CN112094810B - Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody - Google Patents

Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody Download PDF

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CN112094810B
CN112094810B CN202010872160.0A CN202010872160A CN112094810B CN 112094810 B CN112094810 B CN 112094810B CN 202010872160 A CN202010872160 A CN 202010872160A CN 112094810 B CN112094810 B CN 112094810B
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王战辉
沈建忠
温凯
余文博
于雪芝
江海洋
杨玲
李强
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Abstract

The invention discloses a single B cell screening method and application thereof in preparation of a small molecule monoclonal antibody. Transforming small molecules to enable the small molecules to have active groups (namely haptens), carrying out chemical coupling on the small molecules and corresponding fluorescein, and separating and purifying to obtain a hapten-fluorescein compound; b cells after the compound marker (artificial antigen) immunization are used as cells to be screened; and (4) sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells. The invention also provides application of the B cells obtained by screening in preparation of the small molecule monoclonal antibody. The invention marks specific B cells by hapten-fluorescein, utilizes a flow cytometer to carry out rapid sorting, screens fused cells in advance, removes the B cells for identifying carrier protein, and greatly improves the positive rate of identifying the B cells of small molecules.

Description

Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a single B cell screening method and application thereof in preparation of a small molecule monoclonal antibody.
Background
The traditional antibody preparation is to fuse B lymphocytes with myeloma cells, and then to obtain hybridoma cells capable of producing antigen-specific antibodies through multiple rounds of screening by a limiting dilution method. However, B cells isolated from splenocytes were not all effective for fusion, and the effective fusion ratio was only 1:105-106However, only a very small portion of the fused hybridoma cells can secrete antigen-specific antibodies. Particularly in the case of small molecule hapten immunizations, a significant proportion is directed to the carrier protein or "bridge chain" rather than the small molecule itself.
The invention efficiently screens the specific B lymphocytes and the hybridoma cells of the mice based on the flow cytometry and establishes a rapid and efficient monoclonal antibody preparation method. The hapten-fluorescent marker is adopted to enrich the antigen specificity B cells, and the influence of carrier protein and bridge chain antibody is removed through the sorting of a flow cytometer, so that the accurate screening of positive cells is achieved, the effective fusion efficiency is improved, and the accurate preparation of high-performance antibody is realized.
Disclosure of Invention
The invention aims to provide a single B cell screening method and application thereof in preparation of a small molecule monoclonal antibody.
Another objective of the invention is to provide a mouse-poisoning antibody with strong specificity and application thereof.
The invention has the following conception: modifying small molecules to enable the small molecules to have active groups (namely haptens), carrying out chemical coupling on the small molecules and corresponding fluorescein, and separating and purifying to obtain a hapten-fluorescein compound; b cells after the compound marker (artificial antigen) immunization are used as cells to be screened; and (4) sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells. Furthermore, the B cells obtained by the screening method are used for preparing the small molecule monoclonal antibody.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a B cell screening method comprising:
1) designing a hapten with an active group aiming at a small molecular compound A, and chemically coupling the hapten and fluorescein to obtain a hapten-fluorescein compound;
2) coupling the hapten obtained in the step 1) with carrier protein to obtain an artificial antigen;
3) immunizing experimental animals with artificial antigens, collecting blood, and separating to obtain peripheral blood mononuclear cells;
4) labeling peripheral blood mononuclear cells with the hapten-fluorescein complex in the step 1), and then sorting and collecting cells with fluorescence by using a flow cytometer to obtain B cells capable of specifically recognizing the small molecular compound A.
The small molecule compound a may be toxoplasma (TETS).
Preferably, the structure of the murine hapten is of formula I):
Figure BDA0002651464850000021
the rat-poisoning strong hapten shown in the formula I) can be found in CN 109608479A.
The fluorescein can be 5-amino fluorescein and 6-amino fluorescein. .
The carrier protein is selected from Bovine Serum Albumin (BSA), hemocyanin, ovalbumin, thyroid protein, and human serum albumin; bovine serum albumin or hemocyanin is preferred.
