetic stirrer at the temperature of 4 ℃ for 18-24 hours, and filling the fluorescein solution in a beaker.
Preferably, in the step S3, the anti-CD 16/32 antibody has a mass of 2. Mu.g; the first incubation and the second incubation are carried out in an environment at 4 ℃, the time of the first incubation is 5-10 min, and the time of the second incubation is 20min; the culture is performed by using a cell culture box.
Preferably, in the step S3, when the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule are incubated for the second time, the mass of each of the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule is 1 μg; the number of times of the first centrifugal washing, the second centrifugal washing and the third centrifugal washing is 3.
In a second aspect, the invention provides an application of a screening method of antigen-specific plasma cells in preparation of monoclonal antibodies, wherein single antigen-specific plasma cells obtained by screening are subjected to DNA extraction, amplification and sequencing to obtain specific monoclonal antibody genes, and the specific monoclonal antibody genes are subjected to recombinant expression to obtain monoclonal antibodies.
In a third aspect, the present invention provides a method for obtaining a monoclonal antibody, comprising the steps of:
S1, carrying out a first reaction on a specific antibody of an anti-species and a 2-IT solution, filtering and desalting the specific antibody after the reaction, and redissolving the specific antibody by using a buffer solution to filter and desalt the specific antibody;
Carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution;
Uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring uniformly at a low temperature to obtain a bispecific antibody;
s2, diluting an antigen to be marked into an antigen solution of 10mg/ml by using a CB solution, and filling the antigen solution into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; absorbing the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain fluorescent antigen;
S3, extracting PBMC, adding an anti-CD 16/32 antibody to block Fc receptor of the PBMC, and performing first incubation; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells;
S4, separating out the single antigen specific plasma cells by a single cell separation technology to obtain the single antigen specific plasma cells;
s5, extracting DNA of the single antigen specific plasma cells, and performing amplification sequencing to obtain specific monoclonal antibody genes;
S6, carrying out recombinant expression on the specific monoclonal antibody genes to obtain monoclonal antibodies.
Preferably, after the step S6 is completed, the method further includes the steps of: and S7, carrying out pairing verification on the monoclonal antibody.
Compared with the prior art, the screening method of the antigen specific plasma cells sequentially comprises the steps of obtaining the bispecific antibody, obtaining the fluorescent antigen, obtaining the cells with fluorescence and separating the single antigen specific plasma cells, so that the single antigen specific plasma cells can be obtained, and the preparation cost is low, the operation is simple, and the screening time is short.
Drawings
The present invention will be described in detail with reference to the accompanying drawings. The foregoing and other aspects of the invention will become more apparent and more readily appreciated from the following detailed description taken in conjunction with the accompanying drawings. In the accompanying drawings:
FIG. 1 is a flow chart showing the steps of a method for screening antigen-specific plasma cells according to an embodiment of the present invention;
FIG. 2 is a flowchart showing a method for obtaining a monoclonal antibody according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a method for obtaining a monoclonal antibody according to an embodiment of the present invention;
FIG. 4 is a flow chart illustrating an embodiment of the present invention;
FIG. 5 is a flow chart illustrating a second embodiment of the present invention;
FIG. 6 is a flow chart illustrating a third embodiment of the present invention;
FIG. 7 is a diagram of fluorescent staining of cells according to a fourth embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a screening method of antigen-specific plasma cells, which is shown in FIG. 1, and comprises the following steps:
S11, preparation of bispecific antibody
Specifically, the specific antibody of the species to be resisted is reacted with a 2-IT solution for the first time, the specific antibody after the reaction is filtered and desalted, and the specific antibody after the filtration and desalting is re-dissolved by using a buffer solution.
And carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution.
And uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring at a low temperature to uniformly obtain the bispecific antibody.
Wherein the first reaction and the second reaction are respectively carried out by adopting a rotary incubator; the mass of the specific antibody of the species to be anti-and the antibody of the surface marker molecule specific to the plasma cell to be marked is 0.1 mg; the volume of the 2-IT solution is 2 mu L, and the concentration is 6 mg/mL; the temperature of the rotary incubator is 25 ℃ during the first reaction, and the reaction time is 60min; the buffer was 0.05M PBS buffer at pH 7.0.
