Method for isolating single antigen-specific B lymphocytes from monkey peripheral blood
By using specific antibodies for fluorescent labeling and flow cytometry sorting, single antigen-specific B lymphocytes were successfully isolated from monkey peripheral blood, solving the problems of low isolation efficiency and insufficient labeling tools in the prior art, and significantly improving the development efficiency of monoclonal antibodies.
Patent Information
- Application Number
- CN202311274040.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-28
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is difficult to efficiently isolate single antigen-specific B lymphocytes from monkey peripheral blood, and the lack of suitable labeling tools and methods limits the development of monkey-derived monoclonal antibodies.
Memory B lymphocytes that were positive for CD19, CD27, CD23, IgG, IgD and IgM antibodies were sorted for fluorescent labeling and flow cytometry sorting, combined with pre-labeling screening antigens, were sorted for CD19, CD27 and IgG positive and CD23, IgD and IgM negative.
It has achieved efficient isolation of single antigen-specific B lymphocytes from monkey peripheral blood, which has improved the positive rate of antigen-specific B lymphocytes and promoted the development and production of monkey-derived monoclonal antibodies.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of cell sorting technology, and in particular to a method for isolating single antigen-specific B lymphocytes from monkey peripheral blood. Background Art
[0002] B lymphocytes, or B cells for short, are derived from hematopoietic stem cells in the bone marrow. Mature B cells are widely distributed in the spleen, lymph nodes, and peripheral blood of mammals. When stimulated by antigens, they proliferate and differentiate into a large number of plasma cells. Plasma cells synthesize and secrete specific immunoglobulins against antigens, such as IgG, which circulate in the blood and mediate humoral immune responses. As a crucial component of the immune system, B cells secrete antibodies that play a vital role in defending against various pathogens. Monoclonal antibodies are antibodies produced by a single B cell clone that are homogeneous and specific to a particular antigen. Compared to polyclonal antibodies, monoclonal antibodies possess characteristics such as high specificity, high titer, and little or no cross-reactivity, making them effective tools for clinical diagnosis and treatment, and the detection of specific antigens or proteins. Isolating and identifying B cells capable of specifically binding to target antigens is of great significance for the preparation of monoclonal antibodies.
[0003] The early method for obtaining B cells from the immune system that target a specific antigen and preparing monoclonal antibodies was the hybridoma method, which is still widely used in scientific research, clinical diagnosis, and drug development. However, it must be said that this technology still has certain limitations, mainly in the following aspects: 1) The screening process for hybridoma cells is relatively complicated and the development cycle is long, generally requiring 4-6 months; 2) The genes encoding antibodies in the obtained hybridoma cells are unstable and are easily lost after long-term reproduction, thus losing the function of secreting antibodies; 3) It lacks universality: the development of myeloma cells is time-consuming and laborious, and currently only mice, rats, and rabbits have corresponding myeloma cells; it is not applicable to other species.
[0004] Thanks to the rapid development of modern molecular biology and immunology techniques, the in vitro cloning and expression of a specific gene on a single cell at the single-cell level, followed by large-scale production of monoclonal antibodies through in vitro recombination, has attracted increasing attention. This technology does not rely on hybridoma cells, directly isolating antibody-expressing genes from immunized B cells. Therefore, efficiently obtaining specific single B cells targeting the antigen is crucial. B cells are a heterogeneous population, exhibiting different functions and surface markers. To obtain antigen-specific B cells, specific labeling is usually required through BCR molecules (membrane-bound immunoglobulins) on their surface. However, B cells can only produce IgG molecules that specifically bind to antigens on their membrane surface at specific stages. If it is too early, IgM molecules are expressed on the B cell surface; if it is too late, the B cells will have essentially differentiated into plasma cells and lost the ability to bind antigens. Therefore, how to screen for B lymphocytes that express IgG molecules on their membrane epitopes is a current technical challenge. Memory B cells develop from mature B cells after the body's first exposure to an antigen. Their surface contains IgG molecules that recognize the antigen and can remain in the body for extended periods, making them ideal targets for monoclonal antibody production. However, there are very few tool antibodies for labeling the surface of B cells in different species; currently, only humans and mice have well-established labeling protocols, and those for other species have not yet been developed.
