CN112266895A - Screening method of single memory B cell and application of single memory B cell in preparation of small molecule monoclonal antibody - Google Patents

Abstract

The invention provides a screening method of a single memory B cell and application thereof in preparation of a small molecule monoclonal antibody. Based on the flow sorting technology and the microfluidic technology, the invention realizes the organic combination of the precise screening of the antigen specific memory B cells and the efficient preparation of the single cell transcript in a microenvironment, and further performs the pre-amplification of the single cell variable region, the scFv prokaryotic expression and the eukaryotic expression of the monoclonal antibody. A novel single-cell antibody preparation technology is established, and a novel means is provided for preparing the small-molecule rabbit monoclonal antibody.

Description

Screening method of single memory B cell and application of single memory B cell in preparation of small molecule monoclonal antibody

Technical Field

The invention relates to the field of cell biology and immunology, in particular to a screening method of a single memory B cell and application thereof in preparation of a small molecule monoclonal antibody.

Background

The rabbit-derived monoclonal antibody has the advantages of high specificity, high activity, high affinity, capability of recognizing more novel epitopes and the like, and is widely applied to the fields of biology, medicine, environmental detection, food science and the like. With the development of monoclonal antibody technology, Epitomics corporation in the united states prepares rabbit myeloma cells, and a rabbit monoclonal antibody is successfully obtained by a hybridoma technology, but the rabbit myeloma cell line is unstable, so that the technology cannot be widely popularized and used.

The recombinant rabbit monoclonal antibody has the advantages of high consistency, repeatability and the like on the basis of keeping the advantages of the rabbit monoclonal antibody. The technologies of phage display, yeast display, mammalian cell display and the like avoid fussy procedures such as instability and repeated subcloning of fusion hybridomas, are widely applied to screening and preparation of recombinant antibodies of different species, and provide a wide space for development of monoclonal antibodies. However, in most display systems, the antibody library constructed is a random combination of heavy and light chain genes, and therefore it is difficult to screen for antibodies with higher specificity for the original pairing of heavy and light chains.

With the development of single cell sorting technology, the novel high-efficiency precise screening technology of the single B cell provides a better platform for preparing the antibody with higher specificity. The flow cytometry sorting technology can perform multi-parameter fluorescence labeling of single cells, and realize efficient sorting of different types of cells. The flow sorting technology based on the memory B cell surface antigen specific receptor provides good technical support for the precise preparation of the single cell antibody. Traditional single B cell antibody preparation requires artificial single cell RNA extraction and transcript amplification, but single cell RNA is low in quantity, volatile and degradable, and has strict environmental requirements, and some important transcripts are lost after insufficient reverse transcription and low-efficiency amplification.

One of the important features of microfluidic technology (microfluidics) is the unique fluidic properties, such as laminar flow and droplets, in a micro-scale environment. By means of the unique fluid phenomena, the microfluidics can realize the rapid preparation of single cells, meanwhile, the microfluidics can realize a series of micro operations which are difficult to complete by the conventional method, such as single cell RNA extraction, reverse transcription and specific gene amplification or the construction of a single cell cDNA library in a closed micro reaction system, and the microfluidics technology can reduce the micro-scale reaction system to nano-scale or even pico-scale reaction system to furthest reduce the pollution and cross-contamination of exogenous DNA.

Therefore, the development of a single memory B cell antibody preparation technology which combines high-throughput accurate screening and efficient preparation of single cell transcripts is particularly important.

Disclosure of Invention

The invention aims to provide a screening method of a single memory B cell and application thereof in preparation of a small molecule monoclonal antibody.

To achieve the object of the present invention, in a first aspect, the present invention provides a method for screening a single memory B cell, comprising:

1) designing and synthesizing an artificial antigen aiming at the small molecule compound M;

2) immunizing experimental animals by using artificial antigens, and separating to obtain lymphocytes;

3) sorting the lymphocytes obtained in the step 2) by utilizing a flow sorting technology and a microfluidic technology to obtain single memory B cells capable of specifically recognizing the small molecular compound M.

Preferably, the small molecule compound M is Chloramphenicol (CAP).

The artificial antigen is obtained by introducing carboxyl into a chloramphenicol structure to obtain hapten (CAP-HS), and then coupling the hapten and carrier protein through amido bond.

The structure of the hapten CAP-HS is shown in a formula I):

the preparation method comprises the following steps: weighing 2mg of CAP, dissolving in 500 mu L of DMF, adding 2.34g of succinic anhydride, reacting at 60 ℃ for 8h, and concentrating the obtained reactant to obtain CAP-HS.

The carrier protein is selected from Bovine Serum Albumin (BSA), hemocyanin (KLH), ovalbumin, thyroid protein, and human serum albumin; bovine serum albumin or hemocyanin is preferred.