In the step S1, the volume of the SMCC solution is 6 mu L, and the concentration is 7 mg/mL; the temperature of the rotary incubator is 25 ℃ during the second reaction, and the reaction time is 30min; the mixing is carried out by a refrigerator at the temperature of 4 ℃ for 18 hours.
S12, preparation of fluorescent antigen
Specifically, the antigen to be labeled is diluted into an antigen solution of 10mg/ml using a CB solution, and the antigen solution is filled into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; and sucking the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain the fluorescent antigen.
Wherein the concentration of the CB solution is 0.025mol/L, and the pH value is 9.0; the stirring is performed by an electromagnetic stirrer.
The volume of the fluorescein solution is 10 times that of the antigen solution when immersed in the fluorescein solution; after immersing the dialysis bag in the fluorescein solution, stirring the fluorescein solution in an electromagnetic stirrer at the temperature of 4 ℃ for 18-24 hours, and filling the fluorescein solution in a beaker.
S13, fluorescent staining of plasma cells
Specifically, PBMC are extracted, fc receptor of PBMC is blocked by adding anti-CD 16/32 antibody, and the first incubation is performed; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; and incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells.
Wherein the anti-CD 16/32 antibody has a mass of 2 μg; the first incubation and the second incubation are carried out in an environment at 4 ℃, the time of the first incubation is 5-10 min, and the time of the second incubation is 20min; the culture is performed by using a cell culture box.
When the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule are incubated for the second time, the mass of the fluorescent antigen and the mass of the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule are 1 mug; the number of times of the first centrifugal washing, the second centrifugal washing and the third centrifugal washing is 3.
The antibody against the plasma cell-specific surface marker molecule of the fluorescent label is of a kind other than the antibody against the plasma cell-specific surface marker molecule of step S11. Such as anti-plasma cell specific surface marker antibodies include CD38, CD138, B220, etc., where the filtered and desalted anti-plasma cell specific surface marker antibody is one of them (e.g., CD 38), and the fluorescent labeled anti-plasma cell specific surface marker antibody described herein is an anti-plasma cell specific surface marker antibody (e.g., CD 138) against other surface proteins other than the filtered and desalted anti-plasma cell specific surface marker antibody.
S14, separation of plasma cells
Specifically, single antigen-specific plasma cells are isolated by a single cell isolation technique to obtain single antigen-specific plasma cells.
The screening method of the antigen specific plasma cells sequentially comprises the steps of obtaining the bispecific antibody, obtaining the fluorescent antigen, obtaining the cells with fluorescence and separating out the single antigen specific plasma cells, so that the single antigen specific plasma cells can be obtained, and the preparation cost is low, the operation is simple, and the screening time is short.
The invention provides a method for obtaining monoclonal antibodies, which is shown in FIG. 2, and comprises the following steps:
S21, preparation of bispecific antibody
Specifically, carrying out a first reaction on a specific antibody of the species to be resisted and a 2-IT solution, filtering and desalting the reacted specific antibody, and redissolving the filtered and desalted specific antibody by using a buffer solution;
Carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution;
Uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring uniformly at a low temperature to obtain a bispecific antibody;
S22, preparation of fluorescent antigen
Specifically, the antigen to be labeled is diluted into an antigen solution of 10mg/ml using a CB solution, and the antigen solution is filled into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; absorbing the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain fluorescent antigen;
S23, fluorescent staining of plasma cells
Specifically, PBMC are extracted, fc receptor of PBMC is blocked by adding anti-CD 16/32 antibody, and the first incubation is performed; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells;
S24, separation of plasma cells
Specifically, single antigen-specific plasma cells are separated by a single cell separation technology to obtain single antigen-specific plasma cells;
s25, single cell expansion and sequencing
Specifically, DNA extraction and amplification sequencing are carried out on single antigen specific plasma cells (positive cells) to obtain specific monoclonal antibody genes.
S26, antibody Gene list
Specifically, the specific monoclonal antibody gene is subjected to recombinant expression to obtain the monoclonal antibody.
