monoclonal antibody

Biological terminology

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synonym Monoclonal antibody (Monoclonal antibody) generally refers to monoclonal antibody

This entry is made by Compilation and application of scientific encyclopedia entries of "Science Popularization China" to examine.

Monoclonal antibodies are composed of B cell clone The resulting height is uniform, only for a specific Antigenic epitope Of antibody 。 Usually Hybridoma technology To prepare hybridoma antibody Cell fusion technology On the basis of Myeloma cells Blended into B-cell hybridoma 。

Use a single Hybridoma cell The specific antibody against an antigen epitope, namely monoclonal antibody, can be prepared by culturing the cell population.

Chinese name: monoclonal antibody
Foreign name: monoclonal antibody， Abbreviation: mAb
Features: Height uniformity
Type: antibody

inventor: Kohler, Milstein and Jerne
Source cell: Single B cell
Awarded: 1984 Nobel Prize in Medicine
Definition: A highly uniform antibody produced by a single B cell clone that only targets a specific antigen epitope

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definition

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Monoclonal antibodies are produced by artificially prepared hybridoma cells, which are formed by fusion of an antigen activated B cell and a myeloma cell. Advantages of monoclonal antibody: high purity, high sensitivity, strong specificity, cross reaction Less, low preparation cost. Disadvantages: There are certain technical requirements, and it is easy to lose epitopes through chemical treatment of antigens^[1] 。

brief introduction

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1975 Molecular biologist G J.F. Kohler And C. Millstein created Hybridoma technology , which can be cultured and proliferated in vitro mice Myeloma Cells and Meridians antigen The immunized pure line mouse B cells fused to become a hybrid Cell line It not only has the characteristics that tumor cells are easy to proliferate indefinitely in vitro, but also has Antibody forming cell Synthesis and secretion of Specificity Characteristics of antibodies. This hybridoma is treated as a single cell culture , can form a single cell line, that is, monoclone. Using the method of culture or intraperitoneal inoculation of mice, a large number of highly concentrated, very uniform antibodies can be obtained, whose structure, amino acid sequence, specificity, etc. are consistent, and during the culture process, as long as there is no variation, the antibodies secreted at different times can maintain the same structure and function. This monoclonal antibody cannot be obtained by other methods.

This new technology fundamentally solves the long-standing problems of specificity and repeatability in antibody preparation, which can be used to explore

① Fine structure of protein;

② Surface novelty of lymphocyte subsets antigen ；

③ Histocompatibility antigen ；

④ Radioimmunoassay (or enzyme immunoassay) of hormones and drugs;

⑤ Tumor location and classification;

⑥ Purification of microbial and parasite antigens;

⑦ Immunotherapy and drug combined immunity- chemotherapy ("Missile" therapy, using monoclonal antibodies and Target cell Specific binding to bring the drug to the focus. Therefore, monoclonal antibodies can be directly used for the diagnosis, prevention, treatment of human diseases and the research of immune mechanism Malignant tumor The immunodiagnosis and immunotherapy of IgG have opened up a broad prospect.

Preparation process

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1. Immunized animals

Immunized animals are used to immunize mice with target antigen to sensitize mice B lymphocytes Process. Generally, 6-8 week old female BALB/c mice are selected to be immunized according to the pre established immune scheme. Antigen enters through blood circulation or lymphatic circulation Peripheral immune organ Stimulate the corresponding B lymphocyte clone to activate, proliferate and differentiate into sensitized B lymphocytes.

2. Cell fusion

use carbon dioxide The mice were killed by gas, and the spleen was taken out by aseptic operation, and then crushed in a plate to prepare the suspension of spleen cells. The prepared homologous myeloma cells were mixed with mouse spleen cells in a certain proportion, and polyethylene glycol, a fusion promoting agent, was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

3. Selective culture

The purpose of selective culture is to screen the fused hybridoma cells. Generally HAT selective medium 。 In HAT medium, the de novo synthesis of DNA in unfused mouse myeloma cells would be blocked; Unfused myeloma cells are deficient Hypoxanthine - Guanine -Phosphoribosyltransferase (HGPRT), which can not use the remedial pathway to synthesize DNA; In this way, the two DNA synthesis pathways of unfused mouse myeloma cells were blocked, and the DNA of myeloma cells could not replicate and died. Although unfused B lymphocytes have hypoxanthine guanine phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only the fused hybridoma cells can synthesize DNA through the remedial way because they obtained hypoxanthine guanine phosphoribosyltransferase from B lymphocytes, and have the characteristics of unlimited proliferation of myeloma cells. Therefore, hybridoma cells can survive and proliferate in HAT medium.