Adopting an active ester method to synthesize an artificial antigen CAP-KLH/CAP-BSA: 1mg CAP-HS in 500. mu.L DMF, 2.75mg NHS in 200. mu.L DMF, 2.75mg EDC in 400. mu.L DMF, mixing the three, shaking and incubating for 6h at room temperature, slowly dropping 500. mu.L of the reaction solution into 2mg KLH, and shaking and reacting overnight at 4 ℃. Another 500. mu.L of the reaction mixture was added with 2mg of BSA and dialyzed for 72 hours. The solution obtained by coupling is subpackaged and stored at-20 ℃.

In the aforementioned method, the experimental animal is preferably a rabbit, and more preferably lymphocytes are isolated from peripheral blood of a rabbit.

In a second aspect, the present invention provides the use of a single memory B cell obtained according to the above method for the preparation of a monoclonal antibody.

In the application, the cDNA of a single memory B cell is taken as a template, and the nucleotide sequences of the light chain variable region and the heavy chain variable region of the monoclonal antibody are respectively obtained by amplification through a nested PCR reaction.

In a third aspect, the invention provides an anti-chloramphenicol monoclonal antibody, wherein the amino acid sequences of the light chain variable region and the heavy chain variable region are shown as SEQ ID NO 1 and SEQ ID NO 2, respectively.

In a fourth aspect, the invention provides a chloramphenicol detection reagent or kit prepared from the monoclonal antibody.

In a fifth aspect, the invention provides any one of the following uses of the monoclonal antibody:

(1) the application in detecting chloramphenicol;

(2) the application in preparing a detection reagent or a kit for chloramphenicol;

(3) the application in preparing immunochromatographic test strips of chloramphenicol.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the rabbit-derived single-cell chloramphenicol-resistant monoclonal antibody provided by the invention has high affinity with chloramphenicol, can specifically identify chloramphenicol, has detection sensitivity up to 0.01ng/mL (0.01ppb), has high practical value, and has good application prospect in chloramphenicol residue detection.

The existing chloramphenicol detection means mainly comprise colloidal gold test strips and the like, the chloramphenicol is a drug which cannot be detected, the sensitivity requirement on the detection antibody is very high, but the existing mouse monoclonal antibody cannot reach the detection standard, the rabbit antibody can meet the detection requirement due to the characteristics of many antigen epitopes recognized by the rabbit antibody and the like, and the novel technology for preparing the antibody by microfluidics can overcome the technical barrier of the traditional technology.

Drawings

FIG. 1 shows the synthesis of the immunogen CAP-KLH in a preferred embodiment of the present invention.

FIG. 2 is a graph of the titer of rabbit antisera in a preferred embodiment of the invention.

FIG. 3 is a diagram of flow sorting of antigen-specific memory B cells in a preferred embodiment of the invention. Wherein, A: lymphocyte clustering in peripheral blood, B: viable cells of ER tracer +, C: IgG⁺Cell, D: cap⁺A cell.

FIG. 4 is a schematic diagram of the pretreatment of the microfluidic chip and the preparation of the single-cell transcript according to the preferred embodiment of the present invention; wherein, A: chip pretreatment; b: rapidly separating the antigen-specific memory B cells after flow sorting in a chip; c: and (3) cracking and pre-amplifying the single cells in a nanoliter microenvironment of the microfluidic chip.

FIG. 5 is a diagram illustrating the integrity of single cells and their cDNAs on a microfluidic chip according to a preferred embodiment of the present invention; wherein, A: separating single memory B cells in the microfluidic chip (before lysis); b: integrity testing of single cell cDNA transcripts. 4E: the best integrity B cells were selected.

FIG. 6 is a schematic diagram of a rabbit-derived single-cell chloramphenicol single-chain antibody according to a preferred embodiment of the present inventionStandard curves for somatic and monoclonal full antibodies. As can be seen from the figure, IC of the rabbit-derived single-cell chloramphenicol Single-chain antibody₅₀IC of monoclonal full antibody 0.8ng/mL (A)₅₀Is 0.01ng/mL (B).

Detailed Description

Based on the flow sorting technology and the microfluidic technology, the invention realizes the organic combination of the precise screening of the antigen specific memory B cells and the efficient preparation of the single cell transcript in a microenvironment, and further performs the pre-amplification of the single cell variable region, the scFv prokaryotic expression and the eukaryotic expression of the monoclonal antibody. A novel single-cell antibody preparation technology is established, and a foundation is laid for the preparation of the small-molecule rabbit monoclonal antibody. The invention takes chloramphenicol (formula 1) as a target and takes New Zealand white rabbits as model animals.

One of the purposes of the invention is to provide a method for immunizing New Zealand white rabbits by chloramphenicol immunogen.

Another objective of the invention is to provide a method for identifying the affinity of rabbit antiserum.