The method for obtaining the monoclonal antibody in the invention further comprises the following steps after the process of expressing the antibody genes is completed: s26, antibody verification, namely carrying out pairing verification on the monoclonal antibody.
The steps S21 to S24 are the same as the steps S11 to S14 of the antigen-specific plasma cell screening method, respectively, and the specific contents are the same, and are not described herein.
The basic principle of the method for obtaining the monoclonal antibody in the invention is as follows: the antibody of the specific surface marker molecule of the anti-plasma cell is coupled with the specific antibody of the anti-species through a dual-specific antibody coupling technology, and is co-cultured with B cells extracted from immune tissues or peripheral blood of animals, which is equivalent to anchoring the specific antibody of the anti-species to the surface of cell membranes, the antibody secreted by the plasma cells can be captured and enriched by the specific antibody of the anti-species on the membranes, fluorescent staining can be carried out on the plasma cells of the specific antigen after fluorescent antigen is added, namely, the cells with the fluorescent light are the plasma cells of the specific antigen, finally, single plasma cells of the specific antigen are separated through a single cell separation technology, antibody variable region sequencing is carried out, and antibody genes are obtained, and the monoclonal antibody of the specific antigen can be obtained through in vitro expression. Because only the bispecific antibody needs to be coupled, no extra column or expensive consumable materials such as a micropore array chip are needed, so the cost is low; the simple culture and incubation are only needed to be carried out in the culture medium, so that the operation is simple; because the method is equivalent to enriching the secreted antibody on the surface of a cell membrane, the volume is greatly reduced compared with the secretion of the antibody into the supernatant, so that the secretion time can reach the concentration detected only by half an hour, and the screening time is short.
The specific principle is as follows: antigen-specific plasma cells are selected from animal tissues (such as mouse spleen, rabbit spleen and the like) or peripheral blood (such as mouse peripheral blood, rabbit peripheral blood, human peripheral blood and the like) by using a bispecific antibody, single antigen-specific plasma cells are separated by using a single cell separation technology, then antibody genes are obtained by using a single cell antibody gene sequencing technology, and antigen-specific monoclonal antibodies are prepared through expression. One end of the bispecific antibody is an antibody of a surface marker molecule (such as CD38, CD138, CD27, etc.) specific to plasma cells, and the other end is an anti-species specific antibody; the specific principle is shown in FIG. 3, one end of the bispecific antibody is an antibody against plasma cell specific surface marker molecules (such as CD38, CD138, CD27, etc.), and the other end is an anti-species specific antibody. Wherein antibodies against plasma cell specific surface marker molecules can bind to plasma cell specific surface marker molecules such that bispecific antibodies are anchored to plasma cells. Over a period of incubation, the antibody secreted by the plasma cells will be captured and enriched by the anti-species specific antibody on the membrane. After adding fluorescent antigen, fluorescent staining is carried out on plasma cells with antigen specificity, namely, the cells with the fluorescence are the plasma cells with antigen specificity, finally, single antigen-specific plasma cells are separated out through a single cell separation technology, antibody variable region sequencing is carried out to obtain antibody genes, and in vitro expression is carried out to obtain the monoclonal antibody with antigen specificity.
The invention further provides application of the screening method of the antigen specific plasma cells in preparation of monoclonal antibodies, specifically, DNA extraction, amplification and sequencing are carried out on the single antigen specific plasma cells obtained through screening to obtain specific monoclonal antibody genes, and recombinant expression is carried out on the specific monoclonal antibody genes to obtain monoclonal antibodies.
The method for acquiring the monoclonal antibody sequentially comprises the steps of obtaining the bispecific antibody, obtaining the fluorescent antigen, obtaining the cells with fluorescence, obtaining the single antigen specific plasma cells, obtaining the specific monoclonal antibody genes and carrying out recombinant expression on the specific monoclonal antibody genes, so that the monoclonal antibody can be acquired, and the method has the advantages of low preparation cost, simplicity in operation and short screening time.