[0005] Rhesus monkeys, belonging to the genus *Macaque* of the family Cercopithecidae, are widely used in scientific experiments due to their close biological relationship with humans. The gene sequences of rhesus monkey antibodies are highly homologous to those of humans, and using antibodies derived from rhesus monkeys can significantly reduce interspecies rejection reactions, making them excellent candidates for the clinical preparation of monoclonal antibody drugs. However, to date, there are very few cases on the market using monkeys as a source for monoclonal antibody preparation. The main reasons are: there is no literature verifying the application of B cell labeling tools conventionally used in humans and mice to monkeys, and there are no monkey-related target antibodies available on the market. Therefore, developing an efficient technique for isolating and screening single monkey-derived B cells is a major challenge. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides a method for isolating single antigen-specific B lymphocytes from monkey peripheral blood, providing high-quality candidate B cell clones for the preparation of monkey-derived monoclonal antibodies. The aim is to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.
[0007] To achieve the above objectives, the present invention is specifically implemented through the following technical solutions:
[0008] The first aspect of this invention provides a method for isolating single antigen-specific B lymphocytes from monkey peripheral blood, comprising the following steps:
[0009] S1. Peripheral blood mononuclear cells were isolated from the blood of rhesus monkeys immunized with antigens;
[0010] S2. Using CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigens to label peripheral blood mononuclear cells, antigen-specific memory B lymphocytes that are positive for CD19, CD27 and IgG and negative for CD23, IgD and IgM are selected.
[0011] Further, in step S2, the CD19 antibody is a mouse anti-human CD19 antibody, the CD27 antibody is a mouse anti-human CD27 antibody, the CD23 antibody is a mouse anti-human CD23 antibody, the IgG antibody is a goat anti-human IgG antibody, the IgD antibody is a goat anti-human IgD antibody, and the IgM antibody is a goat anti-human IgM antibody.
[0012] Further, in step S2, the CD19 antibody, the CD27 antibody, the CD23 antibody, the IgG antibody, the IgD antibody, and the IgM antibody are all coupled with fluorescence, and the fluorescence includes PE and Alexa. 700, DyLight TM One or more of 405 and FITC.
[0013] Further, in step S2, the method for labeling the peripheral blood mononuclear cells includes: suspending the peripheral blood mononuclear cells in PBS buffer containing 0.5% bovine serum albumin, adding CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigen, mixing by shaking at room temperature for 15 min, then centrifuging at 400g for 5 min, and discarding the supernatant.
[0014] Further, in step S2, the pre-labeled screening antigen is an antigen conjugated with a small molecule fluorescent dye containing avidin and labeled with biotin.
[0015] Furthermore, the pre-labeled screening antigen preparation method includes the following steps: a coupling reaction is carried out by drop addition, in which a small molecule fluorescent dye containing avidin is added dropwise to the biotin-labeled antigen for coupling in multiple batches, and incubation is performed after each drop addition to allow coupling to proceed. The amount of the small molecule fluorescent dye containing avidin is more than 5 times that of the biotin-labeled antigen. After coupling is completed, molecular sieve centrifuge tubes are selected according to the size of the coupling product to remove the uncoupled substrate.
[0016] Furthermore, in step S2, the sorting method includes flow cytometry or immunomagnetic cell sorting.
[0017] Furthermore, the following steps are included after step S2:
[0018] S3. Using 7-AAD for labeling, select the memory B lymphocytes that are biologically active and antigen-specific.
[0019] Furthermore, the antigen is monkeypox protein E8L.
[0020] The second aspect of the present invention provides the application of the single antigen-specific B lymphocytes prepared by the method described above for isolating single antigen-specific B lymphocytes from monkey peripheral blood in the preparation of rhesus monkey monoclonal antibodies.
[0021] The advantages and positive effects of this invention are as follows:
[0022] Based on the kinship between humans and monkeys, this invention innovatively establishes a method for efficiently isolating single antigen-specific B lymphocytes from monkey PBMCs by utilizing the cross-reactivity of anti-human related target antibodies. For the first time, single B lymphocytes have been isolated from monkeys. The obtained B lymphocytes are characterized by high antigen specificity and high viability. Moreover, the sorting process greatly improves the positive rate of B lymphocytes binding to target antigens, with the antigen-specific B lymphocyte positive rate reaching 33.3%. This is beneficial for greatly increasing the number of monoclonal antibodies and shortening the development time of monkey-derived monoclonal antibodies. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 This is a graph showing the ELISA results of the antigen-specific memory B lymphocytes obtained from sorting in Example 1 of the present invention.