The invention also aims to provide a method for separating rabbit peripheral blood lymphocytes.

The fourth purpose of the invention is to provide a flow sorting method of rabbit peripheral blood antigen specific memory B cells.

The fifth purpose of the invention is to provide a method for rapidly preparing the single-cell full-length cDNA transcript based on the microfluidic technology.

The sixth purpose of the invention is to provide a method for measuring the concentration and integrity of single-cell trace cDNA.

The seventh object of the present invention is to provide a method for amplifying rabbit-derived single-cell antibody variable regions.

Another objective of the invention is to provide a method for determining the sequence of the variable region of a single memory B cell antibody by TA cloning.

Another object of the present invention is to provide a method for prokaryotic expression and identification of rabbit scFv derived from single memory B cell.

The tenth object of the present invention is to provide a method for eukaryotic animal expression and identification of rabbit monoclonal antibodies derived from single memory B cells.

The invention is realized by the following technical scheme:

the preparation method of chloramphenicol immunogen CAP-KLH is as follows: 1mg CAP-HS in 500. mu.L DMF, 2.75mg NHS in 200. mu.L DMF, 2.75mg EDC in 400. mu.L DMF, mixed, incubated at room temperature with shaking for 6h, 500. mu.L of the reaction solution was slowly added to 2mg KLH dropwise, and reacted at 4 ℃ with shaking overnight. Another 500. mu.L of the reaction mixture was added with 2mg of BSA and dialyzed for 72 hours. The resulting solution was aliquoted and stored at-20 deg.C (FIG. 1).

The preparation method of the immune preparation comprises the following steps: CAP-KLH (concentration 4mg/mL) was mixed and emulsified in equal volumes with complete Freund's adjuvant/incomplete Freund's adjuvant.

The immunization mode is as follows: multi-point injection immunization method for neck and back. The times of immunization are 8, the first immunization is primary immunization and Freund's complete adjuvant is adopted, and other immunizations all adopt Freund's incomplete adjuvant. In the method, the dose of the immunogen CAP-KLH is 1mg in the first 5 times, and the dose of the immunogen CAP-KLH is decreased by 200 mu g in sequence in the last 3 times. The immunization period is 30 days, and after one week of each immunization, 30mL of blood is collected from the artery in the ear for separation of peripheral blood lymphocytes and detection of serum affinity.

The affinity identification method of rabbit antiserum is as follows: coating an ELISA plate with a coating antigen CAP-BSA, performing gradient dilution on antiserum by PBS, adding a gradient diluted antiserum solution into the coated ELISA plate, incubating the plate at 37 ℃ for 30min in an incubator, adding an enzyme-labeled secondary antibody diluent (horseradish peroxidase-labeled goat-anti-rat secondary antibody/goat-anti-rabbit secondary antibody), adding a color developing solution (2% of 3,3 ', 5, 5' -tetramethylbenzidine solution and 30% of hydrogen peroxide are mixed in equal volume), and performing OD (OD) analysis on the mixture by using the OD (OD) and the OD (concentration) of the mixture of the goat-anti-rat secondary antibody_{450 nm}The OD value of each well was measured by wavelength, and the highest dilution of the antibody was the antibody titer when the OD value was 1.5 (FIG. 2).

The separation method of rabbit peripheral blood lymphocytes comprises the following steps: centrifuging peripheral blood at 18-22 deg.C for 10min at 250g, discarding plasma, adding whole blood and tissue diluent at an amount of 1.5-2 times of the volume of the discarded plasma, and mixing. An appropriate centrifuge tube was taken, the separation medium (the amount added was equal to the volume of the diluted blood sample) was added, 800g was centrifuged for 30min, the middle lymphocyte layer was taken, washed three times, the cells were resuspended in FACS buffer in an ice bath, stored at 4 ℃ and ready for use in flow sorting.

The flow sorting method of rabbit peripheral blood antigen specific memory B cells is as follows: the sorted peripheral blood lymphocytes were resuspended in 1mL of FACS buffer (49.8mL of HBSS +0.2mL of 500mM EDTA +0.25g of BSA), 0.5. mu.g of ER trap (viable cell dye) was added, 10. mu.g of fluorescein PE (Sigma, USA) -labeled goat anti-rabbit secondary antibody (anti-Fab) was added, 20. mu.g of FITC-labeled CAP-BSA was added, and incubated on ice for 30 min. The incubated cell suspension was then centrifuged at 400g for 10min, washed 3 times with FACS buffer and aliquoted to 500. mu.L of FACS buffer (cell concentration 10)⁶About one/mL), optimizing the measuring conditions of the flow cytometer, establishing a proper sorting gate and adjusting the flow rate. Sorting IgG⁺Hapten⁺ER tracer⁺Cells were collected in 5% serum in RFMI 1640 medium in an ice bath for subsequent experiments (fig. 3).