In order to better embody the method for obtaining the monoclonal antibody of the present invention, the following four specific examples will be described:
Detailed description of the preferred embodiments
In the specific embodiment, the plasma cell specific surface marker molecule CD38 is used as an anchor point, and antigen specific plasma cells are obtained from the spleen of a mouse through flow cytometry, and antibody genes are obtained and expressed. The method comprises the following specific steps:
1. Preparation of bispecific antibodies
Taking goat anti-mouse IgG Fc with the mass of 0.1 mg, adding 2-IT solution with the mass of 2 mu L and the concentration of 6mg/mL, and placing the solution on a rotary incubator to react at the temperature of 25 ℃ for 60 min; ultrafiltration desalting the reacted goat anti-mouse IgG Fc, and re-dissolving the desalted goat anti-mouse IgG Fc by using a PBS buffer solution with the pH value of 7.0 and 0; then adding 0.1 mg of anti-mouse CD38 of the rat to be added into 6 mu L of SMCC solution with the concentration of 7 mg/mL, uniformly mixing, and placing on a rotary incubator to react at the temperature of 25 ℃ for 30 min; then carrying out ultrafiltration desalination on the reacted rat anti-mouse CD38, and then re-dissolving the desalted rat anti-mouse CD38 by using a PBS buffer solution with the pH value of 7.0 and 0; and finally, uniformly mixing the desalted goat anti-mouse IgG Fc and the desalted rat anti-mouse CD38, and uniformly stirring and mixing the mixture in a refrigerator reaction at the temperature of 4 ℃ for 18 hours to obtain the bispecific antibody.
2. Preparation of fluorescent antigens
Diluting the new crown N protein to be marked into a solution with the concentration of 10mg/ml by using CB with the concentration of 0.025mol/L, pH and the value of 9.0, putting the solution into a dialysis bag, and fastening the bag with a small gap; preparing FITC into a solution with the concentration of 0.1mg/ml by using the same CB, wherein the required amount is 10 times of the volume of the novel crown N protein solution, and placing the novel crown N protein solution in a beaker; immersing the dialysis bag in FITC liquid, putting the dialysis bag in an environment of 4 ℃, combining the dialysis bag with the FITC liquid for 18-24 hours under slow stirring of an electromagnetic stirrer, and taking out the dialysis bag to finish the marking process; finally, the conjugate in the dialysis bag is sucked, and the free FITC is removed by Sephadex G-25 column chromatography, so as to obtain the fluorescent antigen.
3. Plasma cell fluorescent staining
Extracting PBMC from the spleen cells of the mice by using a gradient centrifugation method, adding a CD16/32 antibody with the mass of 2 mug to block Fc receptor of the PBMC, and incubating for 5-10 min at the temperature of 4 ℃; adding 0.5 mug of bispecific antibody into PBMC, reacting for 30min at 4 ℃, and centrifugally washing for three times; culturing the washed cells in a cell culture box for 2 hours at the density of 10000 cells/mL, and centrifugally washing for three times; then adding 0.5 mug of APC-rat anti-mouse B220 and 1 mug of fluorescent antigen, incubating for 20min at 4 ℃, and centrifugally washing for three times; finally, adding 300 mu L of MACS Buffer for redissolution, adding 5 mu L of 7AAD, and placing in a 44 ℃ environment for light shielding for standby.
4. Plasma cell separation
And (3) carrying out flow separation on the treated sample, and separating and collecting 7AAD-, B220-, fluor 488+ cell groups (Q1 region), wherein a flow analysis chart is shown in FIG. 4.
5. Single cell amplification and sequencing
And respectively extracting DNA from the sorted positive plasma cells, and carrying out amplification sequencing to obtain the specific monoclonal antibody genes.
6. Antibody Gene expression
And carrying out recombinant expression on the obtained antibody genes.
7. Antibody validation
Pairing verification is carried out on the expressed antibody.