[0025] Figure 2 This is a graph showing the ELISA results of the antigen-specific memory B lymphocytes obtained from the sorting in Comparative Example 1 of this invention. Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.
[0027] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.
[0028] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values used in this invention should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. Furthermore, the terms "comprising," "including," "containing," "having," and similar words are non-limiting in meaning, allowing for the addition of other steps and components that do not affect the result.
[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
[0030] This invention provides a method for isolating single antigen-specific B lymphocytes from monkey peripheral blood, comprising the following steps:
[0031] S1. Peripheral blood mononuclear cells were isolated from the blood of rhesus monkeys immunized with antigens;
[0032] S2. Using CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigens to label peripheral blood mononuclear cells, antigen-specific memory B lymphocytes that are positive for CD19, CD27 and IgG and negative for CD23, IgD and IgM are selected.
[0033] This invention first isolates peripheral blood mononuclear cells (PBMCs) from the blood of rhesus monkeys immunized with an antigen. Then, PBMCs are labeled with specific antibodies against B lymphocyte surface markers CD19 and CD27 for positive selection of memory B lymphocytes. Further positive selection is performed using IgG antibody labeling, and negative selection is performed using CD23, IgM, and IgD antibody labeling to increase the abundance of memory B lymphocytes expressing IgG molecules. Combined with antigen labeling for pre-labeling selection, memory B lymphocytes capable of binding to the target antigen are obtained. The target markers for these cells are: CD19 positive, CD27 positive, IgG positive and CD23 negative, IgD negative, and IgM negative, denoted as CD19. + CD27 + CD23 - IgG + IgD - IgM - .
[0034] Due to the current lack of labeling tools for rhesus monkey B lymphocytes, it is difficult to isolate single B lymphocytes from rhesus monkeys for the preparation of monkey-derived monoclonal antibodies. To address this problem, this invention, through extensive experimental testing, discovered that CD19, CD27, CD23, IgG, IgD, and IgM targets are highly homologous in humans and monkeys, and that antibodies against human-related targets specifically bind to these targets. Therefore, based on the species affinity between humans and monkeys, this invention innovatively establishes a method for efficiently isolating single antigen-specific B lymphocytes from monkey PBMCs using the cross-reactivity of anti-human-related target antibodies. This is the first time that highly specific single B lymphocytes have been isolated from monkeys, making the preparation of monkey-derived monoclonal antibodies possible. Furthermore, the monoclonal B lymphocytes isolated through the aforementioned process, as verified by ELISA, exhibit strong antigen-binding capacity, demonstrating high specificity in antigen recognition and significantly improving the positive rate of sorted monoclonal B lymphocytes. The antigen-specific B lymphocyte positive rate can reach 33.3%. Combined with single-cell culture or single-cell PCR technology, the isolated monoclonal B lymphocytes can be used for the efficient preparation of monkey-derived monoclonal antibodies, greatly increasing the quantity of monoclonal antibodies obtained and significantly shortening the development time. In addition, due to the close kinship between monkeys and humans, the prepared monoclonal antibodies can be used directly or with only minimal modification for the treatment of primate-related diseases, providing a more convenient technology for antibody drug development.
[0035] The sorting methods described above include flow cytometry sorting and immunomagnetic cell sorting. In flow cytometry sorting, antibodies used in each step are coupled with fluorescence, thus cells are sorted based on fluorescence signals after antibody labeling. In immunomagnetic cell sorting, antibodies used in each step are bound to magnetic beads, thus magnetic cells are adsorbed and sorted after antibody labeling. This is conventional technology in the field and will not be elaborated further.
[0036] It should be noted that the fluorescent or magnetic beads used with the antibodies in each step can be the same or different, and adjustments can be made according to the specific operation. In some embodiments, when CD19 antibody and CD27 antibody are labeled and sorted simultaneously (in one reaction system) in step S2, the fluorescent or magnetic beads used for the two should be different to distinguish them by different signals. However, when labeling and sorting are performed separately, for example, first using fluorescently coupled CD19 antibody for labeling, then sorting out CD19-positive B lymphocytes, washing away the CD19 antibody, and then further labeling and sorting with fluorescently coupled CD27 antibody, the fluorescent or magnetic beads used for the two can be the same or different.