The fast preparation process of single cell full length cDNA transcript based on micro flow control technology includes the following steps: the antigen-specific memory B cells after flow sorting were diluted to a concentration of about 600 cells/. mu.L in ice-bath medium and stored in an ice box. Firstly, adding a chip pretreatment reagent into the microfluidic chip, placing the microfluidic chip in a C1 machine for vacuumizing pretreatment, and exhausting air in each flow path of the whole chip. In each reaction chamber, cells, lysis solution, reverse transcription reagent and the like are added in sequence (figure 4), reaction is carried out for 9h, and the full-length cDNA transcript product is recovered for subsequent experiments.

The method for measuring the concentration and the integrity of the single-cell trace cDNA comprises the following steps: the method adopts the Qubit 3.0 to measure the cDNA concentration of a single cell, and adopts the Qubit 3.0 without concentration calibration because the Qubit 3.0 is provided with a previously set standard curve^TMdsDNA HS reagents, according to the instructions and cDNA mixed, incubated for 2min, concentration determination. Single cell cDNA integrity determination using Agilent 2100, made by Home AgiThe lens 2100 electrophoresis chip, added Marker and sample, single cell cDNA integrity test (figure 5).

The amplification method of the rabbit-derived single-cell antibody variable region comprises the following steps: primers for the optimized rabbit heavy chain variable region and light chain variable region (Table 1) were used, and high fidelity Pfu enzyme was used at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 20s, 55 ℃ for 40s, and 72 ℃ for 2 min; amplification is carried out at 72 ℃ for 10min, and the sizes of the VH and VL electrophoresis bands are about 300bp respectively.

The method for determining the sequence of the variable region of the single memory B cell antibody by TA cloning is as follows: and (3) respectively cutting and recovering VH and VL, carrying out blunt end connection on pLB vectors (Tiangen Biochemical technology company, China) and sequences through TA cloning, carrying out plate screening, selecting positive clones, sending sequencing, carrying out variable region sequence comparison on an IMGT website, and determining sequence characteristics and species sources.

Prokaryotic expression and identification method of rabbit scFv from single memory B cell is as follows: construction of scFv-PJB33 expression vector (PJB33 vector is gifted by professor Pluckthun, university of Socien, PJB33 vector is referred to Sanlei Xie, Kai Wen, Tao Peng, et al. A novel variable anti-activity fragment diluted by the dHLX peptide with enhanced anti-activity scFv antibody, Food and Agricultural animal immunization, DOI:10.1080/09540105.2017.1368459), electroporation transformation, centrifugation of induced bacterial fluid at 10000rpm for 10min at 4 ℃, ultrasonication of the precipitate, collection of supernatant after ultrasonication, which is the crude scFv extract, ELISA identification of scFv, and evaluation of biological activity (FIG. 6A extract).

The eukaryotic animal expression and identification method of the rabbit monoclonal antibody from a single memory B cell is as follows: connecting VH and VL of a single memory B cell with a heavy chain constant region and a light chain constant region, respectively constructing a heavy chain full-length expression vector and a light chain full-length expression vector on a plasmid pCMV3.0 (Zhuhai happy Rui Biotech, Ltd.) through signal peptide and codon optimization, and transfecting HEK-293 cells in CO₂Shake culturing in constant temperature shaking table. After 3h, adding a proper amount of antibiotics according to the needs, collecting cell supernatant, purifying the antibody, and identifying the antibody performance by ELISA (Fig. 6B).

The invention establishes a technology for efficiently screening and rapidly preparing a single antigen specificity rabbit source memory B cell antibody based on a flow sorting technology and a microfluidic technology, the method has simple and novel operation steps, the flow sorting technology can realize the efficient sorting of high-throughput antigen specificity memory B cells, the rapid separation and RNA extraction of single cells are completed by combining the microfluidic technology, the single cells are reversely transcribed into stable full-length cDNA, and proper primers are designed to realize the in-vitro amplification of the variable region sequence of the single rabbit source memory B cell antibody. The method can obtain the single-cell antibody with high specificity and high sensitivity, overcomes the technical barriers of instability of the traditional rabbit-derived hybridoma, RNA extraction of single cells, in-vitro culture of plasma cell cells and the like, and simultaneously avoids the problems of low repeatability and low affinity of the antibody of the traditional screening technology such as phage display and the like. The flow technology based on electricity and the micro-fluidic technology based on mechanics are organically combined, and the monoclonal antibody with high specificity, high sensitivity and high stability is skillfully applied to the preparation process of the small molecule single cell monoclonal antibody, so that the monoclonal antibody has wider prospects in scientific research and practical application.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 immunization of New Zealand white rabbits

The concentration of the immunogen (CAP-KLH) was adjusted to 4mg/mL using PBS buffer, and the concentration of the immunogen was determined by Bicinchonic Acid method.