Second embodiment
In the specific embodiment, the plasma cell specific surface marker molecule CD138 is used as an anchor point, and antigen specific plasma cells are obtained from the spleen of the mouse through flow cytometry, and antibody genes are obtained and expressed. The method comprises the following specific steps:
1. Preparation of bispecific antibodies
Taking goat anti-mouse IgG Fc with the mass of 0.1 mg, adding 2-IT solution with the mass of 2 mu L and the concentration of 6mg/mL, and placing the solution on a rotary incubator to react at the temperature of 25 ℃ for 60 min; ultrafiltration desalting the reacted goat anti-mouse IgG Fc, and re-dissolving the desalted goat anti-mouse IgG Fc by using a PBS buffer solution with the pH value of 7.0 and 0; then adding 0.1 mg of anti-mouse CD138 to be rat into 6 mu L of SMCC solution with the concentration of 7 mg/mL, uniformly mixing, and placing on a rotary incubator to react at 25 ℃ for 30 min; then carrying out ultrafiltration desalination on the reacted rat anti-mouse CD138, and then re-dissolving the desalted rat anti-mouse CD138 by using a PBS buffer solution with the pH value of 7.0 and 0; and finally, uniformly mixing the desalted goat anti-mouse IgG Fc and the desalted rat anti-mouse CD138, and uniformly stirring and mixing the mixture in a refrigerator reaction at the temperature of 4 ℃ for 18 hours to obtain the bispecific antibody.
2. Preparation of fluorescent antigens
Diluting the new crown N protein to be marked into a solution with the concentration of 10mg/ml by using CB with the concentration of 0.025mol/L, pH and the value of 9.0, putting the solution into a dialysis bag, and fastening the bag with a small gap; preparing FITC into a solution with the concentration of 0.1mg/ml by using the same CB, wherein the required amount is 10 times of the volume of the novel crown N protein solution, and placing the novel crown N protein solution in a beaker; immersing the dialysis bag in FITC liquid, putting the dialysis bag in an environment of 4 ℃, combining the dialysis bag with the FITC liquid for 18-24 hours under slow stirring of an electromagnetic stirrer, and taking out the dialysis bag to finish the marking process; finally, the conjugate in the dialysis bag is sucked, and the free FITC is removed by Sephadex G-25 column chromatography, so as to obtain the fluorescent antigen.
3. Plasma cell fluorescent staining
Extracting PBMC from the spleen cells of the mice by using a gradient centrifugation method, adding a CD16/32 antibody with the mass of 2 mug to block Fc receptor of the PBMC, and incubating for 5-10 min at the temperature of 4 ℃; adding 0.5 mug of bispecific antibody into PBMC, reacting for 30min at 4 ℃, and centrifugally washing for three times; culturing the washed cells in a cell culture box for 2 hours at the density of 10000 cells/mL, and centrifugally washing for three times; then adding 0.5 mug of APC-rat anti-mouse B220 and 1 mug of fluorescent antigen, incubating for 20min at 4 ℃, and centrifugally washing for three times; finally, adding 300 mu L of MACS Buffer for redissolution, adding 5 mu L of 7AAD, and placing in a 44 ℃ environment for light shielding for standby.
4. Plasma cell separation
And (3) carrying out flow separation on the treated sample, and separating and collecting 7AAD-, B220-, fluor 488+ cell groups (Q1 region), wherein a flow analysis chart is shown in FIG. 5.
5. Single cell amplification and sequencing
And respectively extracting DNA from the sorted positive plasma cells, and carrying out amplification sequencing to obtain the specific monoclonal antibody genes.
6. Antibody Gene expression
And carrying out recombinant expression on the obtained antibody genes.
7. Antibody validation
Pairing verification is carried out on the expressed antibody.
Detailed description of the preferred embodiments
In the specific embodiment, the plasma cell specific surface marker molecule CD138 is used as an anchor point, and antigen specific plasma cells are obtained from human peripheral blood through flow cytometry, and antibody genes are obtained and expressed. The method comprises the following specific steps:
1. Preparation of bispecific antibodies
Adding 2-IT solution with a mass of 2 mu L and a concentration of 6mg/mL into goat anti-human IgG Fc with a mass of 0.1 mg, and placing the solution on a rotary incubator to react at 25 ℃ for 60 min; ultrafiltration desalting the reacted goat anti-human IgG Fc, and re-dissolving the desalted goat anti-human IgG Fc by using a PBS buffer solution with the pH value of 7.0 and 0; then adding 0.1 mg of anti-human CD138 of the rat to be added into 6 mu L of SMCC solution with the concentration of 7 mg/mL, uniformly mixing, and placing the mixture on a rotary incubator to react at the temperature of 25 ℃ for 30 min; then carrying out ultrafiltration desalination on the reacted rat anti-human CD138, and then re-dissolving the desalted rat anti-human CD138 by using a PBS buffer solution with the pH value of 7.0 and 0; and finally, uniformly mixing the desalted goat anti-human IgG Fc and the desalted rat anti-human CD138, and uniformly stirring and reacting in a refrigerator at the temperature of 4 ℃ for 18 hours to obtain the bispecific antibody.