[0037] Compared to immunomagnetic cell sorting, flow cytometry can simultaneously label multiple targets, thus significantly improving the positive rate of sorting. Furthermore, the high speed, multi-channel, and high sensitivity of flow cytometers ensure the purity and monoclonal nature of the obtained cells. Therefore, in a preferred embodiment, flow cytometry is used for sorting.
[0038] CD19 is obtained in step S2. + CD27 + CD23 - IgG + IgD - IgM - The antibodies used for the antigen-specific B lymphocyte population were as follows: CD19 antibody (mouse anti-human CD19 antibody), CD27 antibody (mouse anti-human CD27 antibody), CD23 antibody (mouse anti-human CD23 antibody), IgG antibody (goat anti-human IgG antibody), IgD antibody (goat anti-human IgD antibody), and IgM antibody (goat anti-human IgM antibody). These anti-human target antibodies were experimentally verified to have the function of specifically binding to the corresponding targets in monkeys.
[0039] Optionally, the antibody-conjugated fluorescence includes, but is not limited to: PE, Alexa. 700, DyLight TM 405 and FITC.
[0040] In step S2, the method for labeling the peripheral blood mononuclear cells includes: suspending the peripheral blood mononuclear cells in PBS buffer containing 0.5% (wt) bovine serum albumin (BSA), adding CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigen, mixing by shaking at room temperature for 15 min, then centrifuging at 400g for 5 min, and discarding the supernatant.
[0041] Optionally, in step S2, the pre-labeled screening antigen is a biotin-labeled antigen conjugated with a small-molecule fluorescent dye (such as APC) containing avidin, and this antigen is the same as the antigen in step S1. Its preparation method includes the following steps: a coupling reaction is performed by dropwise addition, with the small-molecule fluorescent dye containing avidin added dropwise to the biotin-labeled antigen in multiple additions for coupling. After each addition, the mixture is incubated to ensure sufficient coupling. The amount of the small-molecule fluorescent dye containing avidin is at least five times that of the biotin-labeled antigen. After coupling, a suitable molecular sieve centrifuge tube is selected based on the size of the coupling product to remove the uncoupled substrate. By conjugating the antigen with long-chain biotin and further binding the small-molecule fluorescent dye with avidin, the antigen is fluorescently labeled, facilitating fluorescence signal amplification; simultaneously, this labeling does not affect the binding between the antigen and antibody.
[0042] Optionally, step S2 may include a step of removing dead cells to improve the viability of the final obtained memory B lymphocytes, as follows:
[0043] S3. Using 7-AAD labeling, select the biologically active, antigen-specific memory B lymphocytes. 7-AAD is a nucleic acid dye that can enter dead cells and bind to DNA, thus staining the dead cells. Unstained cells (7-AAD negative, i.e., 7-AAD-negative) can be selected. - This process yields active B lymphocytes, significantly improving cell viability.
[0044] It is understandable that in steps S1 and S2, the antigens used to prepare B lymphocytes are only required to induce an effective immune response in rhesus monkeys and to bind to B lymphocytes, and their types are not specifically limited.
[0045] In some specific embodiments, the antigen is monkeypox protein E8L.
[0046] Another embodiment of the present invention provides the application of the single antigen-specific B lymphocytes prepared by the method described above for isolating single antigen-specific B lymphocytes from monkey peripheral blood in the preparation of rhesus monkey monoclonal antibodies.
[0047] The advantages of the single antigen-specific B lymphocytes prepared by the method of isolating single antigen-specific B lymphocytes from monkey peripheral blood in the preparation of rhesus monkey monoclonal antibodies are the same as the advantages of the method of isolating single antigen-specific B lymphocytes from monkey peripheral blood compared with the prior art, as described above, and will not be repeated here.
[0048] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.
[0049] In the following examples, the monkeypox protein E8L was obtained through our own expression process. The protein uniport number is Q8V4Y0, its amino acid sequence is NP_536532.1, and its nucleic acid sequence is NC_003310.1. The nucleic acid sequence corresponding to amino acids 1-275aa was selected and constructed into the pYURK-his vector. After transfection into 293F cells, the cells were cultured for 72-96 hours, and the culture supernatant was collected. The monkeypox protein E8L was purified using conventional affinity chromatography.