4. Screening and cloning of hybridoma positive clones

Only a few hybridoma cells growing in HAT medium secrete predetermined specific monoclonal antibodies, so screening and cloning must be carried out. Usually Finite dilution method The hybridoma cells were cloned and cultured. Using sensitive, rapid and specific immunological methods, the positive hybridoma cells that can produce the required monoclonal antibodies were screened and cloned for amplification. After comprehensive identification of the monoclonal antibody it secretes immunoglobulin Type, subclass, specificity, affinity, epitope and molecular weight of recognized antigen shall be frozen in time.

5. Mass preparation of monoclonal antibodies

The mass preparation of monoclonal antibody mainly adopts animal in vivo induction method and in vitro culture method.

(1) In vivo induction method, BALB/c mice were injected intraperitoneally with 0.5 ml liquid paraffin or norphytane for pretreatment. After 1-2 weeks, hybridoma cells were inoculated intraperitoneally. Hybridoma cells proliferate in the abdominal cavity of mice and produce and secrete monoclonal antibodies. About 1-2 weeks, the abdomen of the mice was swollen. A large number of monoclonal antibodies can be obtained by extracting ascites with a syringe.

(2) In vitro culture method, hybridoma cells were cultured in culture bottles. In the process of culture, hybridoma cells produce and secrete monoclonal antibodies, collect the culture supernatant, centrifugate the cells and their fragments, and then obtain the required monoclonal antibodies. However, the amount of antibody produced by this method is limited. Various new culture technologies and devices are constantly emerging, which has greatly increased the production of antibodies.

Antigen preparation

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Antigen means that it can stimulate the body to produce (specific) immune response, and can interact with the immune response products, antibodies and Sensitized lymphocyte In vitro binding, occurrence Immune effect (a specific reaction).

There are two basic characteristics of antigen,

One is the ability to induce immune response, that is, immunogenicity,

The second is to react with the products of immune response, that is, antigenicity.

Many substances can become antigens. For specific classification of antigens, see antigen In the process of preparing monoclonal antibodies, many substances can become antigens. In routine scientific research experiments, researchers often choose every mouse/rat to inject 10~50ug each time Recombinant protein 、 coupling Peptides, conjugated small molecules, etc. are used as antigens to produce specific monoclonal antibodies.

Animal immunity

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BALB/c mice are generally used as the host animals for the preparation of monoclonal antibodies. Mouse monoclonal antibodies are widely used. Some domestic and foreign companies have developed rabbit monoclonal antibody preparation technologies.

Immunogen is divided into Soluble antigen And granular (cellular) antigen, diluted with sterile saline and mixed with adjuvant, intraperitoneally injected with the stable emulsion formed after thoroughly mixing the antigen and adjuvant, and enhanced immunity based on the continuous immune response provided by the immunogen

Take 0.2ml blood on the first day of the cycle (obtain 0.1ml pre immune serum)

First immunization (antigenplus Freund's complete adjuvant ）

The second immunization on the 14th day (antigenic addition Freund's incomplete adjuvant ）

Blood collection and ELISA test on the 21st day

The third immunization on the 35th day (antigen plus incomplete Freund adjuvant)

Blood collection and ELISA test on the 42nd day

The fourth immunization on the 56th day (antigen dissolved in PBS or saline)

Day 61 cell fusion

cell fusion

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Fusion agent

Cell fusion methods include physical methods, such as electrofusion, laser fusion, chemical fusion and biological fusion, such as inactivated Sendai virus Here, one of the chemical fusion methods is polyethylene glycol Fusion method. Polyethylene glycol (PEG), at molecular weight When it is 200~700, it is a colorless and odorless viscous liquid; when its molecular weight is greater than 1000, it is a milky white waxy solid, soluble in water, ethanol and many other organic solvents, heat stable, and does not work with many chemicals. be used as cell fusion Polyethylene glycol (PEG) of the agent is generally selected with a molecular weight of 4000 and a common concentration of 50%, pH 8.0~pH 8.2 (adjusted with 10% NaHCO3). PEG with small molecular weight has poor fusion effect and toxicity If the molecular weight is too large, the viscosity is too large and it is difficult to operate. The fusion effect is the highest when the concentration is 50% and the pH is slightly alkaline. PEG of 30%~50% concentration plus 10% DMSO is also used. Different Batch No Even though the molecular weight of PEG is the same, the fusion rate is obviously different, so attention must be paid when selecting PEG. Each batch must be Cytotoxicity It can be applied only after the test. Buy high purity PEG for gas chromatography is generally selected.

