CN110016462B - Method for efficiently separating single antigen-specific B lymphocytes from spleen cells - Google Patents
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Abstract
The invention discloses a method for efficiently separating single antigen specific B lymphocytes from spleen cells, which utilizes negative screening of multiple lymphocyte surface marker antibodies, establishment of a rapid fluorescence labeling antigen substance method and high specificity screening by using fluorescence labeling antigen substances to realize high-throughput rapid screening of the single antigen specific B lymphocytes, and ensures that the positive rate of finally obtained antibody specific B lymphocytes is improved to 30% from less than 5% of that of a traditional hybridoma method. Therefore, the scheme not only can greatly shorten the development time of the monoclonal antibody, but also can greatly improve the quantity and diversity of the obtained monoclonal antibody.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for efficiently separating single antigen specific B lymphocytes from spleen cells.
Background
B lymphocytes, referred to as B cells for short, are derived primarily from pluripotent stem cells in the bone marrow. The differentiation process of mammalian B cells can be mainly divided into five stages, pre-B cells, immature B cells, mature B cells, activated B cells and plasma cells. Wherein the differentiation of pre-B cells and immature B cells is antigen-untrusted, with the differentiation process occurring primarily in the bone marrow; mature B cells will enter the peripheral blood through the circulation of the lymphatic system and, after being stimulated by antigens, can further differentiate into plasma cells that synthesize and secrete antibodies. Plasma cells are capable of producing immunoglobulin IgG specific to an antigen and releasing it into body fluids, i.e., becoming immune antibodies. Single antibodies derived from the same type of B lymphocytes, called monoclonal antibodies, are now widely used in biological and medical research because of their advantages of highly uniform physicochemical properties, high potency, single biological activity, high specificity, and ease of mass production.
The development of monoclonal antibodies now relies to a large extent on the hybridoma technology created by the american molecular biologists g.j.f. kler and c. milstein of 1975, i.e. hybridoma cells are generated after fusion using spleen cells of immunized animals and immortalized myeloma cells, and hybridoma cell lines capable of producing monoclonal antibodies are obtained through multiple rounds of screening. The method has the limitations of long preparation period, gene instability of the generated hybridoma cells, poor screening specificity and positive rate, poor universality (namely, myeloma cells of a certain species obtained through research and development can only be fused with lymphocytes of the species, but cannot be applied to other species), and the like, and is only widely applied to the preparation of the mouse monoclonal antibody at present. However, as known by research, monoclonal antibodies of other species such as rabbit and alpaca have better characteristics than murine monoclonal antibodies, and cannot or are difficult to obtain by the traditional hybridoma cell method.
Therefore, it is an emerging field in immunology and monoclonal antibody development to effectively isolate B lymphocytes capable of specifically binding to an antibody of interest from an immunized animal, thereby effectively isolating genes expressing the antibody, and mass-producing monoclonal antibodies by in vitro recombination. However, since rabbit monoclonal antibodies have many advantages such as good specificity, high affinity, and good diversity, and there is a patent barrier to develop rabbit monoclonal antibodies by using traditional hybridoma methods, it is very important to develop rabbit monoclonal antibodies by using a method of isolating single B lymphocytes. However, the current screening of rabbit single B lymphocyte has the following difficulties:
1. the time point at which antibody-expressing B lymphocytes are enriched in the spleen after boosting immunity is held poorly.
2. B lymphocytes are only able to produce IgG molecules on the membrane surface that bind specifically to antigens at a particular stage; if too early, the IgM molecules are expressed on the surface of B lymphocytes, and if too late, the B lymphocytes will differentiate substantially into plasma cells. Therefore, it is a technical difficulty how to screen the B lymphocytes expressing IgG molecules in membrane epitopes.
3. At present, the types of antibody tools for rabbit B lymphocyte surface CD molecules are few, and the difficulty of sorting B lymphocytes in a specific stage is further increased.