[0050] In the following examples, the antibody used for labeling includes: PE-labeled mouse anti-human CD19 antibody, purchased from Biolegend, catalog number 302254, hereinafter referred to as: PE-CD19; Alexa 700-labeled mouse anti-human CD27 antibody, purchased from Biolegend, catalog number 302814, hereinafter referred to as Alexa. 700-CD27; FITC-labeled mouse anti-human CD23 antibody, purchased from Biolegend, catalog number 338506, hereinafter referred to as: FITC-CD23; DyLight TM 405-labeled goat anti-human IgG antibody, purchased from Jackson, catalog number 109-475-098, hereinafter referred to as DyLight. TM 405-IgG; and FITC-labeled goat anti-human IgD antibody, purchased from Invitrogen, catalog number H15501, hereinafter referred to as FITC-IgD; and FITC-labeled goat anti-human IgM antibody, purchased from Invitrogen, catalog number H15101, hereinafter referred to as FITC-IgM. These targets are highly homologous in humans and monkeys, and their specific binding function has been verified through experimental testing.
[0051] In the following examples, long-chain biotin was purchased from Thermo Fisher Scientific, USA, catalog number A39257. The antigen protein was conjugated according to the manufacturer's recommended standard procedure. After conjugation, the unlabeled biotin was dialyzed off to obtain biotin-labeled monkeypox protein E8L (hereinafter referred to as: E8L antigen or E8L-Biotin). The conjugated biotinylated protein was stored at -20°C for later use.
[0052] In the following examples, the pre-labeled screening antigen is a biotin-labeled antigen conjugated with a small molecule fluorescent dye (APC) containing avidin. The preparation method includes the following steps: the conjugation reaction is carried out by dropwise addition, and the small molecule fluorescent dye containing avidin is added dropwise to the biotin-labeled monkeypox protein E8L for conjugation in multiple batches. After each addition, the mixture is incubated to ensure sufficient conjugation. The amount of small molecule fluorescent dye containing avidin is more than 5 times that of the biotin-labeled monkeypox protein E8L. After the conjugation is completed, a suitable molecular sieve centrifuge tube is selected according to the size of the conjugation product to remove the unconjugated substrate.
[0053] Example 1
[0054] A method for isolating single antigen-specific B lymphocytes from monkey peripheral blood, comprising the following steps:
[0055] S1. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of rhesus monkeys immunized with the antigen; the specific procedures are as follows:
[0056] Animal immunization: To obtain B lymphocytes capable of secreting antibodies that specifically recognize monkeypox protein E8L, recombinant expressed E8L protein (1-275aa) was used as the immunogen. For the first immunization, 300 μg of the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion, which was then injected subcutaneously at multiple sites on the abdomen and back. Subsequently, every 3 weeks, 150 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion, and four booster immunizations were performed.
[0057] Obtaining PBMCs from rhesus monkey blood: Using peripheral blood PBMCs as the source of antigen-specific B lymphocytes, the following steps were performed: One week after the completion of the 5th immunization, 10 mL of anticoagulated blood was collected from the monkey intravenously, and PBMCs were separated according to the following steps:
[0058] 1) Collect fresh blood and dilute it with sterile PBS in equal volumes for later use;
[0059] 2) Take a 15mL sterile centrifuge tube and add 5mL of Ficoll-Paque plus lymphocyte separation medium (purchased from Cytiva, catalog number 17144002). Carefully add 5mL of diluted blood sample to the separation medium interface and centrifuge at 1000g for 30min. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the clear separation medium layer, and the fourth layer is the red blood cell layer.
[0060] 3) Carefully aspirate the ring-shaped milky white lymphocyte layer from the centrifuge tube and transfer it to a new 50mL centrifuge tube;
[0061] 4) Add 40 mL of RMPI-1640 medium containing 100 U / mL penicillin and 100 μg / mL streptomycin to a centrifuge tube containing lymphocytes, mix the cells, centrifuge at 500g for 10 min, and discard the supernatant.
[0062] 5) Repeat step 4) to wash the cells once;
[0063] 6) The obtained lymphocytes can be resuspended in complete culture medium (RMPI-1640 basal medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) for later use, or resuspended in fetal bovine serum containing 10% DMSO and frozen at -80°C. The cells can be transferred to liquid nitrogen the next day and stored for more than 6 months.