6 New Zealand white rabbits (2 months old, 2.5kg in body weight) were assigned the numbers CAP-KLH-1, CAP-KLH-2, CAP-KLH-3, CAP-KLH-4, CAP-KLH-5 and CAP-KLH-6, respectively. The new zealand white rabbit is immunized for eight times, and the immunization mode is 4-8 points of neck and back immunization.

First immunization: each immunization was given 1mL of a priming preparation (1 mg of CAP-KLH per 1mL of priming preparation).

Second to eighth immunizations: counting days from the first immunization, and carrying out immunization every 30 days for 7 times of boosting immunization; the first 3 booster immunizations, each immunization 1mg CAP-KLH; the subsequent 3 booster immunizations are respectively 800 mug, 600 mug and 400 mug, after 1 week of 8 th immunization, the middle ear artery is blood-taken, the supernatant and the lower layer blood cells are collected by centrifugation and are used for separating rabbit peripheral blood lymphocytes.

Example 2 affinity identification of New Zealand white Rabbit antiserum

The antisera obtained one week after immunization were subjected to the measurement of titer and sensitivity. The assay method is indirect ELISA. The specific operation steps are as follows:

the coated CAP-BSA was diluted to 0.1. mu.g/mL with carbonate buffer (pH9.6), coated on ELISA plates at 100. mu.L/well, incubated at 37 ℃ for 2h, and then washed 3 times with PBST solution. The ELISA plates (150. mu.L/well) were blocked with 2% skim milk, incubated at 37 ℃ for 1h, washed 3 times with PBST solution and patted dry. Antiserum was diluted with PBS in a gradient of 8 dilutions starting at a 1:4000 dilution with a 2-fold gradient. 50 μ L of PBS buffer and 50 μ L of a gradient diluted antiserum solution were added to the coated ELISA plates, incubated at 37 ℃ for 30min in an incubator, and then washed 3 times with PBST solution and blotted dry. mu.L of enzyme-labeled secondary antibody diluent (horseradish peroxidase-labeled secondary goat-anti-rat antibody/secondary goat-anti-rabbit antibody) was added to each well, incubated at 37 ℃ for 30min, washed 3 times with PBST solution, and blotted dry. mu.L of color developing solution (2% 3,3 ', 5, 5' -tetramethylbenzidine solution and 30% hydrogen peroxide mixed in equal volume) was added to each well and incubated at 37 ℃ for 15 min. 50 μ L of 2mol/L concentrated sulfuric acid was added to each well. By OD_{450 nm}And measuring the OD value of each well by using the wavelength, wherein the highest dilution factor of the antibody is the antibody titer when the OD value is 1.5. FIG. 2 is a graph showing the variation of serum titer of rabbit serum during the course of immunization. From the 1 st immunization to the 8 th immunization, the antiserum titer showed a tendency to gradually rise and then to stabilize.

Example 3 isolation of Rabbit peripheral blood lymphocytes

1. And (3) selecting a rabbit with the highest serum sensitivity after 8 th immunization, taking 30mL of blood from the artery in the ear, centrifuging to collect supernatant, and diluting the blood cells in the lower layer by using whole blood and tissue diluent or PBS with the same volume.

2. Adding a proper amount of separating medium into a centrifuge tube, spreading the diluted blood above the liquid level of the separating medium, and keeping the interface between the two liquid levels clear, wherein the total volume of the two liquid levels cannot exceed two thirds of that of the centrifuge tube (a Pasteur suction tube can be used for sucking the blood, and then the blood is carefully spread on the separating medium, because of the density difference of the two liquid levels, an obvious layered interface is formed.

3. At room temperature, the horizontal rotor is 500-1000g, and the centrifugation is carried out for 20-30min (the centrifugal force is required to be larger when the volume of the blood is larger, the centrifugation time is longer, the optimal separation condition needs to be found, and the centrifugation rotating speed does not exceed 1200g at most).

4. After centrifugation, significant stratification will occur: the uppermost layer is a diluted plasma layer, the middle layer is a transparent separation liquid layer, a white membrane layer between the plasma and the separation liquid is a lymphocyte layer, and the bottom of the centrifugal tube is red blood cells and granulocytes.

5. The buffy coat cells were carefully pipetted into a 15mL clean centrifuge tube, washed with 10mL PBS or cell wash, and centrifuged at 250g for 10 min.

6. Discarding the supernatant, resuspending the cells with 5mL PBS or cell washing solution, and centrifuging for 10min at 250 g;

7. repeat step 6, discard the supernatant, and resuspend the cells for use.

Example 4 flow sorting of Chloramphenicol-specific peripheral blood memory B cells

1. The peripheral blood lymphocytes with ice bath FACS buffer heavy suspension, the cell concentration is adjusted to 10⁶one/mL.