The structure of the rat virulent hapten-fluorescein complex is shown as the formula II):
Figure BDA0002651464850000022
further, the compounds of formula II) may be prepared by the following method:
(1) dissolving 43mg of the rat virulent hapten shown in the formula I) in 1ml of N, N-Dimethylformamide (DMF), adding 80mg of N, N' -Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the rat virulent hapten solution, and stirring at room temperature for reacting for 2 hours to obtain an activated rat virulent hapten solution;
(2) dissolving 60mg of 5-aminofluorescein in 1ml of DMF to obtain a fluorescein solution;
(3) dropwise adding the activated virulent rat hapten solution into the fluorescein solution under the stirring state, and reacting at room temperature overnight; and then, purifying the reaction solution by TLC, separating bands, and carrying out mass spectrum identification, wherein the identification is successful to obtain the rat virulent hapten-fluorescein complex (hapten fluorescent marker complex).
The invention provides a strong mouse artificial antigen, which is obtained by coupling a strong mouse hapten with carrier protein. Wherein the virulent murine hapten is coupled to the carrier protein by an amide bond. The carrier protein may be bovine serum albumin or hemocyanin. The strong mouse artificial antigen contains a spacer arm between the strong mouse hapten and the carrier protein. The strong artificial antigen of the mouse poison can be used as immunogen and coating antigen.
Optionally, the structure of the strong mouse-poisoning artificial antigen is shown in formula III):
Figure BDA0002651464850000023
further, the compounds of formula III) may be prepared by the following method:
(1) dissolving 43mg of the rat virulent hapten shown in the formula I) in 1ml of N, N-Dimethylformamide (DMF), adding 80mg of N, N' -Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the rat virulent hapten solution, and stirring at room temperature for reacting for 2 hours to obtain an activated rat virulent hapten solution;
(2) 160mg of BSA was dissolved in 20ml of PBS buffer (0.01M, pH7.4) to obtain a BSA protein solution;
(3) dropwise adding the activated virulent rat hapten solution into the BSA protein solution under the stirring state, and reacting at 4 ℃ overnight; then putting the reaction solution into a dialysis bag, dialyzing with a 4 ℃ physiological saline solution for 48h, and changing water for 4 times; the dialysate was dispensed into centrifuge tubes and stored at-20 ℃.
In a second aspect, the present invention provides the use of B cells obtained according to the method for the preparation of a monoclonal antibody.
Alternatively, monoclonal antibodies are prepared using hybridoma technology.
In a third aspect, the invention provides a monoclonal antibody secreted and produced by a hybridoma cell strain (Mus musculus)1G6 with the preservation number of CGMCC NO. 19965. The monoclonal antibody 1G6 can specifically recognize mouse poison.
The hybridoma cell strain is deposited in China general microbiological culture Collection center, West Lu No.1 of the Chaozhou city, Chaozhou, Shangyang district, No. 3, the institute of microbiology, China academy of sciences, zip code 100101, the preservation number CGMCC NO.19965, and the preservation date of 2020, 8 months and 20 days.
In a fourth aspect, the invention provides a mouse poison detection reagent or a 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 the mouse poison strength;
(2) the application in preparing the mouse-poisoning immunochromatographic test strip;
(3) the application in preparing the colloidal gold test strip for strong mouse poisoning.
By means of the technical scheme, the invention at least has the following advantages and beneficial effects:
at present, macromolecule carrier protein is inevitably introduced in the preparation process of the micromolecule antibody, and the interference is generated on cell fusion and detection. The hapten and the fluorescein are coupled and combined on the surface of the cell, and the cell which can only recognize the micromolecule hapten is screened out by utilizing the rapid and accurate sorting of a flow cytometer, so that the positive cell is enriched, the working efficiency is improved, and the workload is reduced.
The hapten-fluorescein is used for marking the specific B cells, the flow cytometer is used for rapid sorting, the fused cells are screened in advance, the B cells for identifying the carrier protein are removed, and the positive rate of the B cells for identifying the small molecular compounds is greatly improved. According to the invention, a plurality of mouse spleen B cells are enriched in a large amount at the early stage, the number of target specific B cells is increased, and non-specific B cells are greatly eliminated, so that the workload is reduced and the working efficiency is improved.