Disclosure of Invention
The invention aims to overcome the defects of the prior art in separating rabbit B lymphocytes and provide a method for efficiently separating single antigen-specific B lymphocytes from spleen cells quickly and with high purity.
In order to achieve the above objects, the present invention provides a method for efficiently isolating single antigen-specific B lymphocytes from spleen cells, comprising the steps of:
1) performing negative screening on spleen cells by using a biomarker non-B cell lymphocyte surface marker antibody, and removing non-B cells by using an avidin magnetic bead and magnetic separation principle so as to improve the relative abundance of the B lymphocytes in the spleen cells;
2) performing further negative screening on the spleen cells obtained in the step 1 by using a fluorescence-labeled IgM polyclonal antibody and a pre-labeled screening antigen, removing B cells without expressing IgG molecules on the surface by using a flow cytometry technology, and reserving target lymphocytes combined with the pre-labeled screening antigen;
3) sorting by 7-AAD to remove dead cells;
4) and (4) screening single cells by using a flow cytometer to obtain target lymphocytes.
Wherein, the pre-marked screening antigen is a common antigen coupled with a small molecular fluorescent dye.
In the above scheme, the pre-labeled screening antigen is a common antigen coupled with a small molecule fluorescent dye containing avidin and labeled with biotin.
Preferably, the preparation method of the pre-labeled screening antigen comprises the following steps: performing coupling reaction in a dropping mode, dropping the small-molecule fluorescent dye containing the avidin into the common antigen marked by the biotin for coupling for multiple times, and incubating after dropping each time to ensure that the coupling is sufficient; wherein the dosage of the small molecular fluorescent dye containing the avidin is more than 5 times of that of the common antigen marked by the biotin.
Preferably, the preparation method of the pre-labeled screening antigen further comprises the following steps: after the coupling of the small-molecule fluorescent dye containing avidin and the common antigen marked by biotin is completed, selecting a proper molecular sieve centrifuge tube according to the size of a coupling product, and removing unconjugated substrate.
Optionally, the step 1) is: the spleen cells are subjected to negative screening by using a CD4 monoclonal antibody, a CD8 monoclonal antibody and a CD14 monoclonal antibody which are labeled by biotin, T lymphocytes and monocytes are removed by using an avidin magnetic bead and magnetic separation principle to obtain the spleen cells which are negative to CD4, CD8 and CD14, and the relative abundance of the B lymphocytes is improved.
Optionally, the fluorescently labeled IgM polyclonal antibody in step 2) is a FITC-labeled IgM polyclonal antibody.
The principle of the invention is as follows:
1. the long-chain biotin is coupled with an antigen substance, and is further combined with small-molecule fluorescein to realize fluorescein labeling of the antigen substance; at the same time, the label does not affect the binding between the antigen and the antibody;
2. the method of magnetic bead sorting is utilized, markers (such as CD4, CD18, CD20 molecules and the like) on the surfaces of lymphocytes (including T lymphocytes and nave B lymphocytes) are utilized, and the obtained spleen cells are subjected to negative screening through corresponding biotin labeled antibodies, so that the screening purity is improved;
3. combining with a flow cytometry screening technology, further removing B lymphocytes of IgM molecules expressed on the surface by using a goat anti-rabbit IgM antibody marked by FITC, and carrying out antigen specificity screening by using an antigen substance marked by fluorescein to obtain high-purity antigen specificity B lymphocytes of IgG molecules expressed on the surface;
4. meanwhile, the flow cytometry screening technology is utilized to complete high-throughput screening of spleen cells and guarantee the monoclonality of the cells obtained by screening.