2. Preparation of fluorescent antigens
Diluting the new crown N protein to be marked into a solution with the concentration of 10mg/ml by using CB with the concentration of 0.025mol/L, pH and the value of 9.0, putting the solution into a dialysis bag, and fastening the bag with a small gap; preparing FITC into a solution with the concentration of 0.1mg/ml by using the same CB, wherein the required amount is 10 times of the volume of the novel crown N protein solution, and placing the novel crown N protein solution in a beaker; immersing the dialysis bag in FITC liquid, putting the dialysis bag in an environment of 4 ℃, combining the dialysis bag with the FITC liquid for 18-24 hours under slow stirring of an electromagnetic stirrer, and taking out the dialysis bag to finish the marking process; finally, the conjugate in the dialysis bag is sucked, and the free FITC is removed by Sephadex G-25 column chromatography, so as to obtain the fluorescent antigen.
3. Plasma cell fluorescent staining
Extracting PBMC from human peripheral blood cells by using a gradient centrifugation method, adding a CD16/32 antibody with the mass of 2 mug to block Fc receptor of the PBMC, and incubating for 5-10 min at the temperature of 4 ℃; adding 0.5 mug of bispecific antibody into PBMC, reacting for 30min at 4 ℃, and centrifugally washing for three times; culturing the washed cells in a cell culture box for 2 hours at the density of 10000 cells/mL, and centrifugally washing for three times; then adding 0.5 mug of APC-rat anti-human B220 and 1 mug of fluorescent antigen, incubating for 20min at 4 ℃, and centrifugally washing for three times; finally, adding 300 mu L of MACS Buffer for redissolution, adding 5 mu L of 7AAD, and placing in a 44 ℃ environment for light shielding for standby.
4. Plasma cell separation
And (3) carrying out flow separation on the treated sample, separating and collecting 7AAD-, B220-, fluor 488+ cell groups (Q1 region), wherein a flow analysis chart is shown in FIG. 6.
5. Single cell amplification and sequencing
And respectively extracting DNA from the sorted positive plasma cells, and carrying out amplification sequencing to obtain the specific monoclonal antibody genes.
6. Antibody Gene expression
And carrying out recombinant expression on the obtained antibody genes.
7. Antibody validation
Pairing verification is carried out on the expressed antibody.
Detailed description of the preferred embodiments
In the specific embodiment, a plasma cell specific surface marker molecule CD138 is used as an anchor point, and single-cell micromanipulation is used for obtaining antigen specific plasma cells from the spleen of a mouse, obtaining antibody genes and expressing antibodies. The method comprises the following specific steps:
1. preparation of bispecific antibodies.
Taking goat anti-mouse IgG Fc with the mass of 0.1 mg, adding 2-IT solution with the mass of 2 mu L and the concentration of 6mg/mL, and placing the solution on a rotary incubator to react at the temperature of 25 ℃ for 60 min; ultrafiltration desalting the reacted goat anti-mouse IgG Fc, and re-dissolving the desalted goat anti-mouse IgG Fc by using a PBS buffer solution with the pH value of 7.0 and 0; then adding 0.1 mg of anti-mouse CD138 to be rat into 6 mu L of SMCC solution with the concentration of 7 mg/mL, uniformly mixing, and placing on a rotary incubator to react at 25 ℃ for 30 min; then carrying out ultrafiltration desalination on the reacted rat anti-mouse CD138, and then re-dissolving the desalted rat anti-mouse CD138 by using a PBS buffer solution with the pH value of 7.0 and 0; and finally, uniformly mixing the desalted goat anti-mouse IgG Fc and the desalted rat anti-mouse CD138, and uniformly stirring and mixing the mixture in a refrigerator reaction at the temperature of 4 ℃ for 18 hours to obtain the bispecific antibody.