Fusion process

There are many fusion methods, including Rotation method And centrifugation. Spleen cells and Myeloma cells The scale of is from 1:1 to 10:1. 3: 1 or 5:1 is most commonly used.

1. Reagents and materials

(1) Spleen cells and myeloma cells for fusion.

(2)1640 Culture medium 100ml。

(3) Complete 1640 liquid 100ml.

(4) 2.5% FCS-1640 solution 50ml.

(5) HAT culture medium 100ml。

(6) 50% PEG: Take 4000 molecular weight, high purity (imported from Japan or Serva) PEG 10g and put it into a 25ml bottle for high-pressure sterilization. Before use, mix 10ml of 1640 solution preheated at 40 ℃ with the same amount (W/V), and check the pH with phenol red. Generally, it is unnecessary to adjust the pH value. If the pH changes, it can be adjusted with HCl or NaHCO3.

(7) 10ml and 50ml sterile precipitation tubes or bottles.

(8) 40 hole plastic culture plate.

2. Operation method

⑴ Prepare spleen cells.

⑵ Mix 108 spleen cells and 107 mouse myeloma cells in a 50ml sedimentation tube, and add 50ml 2.5% FCS-1640 solution.

⑶ Centrifuge at room temperature of 400g for 3min to precipitate cells.

(4) Remove supernatant 。

(5) Tap the bottom of the pipe to make the sediment flow Settling tube Put it in a 40 ℃ water bath to reach the fusion temperature.

(6) Add 0.8ml of 50% PEG preheated to 40 ℃, drip slowly with 1ml pipette, shake the sedimentation tube while adding, and particles can be seen by naked eye. The drip process is required to last for 2min.

(7) Add 1ml of 1640 liquid and shake it for 1min.

(8) Repeat (7) once.

(9) Add 1ml 1640 liquid, shake while adding, and continue for 0.5min.

(10) Repeat (9) once.

(11) Add 15ml 1640 solution.

(12) At room temperature, centrifugate 400g for 1min to precipitate cells.

(13) Remove the supernatant, tap the bottom of the tube gently, and add 25ml of complete 1640 liquid containing HAT.

(14) Add fused Cytosol , 1 drop per hole (about 0.05ml).

(15) After gently shaking, put into 37 ℃ of 5% CO2 saturation humidity Hotbox Medium cultivation.

(16) On the 3rd, 6th, 9th and 10th days, replace with complete 1640 culture medium containing HAT. Pay attention to gently suck the supernatant, do not suck out the cells fixed at the bottom of the hole, and add appropriate amount of Feeder cell 。

(17) Add complete 1640 culture medium containing HAT on the 12th and 15th days. Used before each fluid change inverted microscope The growth of hybridoma cells can be observed in about 10 days. Most hybridoma cells appear within 10~20 days, but some can only appear within 1 month. After hybridoma cells appear, aspirate the supernatant and check the antibody.

(18) Hybridoma cells that continue to grow shall be proliferated and passaged. In the process of passage, HAT solution and complete 1640 solution are canceled and replaced by 10% FCS-1640 solution according to the situation. Also saved in liquid nitrogen And Cloning During this period, each generation should check the antibody to prevent the mutation and loss of antibody producing cells.

Cell fusion key

1. Technical errors often lead to the failure of fusion. For example, donors lymphocyte No immune response 。 This is bound to fail.

2. The biggest failure reason of fusion test is pollution. The key to successful fusion is to provide a non-toxic and sterile operating environment.

Hybridoma cell

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Cloning A large number of single clonal antibodies can be obtained by collecting the supernatant from a large number of cells cultured in vitro. however in vitro culture The monoclonal antibody obtained by the method is limited, which cannot exceed the specific cell concentration, and the culture medium needs to be changed every day. Hybridoma cell reproduction in vivo can overcome these limitations. Hybridoma cells have the genetic characteristics of tumor cells derived from parental lymphocytes. If inoculated Histocompatibility Of homologous mice or mice that cannot reject hybridoma (none thymus The hybridoma cells began to multiply indefinitely until the host died. The ascites and serum of mice producing tumor cells contain a large number of monoclonal antibodies secreted by hybridoma cells, and the titer of such antibodies is often 100~1000 times higher than the supernatant of cultured cells.

utilize Immunosuppressant , such as Norphytane , Liquid paraffin wax Anti lymphocyte serum can accelerate and promote the growth of tumor.