According to the invention, the traditional hybridoma method is completely abandoned to obtain the rabbit monoclonal antibody, the limited dilution is not needed for subcloning, and the development time of the rabbit monoclonal antibody is shortened to 1 week from the original 6-8 weeks; and completely solves the problems of instability of rabbit hybridoma cells and the like. Compared with the prior art that the antigen-specific cells are separated by the magnetic bead method, the positive rate of the screened antigen-specific cells can be further improved because more cell surface marker antibodies are added; meanwhile, 7-AAD is introduced for live cell screening so as to ensure the activity of finally obtained B lymphocytes; moreover, flow cytometry ensures the monoclonality of the B lymphocytes obtained by screening, and reduces the complexity of molecular cloning at the later stage.
The invention has the beneficial effects that: the negative screening of multiple lymphocyte surface marker antibodies, the establishment of a rapid fluorescence-labeled antigen substance method and the high-specificity screening of fluorescence-labeled antigen substances are utilized, so that the high-throughput and rapid screening of single antigen-specific B lymphocytes is realized, and the positive rate of finally obtained antibody-specific B lymphocytes is improved to 30% from less than 5% of that of the traditional hybridoma method. Therefore, the scheme not only can greatly shorten the development time of the monoclonal antibody, but also can greatly improve the quantity of the obtained monoclonal antibody.
Drawings
FIG. 1 is a schematic diagram of a test example of the present invention.
FIG. 2 is a flow cytometric analysis quality control diagram of a fluorescence labeled antigen without removing free fluorescein coupled avidin
FIG. 3 is a flow cytometric quality control diagram of a fluorescently labeled antigen with free fluorescein removed.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments. The following examples are carried out on the premise of the technical scheme of the invention, and detailed embodiments and specific operation procedures are given, but the scope of the invention is not limited to the following examples.
Example 1
1. Pre-labeling of antigenic material:
1.1 if the antigen substance is protein, coupling the protein by using long-chain biotin purchased from Pierce company in America according to a standard process recommended by a manufacturer, and dialyzing to remove the unmarked biotin after the coupling is finished; if the antigenic substance is a polypeptide or a small molecule compound, the biotin molecule is pre-conjugated during the synthesis. The antigen in this example was human C-reactive protein (CRP) (available from Fitzgerald Industries International)
1.2 coupling of biotinylated antigen with a small fluorescent dye, in this example the antigen substance was coupled with Cy 5-coupled avidin; the amount of the Cy 5-coupled avidin required was calculated according to the size of the antigen substance, and it was suggested that the amount of Cy 5-coupled avidin and the amount of biotin-coupled antigen were used in such a manner that the coupling reaction was carried out in stepwise dropping manner, i.e., the volume of the liquid containing 50. mu.g of biotin-coupled antigen was adjusted to 100. mu.l with 1 XPBS buffer, after dropping 1/5 total amount of the required Cy 5-coupled avidin each time, incubation was carried out for 10 minutes, and then 1/5 of Cy 5-coupled avidin was dropped again, and this was repeated five times.
1.3 after the coupling is finished, selecting a molecular sieve centrifuge tube with a proper size according to the molecular weight of Cy5 coupled avidin and biotin-labeled antigen, and removing unconjugated Cy5 coupled avidin and biotin-labeled antigen; if the size of the biotin-labeled antigen is 30KD and the size of the Cy5 coupled avidin is about 55KD, a molecular sieve centrifuge tube with 50KD is adopted to remove the redundant unlabeled substances.
1.4 the labeled antigen molecules can be stored at 4 degrees for later use; the storage period is 4-6 weeks.
2. Before 3-6 days after the sacrifice of the immunized rabbit, 400 micrograms of antigen (the antigen used here may be any antigen such as protein, conjugated polypeptide, and small molecule compound, and in this example, the CRP antigen) was injected intravenously from the auricular vein of the rabbit without any adjuvant.