[0064] S2. To isolate memory B lymphocytes capable of secreting antibodies that specifically recognize the E8L antigen from obtained PBMCs, fluorescently labeled antibodies (PE-CD19, Alexa) were used. 700-CD27, FITC-CD23, DyLight TM 405-IgG, FITC-IgD, and FITC-IgM) and antigens were used to label specific lymphocyte populations in PBMCs. The specific procedure is as follows:
[0065] 1) Centrifuge the prepared PBMC at 400g for 5 minutes and discard the culture medium;
[0066] 2) Resuspend the cells in 1 mL of PBS buffer containing 0.5% BSA, transfer to a 1.5 mL EP tube, centrifuge at 400 g for 5 min, and discard the supernatant;
[0067] 3) Resuspend the cells in 1 mL of PBS buffer containing 0.5% BSA, add 2 μg of biotin-labeled E8L antigen (E8L-Biotin), mix well at room temperature with shaking for 15 min, centrifuge at 400g for 5 min, and discard the supernatant.
[0068] 4) Resuspend the cells in 1 mL of PBS buffer containing 0.5% BSA, then add APC-Neutravidin, PE-CD19, and Alexa in sequence. 700-CD27, FITC-CD23, DyLight TM 405-IgG, FITC-IgD and FITC-IgM were mixed by shaking at room temperature for 15 min, then centrifuged at 400g for 5 min, the supernatant was discarded, the cells were resuspended in PBS buffer containing 0.5% BSA, centrifuged at 400g for 5 min, and the supernatant was discarded.
[0069] S3. Perform dead cell labeling, the specific steps are as follows:
[0070] Resuspend the cells in 1 mL of PBS buffer containing 2% fetal bovine serum, add 1 μL of 7-AAD dye, filter through a flow cytometer, and then run on a flow cytometer (Beckman SRT).
[0071] Set up a flow sorting strategy to sort E8L + CD19 + CD27 + CD23 - IgG + IgD - IgM - 7-AAD - The cell populations were sorted into 96-well plates at a rate of 1 cell / well, and a suitable storage solution was selected according to the requirements of subsequent experiments.
[0072] To verify whether the sorted B lymphocytes were antigen-specific memory B lymphocytes, single-cell gene amplification technology was used to amplify the variable region sequence of the antibody, construct it into a recombinant antibody expression vector, and obtain recombinant monkey monoclonal antibodies through a 293 cell expression system. A total of 96 single B lymphocytes were isolated, and 61 recombinant antibodies were expressed. Then, by coating the corresponding antigens, an indirect ELISA method was used to verify whether the antibodies could specifically bind to the antigens. The steps are as follows:
[0073] 1) Dilute the E8L antigen protein to 1 μg / mL with 1×PBS buffer, and add 25 μL / well to the ELISA plate. Incubate overnight at 4°C.
[0074] 2) Discard the supernatant, add 100 μL of blocking buffer (PBS containing 5% Skim milk, 1% BSA and 0.05% Tween-20) for blocking, react and bind for 1 h at room temperature, then add 100 μL of washing buffer (PBS containing 1% BSA and 0.05% Tween-20) to wash the plate, and repeat five times.
[0075] 3) Add 100 μL of the antibody sample to be tested (mixed with washing buffer at a 1:1 ratio and then serially diluted 3-fold, for a total of 4 gradients). React at room temperature for 1 hour, discard the supernatant, then add 100 μL of washing buffer to wash the plate, and repeat five times.
[0076] 4) Add 25 μL of Goat anti Human IgG-HRP secondary antibody (1:5000 dilution) to each well, protect from light, react and bind at room temperature for 1 h, discard the supernatant, add 100 μL of washing buffer to wash the plate, and repeat five times;
[0077] 5) Add 100 μL of TMB substrate, gently tap the plate to ensure mixing, and react at room temperature in the dark for 3 min;
[0078] 6) When a clear gradient appears in the standard wells or the positive sample wells change color, add 100 μL of oxalic acid stop solution to each well to terminate the reaction.
[0079] 7) The absorbance was measured at 450 nm and 630 nm, respectively, and the result was expressed as OD. 450nm Subtract OD 630nm This is the corrected absorbance value.