Drawings
FIG. 1 is a mass spectrum of a rat hapten-fluorescein complex in a preferred embodiment of the invention.
FIG. 2 is the mass spectrum detection result of the rat superantigen-fluorescein complex under TETS-2j-AF negative ion mode [ M-H ] -, in the preferred embodiment of the present invention.
FIG. 3 is a mass spectrum of a strong mouse-poisoning artificial antigen in a preferred embodiment of the present invention. Wherein, A and B are respectively BSA and TETS-2j-BSA mass spectrograms.
FIG. 4 is a graph showing the comparison of the characteristics of hybridomas screened in two steps by the new method for preparing hybridomas based on flow cytometry (BCEM method) and the conventional method (Control method) according to the preferred embodiment of the present invention. Wherein, A: IC (integrated circuit)50B, the following steps: best standard curves for the BCEM and Control methods, C: ab titer, D: the Ag titer.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
The flow cytometer used was purchased from Beckman-Coulter.
Example 1 Synthesis and characterization of the murine Strong hapten-fluorescein Complex
1. Dissolving 43mg of the rat virulent hapten shown in the formula I) in 1ml of N, N-Dimethylformamide (DMF), adding 80mg of N, N' -Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the rat virulent hapten solution, and stirring at room temperature for reacting for 2 hours to obtain an activated rat virulent hapten solution;
(2) dissolving 60mg of 5-aminofluorescein in 1ml of DMF to obtain a fluorescein solution;
(3) dropwise adding the activated virulent rat hapten solution into the fluorescein solution under the stirring state, and reacting at room temperature overnight; and then, purifying the reaction solution by TLC, separating bands, and carrying out mass spectrum identification, wherein the identification is successful to obtain the rat virulent hapten-fluorescein complex (hapten fluorescence labeled complex), the structure of which is shown in a formula II), and a mass spectrum chart is shown in a figure 1. The mass spectrometric detection under TETS-2j-AF negative ion mode [ M-H ] -was 674.06598 (FIG. 2).
Example 2 Synthesis and identification of potent murine Artificial antigen
1. Dissolving 43mg of the rat virulent hapten shown in the formula I) in 1ml of N, N-Dimethylformamide (DMF), adding 80mg of N, N' -Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the rat virulent hapten solution, and stirring at room temperature for reacting for 2 hours to obtain an activated rat virulent hapten solution;
2. 160mg of BSA was dissolved in 20ml of PBS buffer (0.01M, pH7.4) to obtain a BSA protein solution;
3. dropwise adding the activated virulent rat hapten solution into the BSA protein solution under the stirring state, and reacting at 4 ℃ overnight; then putting the reaction solution into a dialysis bag, dialyzing with a 4 ℃ physiological saline solution for 48h, and changing water for 4 times; the dialysate was dispensed into centrifuge tubes and stored at-20 ℃.
The strong mouse artificial antigen is prepared into a solution by PBS for flight mass spectrometry, and BSA is used as a control. The flight mass spectrum result shows that: the rat hapten and BSA have a coupling reaction (the mass ratio of the rat hapten to the BSA is 6.0:1), so that a rat hapten BSA conjugate shown in the formula III) is obtained (a mass spectrogram is shown in a figure 3).
Example 3 method for screening potent mouse B cells
First, preparation of immune sample
1. Mouse immunization
Mice 9 weeks old, 25-35 g in weight and female Balb/c were immunized with the artificial antigen prepared in example 2, and the mice were immunized at a dose of 100. mu.g/time, once a month, for a total of 4 times, and peripheral blood of the mice was obtained after each immunization and used as a test immune sample.
2. Isolation of mouse peripheral lymphocytes
Adding peripheral blood, phosphate buffer solution and lymphocyte separation solution with equal volume into a 15ml centrifuge tube, keeping obvious liquid level boundary, centrifuging for 30min at room temperature of 800g to obtain a middle lymphocyte layer, adding 5ml phosphate buffer solution of 250g, centrifuging for 10min, and separating to obtain peripheral blood mononuclear cells.