2. Mu.g of ER tracer, 10. mu.g of PE-labeled goat anti-rabbit secondary antibody (anti-Fab) and 20. mu.g of FITC-labeled CAP-BSA were added in this order and they were stained on ice for 30 min.

3. After staining was complete, 400g was centrifuged for 10min, resuspended in ice-bath FACS buffer, washed 3 times, and aliquoted to 500 μ L FACS buffer, and the cell suspension was transferred to a sterile flow tube and placed on ice for sorting. Another 10. mu.L of cell suspension was taken for laser confocal imaging.

4. Optimizing the measuring conditions of the flow cytometer, establishing a proper sorting gate, adjusting the flow rate, and sorting the IgG⁺Cap⁺ER tracer⁺The cells were collected in 5% serum in RFMI 1640 medium in ice bath, and FIG. 3A represents lymphocyte subpopulation in peripheral blood, FIG. 3B represents live cells of ER concentrator +, FIG. 3C represents IgG⁺Cells, FIG. 3D represents Cap⁺A cell.

The cells with the best integrity number 4E were finally selected for heavy and light chain amplification.

Example 5 Rapid preparation of Single-cell full-Length cDNA transcripts based on microfluidic technology

1. And (4) preprocessing a 96-pore plate microfluidic chip. Chip pretreatment was performed as shown in FIG. 3A, by adding 200 μ L C1Harvest Reagent (Fluidigm, USA) to the wells marked with red circles, 20 μ L C1Harvest Reagent to 40 solid red circles, 20 μ L C1 Preloading Reagent to purple solid circles, 15 μ L C1 Blocking Reagent to white solid circles (Fluidigm, USA), 20 μ L C1 Wash Reagent to gray solid circles (Fluidigm, USA), and placing the chips in C1 and applying vacuum for 20 min.

2. And (4) preparing single cells. The antigen-specific memory B cell concentration after flow sorting was diluted to 66,000-333,000cells/mL, then the cell Suspension was mixed with the Suspension Reagent (Takara, japan) at a volume ratio of 3:2, 60 μ L was added to the green solid circle (fig. 4B), the C1 Blocking Reagent of the white solid circle in step (1) was discarded, the chip was put into the C1 single cell preparation system, and the single cells were captured in a separate reaction chamber of the microfluidic chip (fig. 4A).

3. Preparation of full-length cDNA transcript of single cell. Add 180. mu. L C1Harvest Reagent to the red rectangle, 9. mu.L of lysine mix A to the orange solid circle (Table 1), 9. mu.L of RT mix B to the yellow solid circle (Table 2), 24. mu.L of PCR mix C to the inlets 7 and 8 (Table 3), place the chip in the C1 machine, perform Lysis and reverse transcription of the cells and pre-amplification of the full-field cDNA, approximately for 9h (FIG. 4C).

4. And (3) recovering the single-cell cDNA preamplification product. The PCR products in the chip were recovered to a 96-well PCR plate in the order of the chip.

TABLE 1 Single-cell lysis System mix-mix A

TABLE 2 micro-fluidic chip reverse transcription system mix-mix B

TABLE 3 microfluidic chip PCR Pre-amplification System

Example 6 concentration and integrity determination of Single cell Trace cDNA

1. mu.L of the single-cell cDNA was incubated for 2min in a mixed manner with 200. mu.L of the reaction solution.

2. The sample tube is inserted into the Qubit 3.0 sample chamber and read by click. The quantitative analysis took about 3s, and 10 cells were detected at a concentration of 0.2-0.4 ng/. mu.L.

3. The cDNA of the 10 cells is selected to carry out Agilent 2100 integrity detection, the glue mixed solution and the reagent in the reagent box are balanced for 30min at room temperature, then operation is carried out, a new chip is taken out, the chip is fixed in a chip groove, and 9 mu L of the cDNA is added at the glue injection position (note that each time the sample is added, the sample is added at the bottom of the tube and cannot be added on the wall of the tube, and the pipettor is used for the first sample during each sample adding, so that bubbles are prevented from being generated).

4. And setting a timer for 60s for countdown, placing the injector on the injection machine at a position of 1mL, covering the injection machine, slowly pushing the injector down, and fixing the injector at the lowest gear to start countdown for 60 s. After the countdown is finished, the syringe is carefully removed from the gear position, and after the default time of 5s, the syringe is slowly lifted up to 1mL, and the process is stopped. The chip maker was opened and 9. mu.L of each of the mixed gels was added to the wells. Marker was added and 5 μ L Marker (green cap) was added to all 12 wells (11 sample wells and 1 ladder well).

5. mu.L of the sample was added to the sample well, and if there were less than 11 samples, 1. mu.L of ultrapure water was added to the wells without the sample instead, and the results are shown in FIG. 4B.