[0080] A total of 32 positive clones were identified through the above tests. (See attached...) Figure 1 The samples marked in dark gray indicate positive clone numbers (AH). The total positive rate of antigen-specific memory B lymphocytes isolated was 33.3%, significantly higher than the positive rate of hybridomas (approximately 5%).
[0081] Comparative Example 1
[0082] To verify the high specificity of the marker combination of the present invention, the CD19 marker combination based on the memory B lymphocyte marker was simultaneously sorted. + CD27 + CD23 - 7-AAD - And antigen E8L + A total of 96 B lymphocytes were detected (it should be noted that this number is a manually set target cell sorting quantity; sorting was stopped once 96 were obtained, and does not mean that the sorted sample contained only 96 B lymphocytes). 64 recombinant antibodies were expressed. Antigen specificity was detected using indirect ELISA, yielding 2 positive clones. The memory B lymphocyte positivity rate was only 2.08% (see...). Figure 2It is evident that screening solely using CD19, CD27, and CD23 targets significantly reduces the sorting efficiency and positive clone rate of antigen-specific memory B lymphocytes. This may be because the E8L antigen could bind to other receptors on the surface of B lymphocytes, leading to false positives. However, this invention, through further labeling with IgG, IgD, and IgM antibodies, eliminates this binding, ensuring that the final sorted memory B lymphocytes are obtained through the specific binding of membrane IgG molecules to the E8L antigen, thereby greatly improving the final positive screening rate.
[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for isolating single antigen-specific B lymphocytes from monkey peripheral blood, characterized in that: The following steps are involved: S1, peripheral blood mononuclear cells were isolated from the blood of rhesus monkeys immunized with antigens; S2. Label the peripheral blood mononuclear cells with CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigens to sort out antigen-specific memory B lymphocytes that are positive for CD19, CD27 and IgG and negative for CD23, IgD and IgM.
2. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 1, characterized in that: In step S2, the CD19 antibody is a mouse anti-human CD19 antibody, the CD27 antibody is a mouse anti-human CD27 antibody, the CD23 antibody is a mouse anti-human CD23 antibody, the IgG antibody is a goat anti-human IgG antibody, the IgD antibody is a goat anti-human IgD antibody, and the IgM antibody is a goat anti-human IgM antibody.
3. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 1, characterized in that: In step S2, the CD19 antibody, the CD27 antibody, the CD23 antibody, the IgG antibody, the IgD antibody and the IgM antibody are all coupled with fluorescent molecules, and the fluorescent molecules include PE, Alexa Fluor, 700、DyLight TM 405 and FITC.
4. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to any one of claims 1 to 3, characterized in that: In step S2, the method for labeling the peripheral blood mononuclear cells comprises: The peripheral blood mononuclear cells were suspended in PBS buffer containing 0.5% bovine serum albumin, and CD19 antibody, CD27 antibody, CD23 antibody, IgG antibody, IgM antibody, IgD antibody and pre-labeled screening antigen were added, and the mixture was shaken and mixed at room temperature for 15 minutes, then centrifuged at 400g for 5 minutes, and the supernatant was discarded.
5. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to any one of claims 1 to 3, characterized in that: In step S2, the pre-labeled screening antigen is an antigen coupled to a small molecule fluorescent dye including avidin and labeled with biotin.
6. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 5, characterized in that: The method for preparing the pre-marked screening antigen comprises the following steps: The coupling reaction is carried out by dripping, and the small molecule fluorescent dye containing avidin is dripped into the antigen labeled with biotin for coupling in multiple times. After each dripping, the coupling is incubated, and the amount of the small molecule fluorescent dye containing avidin is more than 5 times that of the antigen labeled with biotin. After the coupling is completed, a molecular sieve centrifuge tube is selected according to the size of the coupling product to remove the uncoupled substrate.
7. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 1, characterized in that: In step S2, the sorting method includes flow cytometry sorting or immunomagnetic cell sorting.
8. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 1, characterized in that: The step S2 further includes the following steps: S3. Labeling with 7-AAD to sort out the antigen-specific memory B lymphocytes with biological activity.
9. The method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to claim 1, characterized in that: The antigen is monkeypox protein E8L.
10. Use of the single antigen-specific B lymphocytes prepared by the method for isolating single antigen-specific B lymphocytes from monkey peripheral blood according to any one of claims 1 to 9 in the preparation of monoclonal antibodies in rhesus monkeys.