2. Preparation of fluorescent antigens
Diluting the new crown N protein to be marked into a solution with the concentration of 10mg/ml by using CB with the concentration of 0.025mol/L, pH and the value of 9.0, putting the solution into a dialysis bag, and fastening the bag with a small gap; preparing FITC into a solution with the concentration of 0.1mg/ml by using the same CB, wherein the required amount is 10 times of the volume of the novel crown N protein solution, and placing the novel crown N protein solution in a beaker; immersing the dialysis bag in FITC liquid, putting the dialysis bag in an environment of 4 ℃, combining the dialysis bag with the FITC liquid for 18-24 hours under slow stirring of an electromagnetic stirrer, and taking out the dialysis bag to finish the marking process; finally, the conjugate in the dialysis bag is sucked, and the free FITC is removed by Sephadex G-25 column chromatography, so as to obtain the fluorescent antigen.
3. Plasma cell fluorescent staining
Extracting PBMC from the spleen cells of the mice by using a gradient centrifugation method, adding a CD16/32 antibody with the mass of 2 mug to block Fc receptor of the PBMC, and incubating for 5-10 min at the temperature of 4 ℃; adding 0.5 mug of bispecific antibody into PBMC, reacting for 30min at 4 ℃, and centrifugally washing for three times; culturing the washed cells in a cell culture box for 2 hours at the density of 10000 cells/mL, and centrifugally washing for three times; then adding 0.5 mug of APC-rat anti-mouse B220 and 1 mug of fluorescent antigen, incubating for 20min at 4 ℃, and centrifugally washing for three times; finally, adding 300 mu L of MACS Buffer for redissolution, adding 5 mu L of 7AAD, and placing in a 44 ℃ environment for light shielding for standby.
4. Plasma cell separation
And observing the treated sample under a fluorescence microscope, and picking out cells emitting fluorescence by using a single cell picker, wherein a cell fluorescence staining chart is shown in fig. 7.
5. Single cell amplification and sequencing
And respectively extracting DNA from the sorted positive plasma cells, and carrying out amplification sequencing to obtain the specific monoclonal antibody genes.
6. Antibody Gene expression
And carrying out recombinant expression on the obtained antibody genes.
7. Antibody validation
Pairing verification is carried out on the expressed antibody.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
While the embodiments of the present invention have been illustrated and described in connection with the drawings, what is presently considered to be the most practical and preferred embodiments of the invention, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, is intended to cover various equivalent modifications and equivalent arrangements included within the spirit and scope of the appended claims.
Claims (10)
1. A method of screening for antigen-specific plasma cells, comprising the steps of:
S1, carrying out a first reaction on a specific antibody of an anti-species and a 2-IT solution, filtering and desalting the specific antibody after the reaction, and redissolving the specific antibody by using a buffer solution to filter and desalt the specific antibody;
Carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution;
Uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring uniformly at a low temperature to obtain a bispecific antibody;
s2, diluting an antigen to be marked into an antigen solution of 10mg/ml by using a CB solution, and filling the antigen solution into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; absorbing the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain fluorescent antigen;
S3, extracting PBMC, adding an anti-CD 16/32 antibody to block Fc receptor of the PBMC, and performing first incubation; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells;
S4, separating out the single antigen specific plasma cells by a single cell separation technology to obtain the single antigen specific plasma cells.
2. The method for screening antigen-specific plasma cells according to claim 1, wherein in the step S1, the first reaction and the second reaction are performed using a rotary incubator, respectively; the mass of the specific antibody of the species to be anti-and the antibody of the surface marker molecule specific to the plasma cell to be marked is 0.1 mg; the volume of the 2-IT solution is 2 mu L, and the concentration is 6 mg/mL; the temperature of the rotary incubator is 25 ℃ during the first reaction, and the reaction time is 60min; the buffer was 0.05M PBS buffer at pH 7.0.
3. The method of claim 2, wherein in step S1, the SMCC solution has a volume of 6 μl and a concentration of 7 mg/mL; the temperature of the rotary incubator is 25 ℃ during the second reaction, and the reaction time is 30min; the mixing is carried out by a refrigerator at the temperature of 4 ℃ for 18 hours.