3. Rabbits were sacrificed, spleens were collected and splenocytes isolated according to relevant standard procedures.
4. Take 5X 107Spleen cells, 400g after centrifugation for 3 min; resuspend spleen cells in 1 ml of 1 XPBS + 2% FBS buffer, centrifuge at 400g for 3 min, discard the supernatant; repeating the above steps once, then using 100 microliter1 XPBS + 2% FBS buffer resuspended spleen cells, then 1 microgram each of biotin-labeled mouse anti-rabbit CD4 monoclonal antibody (purchased from Bio-Rad), mouse anti-rabbit CD8 monoclonal antibody (purchased from Bio-Rad), and mouse anti-rabbit CD14 monoclonal antibody (purchased from Novus Biologicals) was added and incubated for 10 minutes.
5. After centrifugation at 400g for 3 minutes, spleen cells were resuspended in 1 ml of 1 XPBS + 2% FBS buffer, and then centrifuged at 400g for 3 minutes again, and the supernatant was discarded; the above procedure was repeated once, and then spleen cells were resuspended in 100. mu.l of 1 XPBS + 2% FBS buffer, and 10. mu.l of avidin-coupled magnetic nanospheres (purchased from Miltenyi Biotec, USA ) were added.
6. After incubation for 10 minutes, 1 × PBS + 2% FBS buffer was added to a final volume of 500 μ l, followed by separation using a magnetic separation column of meitian ; the flow-through was collected as spleen cells negative for CD4/CD8/CD14 and counted.
7. After centrifugation at 400g for 3 min, at 0.5X 107Cells were resuspended in spleen cells using 1 XPBS + 2% FBS buffer at a density of 100. mu.l, and FITC-labeled goat anti-rabbit IgM polyclonal antibody (from Bio-Rad) and pre-labeled screening antigen as described in step 1 were added to the cell suspension at a volume of 1. mu.g per 100. mu.l.
8. After 20 minutes of incubation at 4 degrees, 400g were centrifuged for 3 minutes; then, 1 ml of 1 XPBS + 2% FBS buffer was added to wash the cells, and then 400g of the cells were centrifuged for 3 minutes. This procedure was repeated once and then again according to 0.5X 107Cells were resuspended in 1 XPBS buffer per 100 microliters of density and 1 microliter of 7-AAD was added for viable cell screening.
9. Screening of single cells was performed by flow cytometry. The cell populations selected were: 7-AAD negative (viable cells), FITC-IgM negative, and antigen positive.
Test examples
1. In order to verify that the improvement of the method for labeling antigen with fluorescein is superior to other traditional methods in fluorescence intensity, we specially designed a test method for verification (the principle is shown in figure 1).
In this example, the CRP protein antigen was used, in accordance with example 1 above.
We first chemically couple a reference antibody (a known antibody that can recognize an antigen) to Protein a-coated nanospheres (2-4 microns in diameter) to mimic antibody-expressing B cells on the inner surface of a body; and then, incubating the nano-microspheres coupled with the reference antibody with antigen proteins pre-labeled by fluorescein in different processes, and analyzing by using flow cytometry to evaluate the effect of labeling the antigen proteins in different processes.
2. Comparison of pre-labeling and non-pre-labeling: the antigen is not pre-marked, namely the antigen is not pre-marked according to the method of the invention, but the biotin-marked antigen, fluorescein coupled avidin and suspension containing B lymphocyte or nano microsphere coupled with reference antibody (in the evaluation experiment) are incubated for 20 minutes; the pre-labeling method is to incubate the antigen protein pre-labeled with fluorescent group by the above method with a suspension containing B lymphocytes or reference antibody-coupled nanospheres (for evaluation experiments), and the incubation time is also 20 minutes. The incubated sample was finally analyzed by flow cytometry, the results are shown in FIG. 2.
From the results, it is obvious that the fluorescence intensity of the antigen protein prepared by the pre-labeling method and the antigen protein prepared by the non-pre-labeling method is obviously higher than that of the antigen protein prepared by the non-pre-labeling method after the antigen protein is incubated with the reference antibody-coupled nanospheres and analyzed by a flow cytometry.