Example 7 amplification of the complete variable regions of the heavy and light chains

Selecting a cell with good integrity of cDNA, and optimizing rabbit heavy chain and light chain variable region primers (table 4) at 95 deg.C for 3min by using cDNA as template; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 2 min; extension at 72 ℃ for 10 min. And (3) after the PCR reaction is finished, carrying out 1% agarose gel electrophoresis identification on the product, and recovering the target fragment.

TABLE 4 Rabbit variable region amplification primer sequences

Note: r is A/G, Y is C/T, and S is C/G.

EXAMPLE 8TA cloning assay of Individual memory B cell antibody variable region sequences

1. After the target fragment gel was recovered, the ingredients were added to a sterile centrifuge tube according to table 3, the tube was flicked gently to mix the contents, and centrifuged briefly for 3-5 s. The mixed reaction solution was left at room temperature for 5 min. After the reaction, the centrifuge tube was placed on ice to perform the subsequent conversion reaction.

2. Adding part of the ligation product into 100 μ L DH5 α competent cells (the competent cells should be taken out from a refrigerator at-70 deg.C and placed on ice, the ligation product should be added when just thawing, the addition of the ligation product is not more than one tenth of the volume of the competent cells), gently flicking and mixing, ice-cooling for 30min, placing the centrifuge tube in a water bath at 42 deg.C for 90s, taking out the tube and immediately placing in the ice-cooling bath for 2-3min without shaking the centrifuge tube. Add 350. mu.L of pre-heated SOC (without antibiotics) medium at 37 ℃ to the centrifuge tube and shake culture at 180rpm and 37 ℃ for 45-60 min. The bacterial solution in the centrifuge tube is mixed evenly, 200 mul is absorbed and added on LB solid agar culture medium containing ampicillin, and the cells are evenly smeared by using a sterile elbow glass rod or glass beads. After the surface of the plate is dried, the plate is inverted and cultured at 37 ℃ for 12-16 h.

3. The resulting white colonies were inoculated into 2 XYT medium (containing ampicillin at a final concentration of 50-100. mu.g/mL), shaking-cultured overnight at 37 ℃ with a shaker, and the resulting bacterial suspension was sequenced to determine sequence information.

4. The optimal VH and VL amino acid sequences (SEQ ID NOS: 1-2) were screened by sequencing. The amino acid sequences of the corresponding monoclonal full antibodies (scFv including IgL and IgH) are shown in SEQ ID NO 3 and 4.

Example 9 prokaryotic expression and ELISA identification of Single memory B cell derived Rabbit scFv

1. The scFv constructed in the example 8 is connected to an PJB33 expression vector, dialyzed in pure water for 30min under aseptic condition, the electrochemical competence of 50 μ L RV308 (American Standard Biometrics Collection center) is taken out from an ultra-low temperature refrigerator at minus 80 ℃, placed on ice for melting, the dialyzed product is added into RV308 competent cells, mixed gently, transferred to a clean, dry and precooled click transformation cup for electric shock transformation. Immediately after the electric shock transformation, 950. mu.L of 2 XYT medium was added to resuspend the cells, and the cells were shake-cultured at 37 ℃ and 250rpm for 1 hour. 100. mu.L of the bacterial suspension was applied to a 34. mu.g/mL chloramphenicol-containing 2 XYT plate, and cultured overnight by inversion at 37 ℃.

2. Picking single colony from the plate standing overnight, shaking and culturing at 37 ℃ and 250rpm for 1h, and then carrying out PCR identification on the bacterial liquid.

3. The positive colonies were inoculated into 100mL of 2 XYT medium at a ratio of 1:100, and the OD of the inoculum was measured after 8 hours₆₀₀Value, OD₆₀₀At values between 0.6 and 0.8, overnight inducible expression of IPTG was performed (final concentration of IPTG was 0.25 mM). Centrifuging the induced bacteria liquid at 4 deg.C 10000rpm for 10min, ultrasonically crushing thallus precipitate, collecting supernatant after ultrasonic treatment to obtain scFv crude extractAnd (4) liquid.

4. Taking scFv crude extract for gradient dilution, performing chessboard ELISA with corresponding coating antigen (CAP-BSA) gradient dilution solution, and selecting OD_{450 nm}The maximum dilution factor of scFv of 1.5-2.0 is the optimal working concentration of antibody, and the corresponding concentration of coating source is the optimal working concentration of coating source. ELISA identification of scFv at the above-described optimal working concentration and evaluation of its biological activity (FIG. 5A), sensitivity IC₅₀It was 0.8 ng/mL.

EXAMPLE 10 eukaryotic expression and identification of Single memory B cell derived Rabbit monoclonal antibodies

1. VH and VL of scFv in example 9 were linked to heavy and light chain constant regions, respectively (SEQ ID NOS: 3-4), and full-length heavy and light chain expression vectors were constructed on pCMV3.0 by signal peptide and codon optimization, respectively.