Resuspending peripheral blood mononuclear cells, and adding 106And adding 1-3 mu g of the hapten fluorescence labeling compound prepared in the example 1 into each cell, blowing, stirring uniformly, and reacting at room temperature in a dark place. A small number of cells were left without hapten fluorescent-labeled complex as negative control. After the reaction, phosphate buffer solution is added for resuspension, 400g of the suspension is centrifuged for 5min, the supernatant is discarded, and the suspension is resuspended and precipitated by phosphate buffer solution again to prepare a sample.
3. Flow cytometric sorting
The sample is filtered by a filter screen before being put on the machine. Selecting a sorting and collecting mode, placing a proper collector gate, acquiring data and sorting. The peripheral blood mononuclear cell area is marked out through a two-dimensional scatter diagram to exclude dead cells, cell debris, granulocytes and other cells, and the peripheral blood mononuclear cells are obtained.
Positive cells were then judged by two-dimensional peak row plots of the FL1 channel/histogram interface, defining the boundary between antigen positive and negative. Antigen-specific B cells were obtained.
Example 4 hybridoma and monoclonal antibody preparation
1. Cell fusion
The positive B cells were fused with the bone marrow hybridoma SP2/0, and 1 week later, a monoclonal cell line was obtained by dilution.
2. Identification of monoclonal cell lines secreting monoclonal antibodies
Culturing the monoclonal cell strain, detecting the antibody secreted by the monoclonal cell strain, and displaying the expression of the TETS monoclonal antibody according to the detection result.
The specific method comprises the following steps:
1.1 method for calculating hybridoma formation efficiency (formula 1): the number of growth wells was multiplied by two values, corresponding to the highest, lowest, respectively, number of cell clumps in more than half of the wells, and then divided by the total number of B cells. The positive rate was calculated as the ratio of the number of wells with screening OD greater than 0.4 to the total number of wells.
Figure BDA0002651464850000051
In the formula, H: efficiency of hybridoma formation, A1: highest cell mass in more than half of the wells, A2: lowest cell mass in more than half of the wells, C: number of 96-well plate, D: number of splenic B cells 1, E: the number of spleens.
1.2 preparation of ascites
One part of the hybridoma was cryopreserved, and the other part was injected intraperitoneally to produce ascites.
1.3 antibody purification
Mouse ascites was purified using a HiTrap Protein A HP purification column.
1.4 characterization of antibody Properties
1.4.1 specific identification
Determination of IC of antibodies by ICELISA50Concentration values, evaluation of antibody sensitivity, detection of antibody cross-reactivity with other structural and functional analogs, such as heptachloro, fipronil, toxaphene, hexamethylenetetramine, amantadine, atropine, hexamethylenetetramine tribromide, and N- (4-nitrobenzyl) -1-adamantanamine hydrobromide, and the like, and identification of antibody specificity.
1.4.2 subtype identification
The purified antibody subtypes were characterized using Rapid ELISA Mouse mAb Isotyping Kit.
2.1 hybridoma Generation efficiency calculation
The hybridoma generation efficiency of the two methods (namely the novel method based on the flow cytometry and the traditional hybridoma preparation technology) is calculated by the formula 1, and the positive rate of the hybridoma cell strain of the novel method based on the flow cytometry reaches 76 percent which is 2.75 percent higher than that of the traditional method. Comparing the generation efficiency of the hybridomas after 7d fusion, and the new hybridoma preparation method based on flow cytometry is (4-12)/105Compared with the conventional method (5-10)/105The difference was not significant.