3. For the antigen protein which is pre-marked, the method is further improved, namely, the antigen protein which is not marked with the fluorescence and is pre-marked with the fluorescein is separated by using a molecular sieve centrifuge tube. In principle, any biological or chemical reaction is a dynamic process, that is, the combination between fluorescein-conjugated avidin and avidin-labeled antigen has a dynamic equilibrium, and in a certain time, the reaction system must have antigen protein labeled with fluorescein and antigen protein not labeled with fluorescein; the antigen protein not labeled with fluorescein will have an effect on the final detection, since it can also bind to the corresponding antibody without a detectable fluorescent signal. To demonstrate the validity of this theory, and our improved effectiveness for this method, we also validated the method described above, with the results shown in FIG. 3.
The results clearly show that the fluorescence intensity of the antigen protein which is not marked with fluorescein is obviously higher than that of the antigen protein which is not marked with fluorescein through centrifugal treatment of a molecular sieve centrifuge tube after the antigen protein is removed through centrifugal treatment of the pre-marked antigen through the molecular sieve centrifuge tube and the nano-microsphere coupled with the reference antibody are analyzed through a flow cytometry.
Claims (4)
1. A method for efficiently isolating single antigen-specific B lymphocytes from spleen cells immunized with a common antigen, comprising the following steps:
1) performing negative screening on spleen cells by using a CD4 monoclonal antibody, a CD8 monoclonal antibody and a CD14 monoclonal antibody which are labeled by biotin, removing T lymphocytes and monocytes by using an avidin magnetic bead and magnetic separation principle to obtain CD4, CD8 and CD14 negative spleen cells, and improving the relative abundance of B lymphocytes;
2) performing further negative screening on the spleen cells obtained in the step 1 by using a fluorescence-labeled IgM polyclonal antibody and a pre-labeled screening antigen, removing B cells without expressing IgG molecules on the surface by using a flow cytometry technology, and reserving target lymphocytes combined with the pre-labeled screening antigen;
3) sorting by 7-AAD to remove dead cells;
4) screening single cells by using a flow cytometer to obtain target lymphocytes;
the preparation method of the pre-labeled screening antigen comprises the following steps: performing coupling reaction in a dropping mode, dropping the small-molecule fluorescent dye containing the avidin into the common antigen marked by the biotin for coupling for multiple times, and incubating after dropping each time to ensure that the coupling is sufficient; wherein the dosage of the small molecular fluorescent dye containing the avidin is more than 5 times of that of the common antigen marked by the biotin.
2. The method of claim 1 for efficiently isolating single antigen-specific B lymphocytes from spleen cells, wherein: the pre-marked screening antigen is a common antigen which is coupled with a small molecular fluorescent dye containing avidin and is marked by biotin.
3. The method of claim 2 for efficiently isolating single antigen-specific B lymphocytes from spleen cells, wherein: the preparation method of the pre-labeled screening antigen further comprises the following steps: after the coupling of the small-molecule fluorescent dye containing avidin and the common antigen marked by biotin is completed, selecting a proper molecular sieve centrifuge tube according to the size of a coupling product, and removing unconjugated substrate.
4. The method of claim 1 for efficiently isolating single antigen-specific B lymphocytes from spleen cells, wherein: the IgM polyclonal antibody fluorescently labeled in the step 2) is an FITC-labeled IgM polyclonal antibody.
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| CN112094810B (en) * | 2020-08-26 | 2022-07-05 | 中国农业大学 | Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody |
| CN114317432A (en) * | 2021-12-31 | 2022-04-12 | 河南赛诺特生物技术有限公司 | Screening method of rabbit B lymphocytes capable of recognizing multiple antigens and preparation method of multiple rabbit-derived engineered antibodies |
| CN114686427B (en) * | 2022-05-23 | 2022-07-29 | 中国人民解放军总医院第一医学中心 | Spleen-regulating B lymphocyte and preparation method and application thereof |
| 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 |
| CN115466731B (en) * | 2022-09-29 | 2023-06-20 | 深圳市福田区格物智康病原研究所 | Hybridoma cell strain screening method |
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