2. HEK-293 cells were placed in 5% CO₂Shaking culturing at constant temperature of 120rpm in constant temperature shaking table at 37 deg.C, and allowing cells to grow to density of 2 × 10⁶～4×10⁶At one/mL, the cell can be used for subsequent transfection, and the cell survival rate is required to be more than 98% in order to ensure the transfection effect.

3. Preparing two 15mL sterile centrifuge tubes, adding 5mL culture medium and 100 mu g sterile plasmid DNA into one tube, and gently blowing, beating and mixing uniformly; and adding 5mL of culture medium and 500 mu L of TA-293 transfection reagent into the other centrifugal tube, gently blowing, uniformly mixing, and standing at room temperature for 10min to prepare the plasmid-vector compound.

4. Taking out the cells from the constant temperature shaking table, adding the prepared plasmid-vector complex while shaking, and returning CO₂Shake culturing in constant temperature shaking table. After 3h, a proper amount of antibiotic can be added according to the requirement.

5. Cell supernatants were collected and antibody purified.

Example 11 identification of Rabbit monoclonal antibodies

1. Determination of antibody affinity

Performing ELISA optimization test on purified rabbit total antibody and corresponding coating antigen (CAP-BSA) gradient dilution solution, evaluating bioactivity and optimal sensitivity IC₅₀Is 0.01ng/mL (FIG. 5B))。

2. Antibody tolerance assay

Determination of antibody salt ion tolerance concentration: preparing buffer solutions with different NaCl concentrations to make the final concentration of NaCl in the detection system be 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0M respectively, and adopting conventional ELISA detection steps to read OD value of each well and calculate IC₅₀Values (table 5). The table shows that the antibody can tolerate 6.0M NaCl and has good salt ion tolerance.

TABLE 5 determination of the tolerance of the antibodies to salt ions

Determination of methanol tolerance content of antibody: preparing dilutions with different methanol contents to give final concentrations of 0%, 10%, 15%, 20%, 30%, 40%, 50%, 60% and 70% methanol in the assay, and performing conventional ELISA assay procedures for the remainder, reading OD values of the wells and calculating IC₅₀Values (table 6). As is clear from the table, the antibody was excellent in methanol tolerance and can tolerate methanol at 60% or more.

TABLE 6 determination of the methanol tolerance of the antibodies

Antibody pH tolerance range assay: the antibody dilutions were adjusted to pH 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5, 9.0 using concentrated acid or concentrated base. The antibodies were diluted individually to optimal concentrations using the serial dilutions, and the remaining steps were performed by conventional ELISA, reading the OD of each well and calculating the IC₅₀Values (table 7). As can be seen, the antibody was well tolerated at pH, and showed good tolerance between pH4.5 and 8.5.

TABLE 7 determination of the tolerance of the antibodies to pH values

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<120> screening method of single memory B cell and application thereof in preparation of small molecule monoclonal antibody

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Claims (8)

1. A method for screening for individual memory B cells, comprising:

1) designing and synthesizing an artificial antigen aiming at the small molecule compound M;

2) immunizing experimental animals by using artificial antigens, and separating to obtain lymphocytes;

3) sorting the lymphocytes obtained in the step 2) by utilizing a flow sorting technology and a microfluidic technology to obtain single memory B cells capable of specifically recognizing the small molecular compound M.

2. The method according to claim 1, wherein the small molecule compound M is chloramphenicol;

the artificial antigen is obtained by introducing carboxyl into a chloramphenicol structure to obtain a hapten and coupling the hapten and carrier protein through amido bond;

preferably, the hapten is of the formula I):

the carrier protein is selected from bovine serum albumin, hemocyanin, ovalbumin, thyroid protein and human serum albumin.

3. The method of claim 2, wherein the test animal is a rabbit and the lymphocytes are isolated from peripheral blood of the rabbit.

4. Use of a single memory B-cell obtained according to the method of any one of claims 1 to 3 for the preparation of a monoclonal antibody.

5. The use of claim 4, wherein the cDNA of a single memory B cell is used as a template, and the nucleotide sequences encoding the variable regions of the light chain and the heavy chain of the monoclonal antibody are obtained by amplification respectively by nested PCR.

6. The monoclonal antibody against chloramphenicol is characterized in that the amino acid sequences of the variable regions of the light chain and the heavy chain are respectively shown as SEQ ID NO 1 and SEQ ID NO 2.

7. A chloramphenicol detecting reagent or kit produced from the monoclonal antibody of claim 6.

8. The monoclonal antibody of claim 6 for any one of the following uses:

(1) the application in detecting chloramphenicol;

(2) the application in preparing a detection reagent or a kit for chloramphenicol;

(3) the application in preparing immunochromatographic test strips of chloramphenicol.

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