4. The method of claim 1, wherein in step S2, the CB solution has a concentration of 0.025mol/L and a pH of 9.0; the stirring is performed by an electromagnetic stirrer.
5. The method for screening antigen-specific plasma cells according to claim 4, wherein in the step S2, the volume of the fluorescein solution is 10 times that of the antigen solution when immersed in the fluorescein solution; after immersing the dialysis bag in the fluorescein solution, stirring the fluorescein solution in an electromagnetic stirrer at the temperature of 4 ℃ for 18-24 hours, and filling the fluorescein solution in a beaker.
6. The method of claim 1, wherein in step S3, the anti-CD 16/32 antibody has a mass of 2 μg; the first incubation and the second incubation are carried out in an environment at 4 ℃, the time of the first incubation is 5-10 min, and the time of the second incubation is 20min; the culture is performed by using a cell culture box.
7. The method according to claim 6, wherein in the step S3, the mass of the fluorescent antigen and the fluorescent labeled antibody against the plasma cell-specific surface marker molecule is 1 μg when the fluorescent antigen and the fluorescent labeled antibody against the plasma cell-specific surface marker molecule are subjected to the second incubation; the number of times of the first centrifugal washing, the second centrifugal washing and the third centrifugal washing is 3.
8. The application of the screening method of the antigen specific plasma cells in the preparation of monoclonal antibodies is characterized in that the single antigen specific plasma cells obtained by screening according to any one of claims 1-7 are subjected to DNA extraction, amplification and sequencing to obtain specific monoclonal antibody genes, and the specific monoclonal antibody genes are subjected to recombinant expression to obtain monoclonal antibodies.
9. A method for obtaining a monoclonal antibody, comprising the steps of:
S1, carrying out a first reaction on a specific antibody of an anti-species and a 2-IT solution, filtering and desalting the specific antibody after the reaction, and redissolving the specific antibody by using a buffer solution to filter and desalt the specific antibody;
Carrying out a second reaction on the antibody of the anti-plasma cell specific surface marker molecules to be marked and the SMCC solution, filtering and desalting the reacted antibody of the anti-plasma cell specific surface marker molecules, and carrying out filtering and desalting on the antibody of the anti-plasma cell specific surface marker molecules by using buffer solution for redissolution;
Uniformly mixing the specific antibody after the reconstitution and the antibody of the specific surface marker molecule of the plasma cell after the reconstitution, and reacting and stirring uniformly at a low temperature to obtain a bispecific antibody;
s2, diluting an antigen to be marked into an antigen solution of 10mg/ml by using a CB solution, and filling the antigen solution into a dialysis bag; diluting fluorescein to 0.1mg/ml of fluorescein solution using CB solution; sealing the dialysis bag, immersing the dialysis bag into the fluorescein solution, and stirring; absorbing the solution in the dialysis bag, and removing free fluorescein by using Sephadex G-25 column chromatography to obtain fluorescent antigen;
S3, extracting PBMC, adding an anti-CD 16/32 antibody to block Fc receptor of the PBMC, and performing first incubation; adding the bispecific antibody into the PBMC after the first incubation for reaction, and performing first centrifugal washing on the PBMC after the reaction; culturing the PBMC subjected to the first centrifugal washing at a density of 10000-1000000/mL, and performing the second centrifugal washing on the cultured PBMC; incubating the fluorescent antigen and the antibody of the fluorescent-labeled anti-plasma cell specific surface marker molecule for the second time, and performing centrifugal washing on the antibody of the anti-plasma cell specific surface marker molecule after the second incubation for the third time to obtain cells with fluorescence, wherein the cells with fluorescence are the antigen specific plasma cells;
S4, separating out the single antigen specific plasma cells by a single cell separation technology to obtain the single antigen specific plasma cells;
s5, extracting DNA of the single antigen specific plasma cells, and performing amplification sequencing to obtain specific monoclonal antibody genes;
S6, carrying out recombinant expression on the specific monoclonal antibody genes to obtain monoclonal antibodies.
10. The method of claim 9, wherein after the step S6 is completed, the method further comprises the steps of: and S7, carrying out pairing verification on the monoclonal antibody.
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