2.2 characterization of antibody Properties
2.2.1 affinity identification
The traditional hybridoma preparation groups, i.e., monoclonal antibodies of series 2, 2B6, 2J05, 2D4, 2G6, 2E8, 2a6, 2C3 and 2D9, and the new hybridoma preparation groups, i.e., monoclonal antibodies of series 1, 1E10, 1G6, 1H5, 1F4, 1D4, 1B4, 1C6 and 1B5, based on flow cytometry. Determination of IC of antibodies by the ICELISA method50Concentration values, evaluation of sensitivity, affinity and specificity of the antibodies, FIG. 4, New method set IC for hybridoma preparation based on flow cytometry5016.29ng/mL, group IC for traditional hybridoma preparation50Is 33.35ng/mL, IC50The sensitivity is improved by 2 times; hybridoma preparation based on flow cytometry novel method for preparing most sensitive antibody 1G6, IC50Is 1.98ng/mL, the most sensitive antibody 2B6, IC of the traditional hybridoma preparation group50Is 11.49ng/mL, and the sensitivity is improved by 5 times. This study also compared the antibody and antigen titers of sequence 1 and sequence 2, with significant differences in antigen dilution (p)<0.05), indicating that specific B cell enrichment increases the efficiency of efficient B cell fusion.
2.2.2 identification of specificity
Determination of IC of antibodies by the ICELISA method50Concentration values, evaluation of antibody sensitivity, detection of antibody cross-reactivity with other structural and functional analogs, and cross-reactivity of 9 other compounds (6 functional analogs and 3 structural analogs) with 1G6 and 2B6 was determined for antibodies prepared by the two methods, and the results are shown in Table 1. The monoclonal antibodies prepared by the two methods have no cross reaction with other compounds, which shows that the antibodies prepared by the two methods are not significant in specificity.
TABLE 1 specificity of functional and structural analogs CRs calculated by the ic-ELSA method
Figure BDA0002651464850000071
2.2.3 subtype identification
The purified antibody subtype is characterized and identified by a Rapid ELISA Mouse mAb Isotyping Kit, and the Kit is combined with a primer and a primerAs shown in table 2. The light chain kappa/lambda ratio of the novel process is 5/3, the kappa/lambda ratio of the conventional process is 1/1; preparation of antibody heavy chain IgG1The subtype is the most, and only one strain of IgG2aThe subtype is. Most sensitive antibody 1G6, IC prepared by the novel method50Is 1.98ng/mL, the most sensitive antibody 2B6, IC prepared by the traditional method50Is 11.49ng/mL, and the sensitivity is improved by 5 times.
2.2.4 comparison of other characteristics
Table 3 compares other characteristics of the two methods. The new hybridoma preparation method based on flow cytometry can complete the screening process within 10d, and compared with the traditional hybridoma preparation method with the method of 35d, the method shortens 25 d. The positive rate of the hybridoma cell strain in the novel method reaches 76 percent, which is 2.75 percent higher than that of the hybridoma cell strain in the traditional method. Fetal calf serum and cell culture solution are the most main experimental materials in the hybridoma preparation process, in order to obtain 8 hybridoma cells, the novel method does not need subclone culture, only 2 fetal calf serum culture media (20 percent of which are 60 mL) are paved in the fusion process (300 mu L/hole of a 96-hole cell culture plate), and the traditional method needs to be paved with 112 fetal calf serum culture media (8 fused and 8 subcloned in 4 rounds and 8 in each round) which are 3360mL, so that the consumable material is greatly reduced to 2 percent. The method does not need subcloning and ic-ELISA detection, so that the monitoring and development of toxic, harmful and expensive small molecule antibodies are possible.
TABLE 3 novel method for hybridoma preparation based on flow cytometry in comparison with conventional hybridoma preparation methods
Figure BDA0002651464850000072
Note: the cell culture medium was DMEM medium containing 20% fetal bovine serum (Gibco Corp.); serum was priced $1200 per bottle.
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.
Figure BDA0002651464850000091

Claims (3)

1. The monoclonal antibody is secreted and produced by hybridoma cell strain 1G6 with the preservation number of CGMCC NO. 19965.
2. A reagent or kit for detecting mouse toxicity prepared from the monoclonal antibody of claim 1.
3. The monoclonal antibody of claim 1 for any one of the following uses:
(1) the application in detecting the mouse poison strength; the use is for non-disease diagnostic purposes;
(2) the application in preparing the mouse-poisoning immunochromatographic test strip;
(3) the application in preparing the colloidal gold test strip for strong mouse poisoning.
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