Rat neuroglial cell line tumor hybridoma cell line

1. Materials

(1) Autoclaved Norphytane 。

(2) Balb/c mice: 5~8 Weeks old 。

(3) Hybridoma cells: Logarithmic growth period Cells.

(4) RPMI-1640 culture medium

(5) Newborn bovine serum

2. Operation method

(1) 0.5 ml of norphytane was intraperitoneally injected into mice, and cells were implanted within 1 to 9 weeks after injection.

(2) Hybridoma cells in logarithmic growth phase were collected, washed once with 5% FCS - 1640 solution, centrifuged at 1000 r/min for 10 min.

(3) Sampling Trypan blue Dyeing Living cell 5% FCS - 1640 solution was used again to prepare 1.0 × 107 cells/ml suspension.

(4) I got an injection Norphytane Of Duplicant Hybridoma cells were inoculated, and 1ml (including 1.0 × 107 cells/ml) was injected intraperitoneally into each mouse.

(5) About 10 days after inoculation, the tumor volume is the largest. At this time, ascites can be extracted from the abdominal cavity, once every 1-3 days, 10 times. The serum can be determined by the axillary artery or heart Blood was collected and separated.

Purification experiment

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Monoclonal antibody preparation process

1. Material (1) 20mMol/LpH7.8~7.9Tris HCl buffer solution

20mMol/LNaCl

(2) 20mMol/LpH7.8~7.9 Tris HCl buffer solution

40mMol/LNaCl

(3) 20mMol/LpH7.8~7.9 Tris HCl buffer solution

80mMol/LNaCl

(4) 20mMol/LpH7.8~7.9 Tris HCl buffer solution

(5) Saturation ammonium sulphate liquid

(6) DEAE cellulose column

2. Operation method

(1) The mouse ascites were diluted 4 times with cold PBS solution, centrifuged at 1.00 × 105 for 30 min, and precipitated.

(2) Add saturated ammonium sulfate solution slowly in the supernatant at 4 ℃, stir while adding, so that the solution finally reaches 50% ammonium sulfate concentration.

(3) The solution was placed in ice for 30min~60min, and then centrifuged at 5000r/min for 10min to remove the supernatant.

(4) Dissolve the precipitate in Tris HCl buffer solution (40mmol/LNaCl) (the solution may be turbid).

(5) Mount Dialysis bag In Tris HCl Buffer (20mmol/L NaCl).

Monoclonal antibody preparation process

(6) Centrifuge to precipitate. (7) After the solution is diluted (1:100 or higher dilution), the protein content is measured at 280nm. It is estimated that the protein content 1A280unit=0.8mgProtein generally contains about 25mg~36mg of total protein per ml of ascites.

(8) Passing DEAE cellulose column: the cellulose column is 40cm high and balanced with 20mmol/L NaClTris buffer solution. dialysis sample Dilute with Tris buffer in equal amount.

The speed of the sample entering the column bed is 1ml~2ml/min, with NaCl linear gradient Elution. Most of the monoclonal IgG was eluted at 40mmol/L and 80mmol/L NaCl, with few exceptions, the monoclonal antibody was eluted at 120mmol/L~150mmol/L NaCl. Measure the OD280nm to collect the protein peak, and store the monoclonal IgG for standby.

Hybridoma produces various immunoglobulin However, it is mainly IgG and IgM, which is the main one depends on the immune procedure of antigen. Immune once, and take the spleen 3 days later, most of which produce IgM B lymphocytes 。 Most of the patients immunized for many times were IgG.

Identification experiment

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McAb production

1. Hybridoma cell chromosome examination Colchicine Pyrolysis method.

2. Monoclonal antibody type Determination of subtype: purchase the standard antiserum of rabbit anti mouse Ig type and subtype, and use Agar diffusion method Or ELISA sandwich method.

Instruction of kit for determination of Ig type and subtype of monoclonal antibody by ELISA sandwich method

Reagent principle

This kit uses the double antibody sandwich method to identify mice Lymphocyte hybridoma Classes and subclasses of monoclonal antibodies or monoclonal antibodies purified by specific affinity in the culture supernatant (IgG1 IgG2a IgG2b IgG3 IgM IgA can be distinguished). Use the secondary antibody of the common site of mouse antibody to coat the microplate, combine it with the mouse antibody in the added culture supernatant, add HRP labeled anti mouse antibodies of various types and subclasses to react respectively, finally use the TMB substrate system to develop color and terminate with dilute sulfuric acid, and then use the enzyme microplate meter to detect the absorbance to determine the type or subclass of the tested monoclonal antibody. This kit has extremely high resolution, and is a reliable tool for you to identify mouse monoclonal antibodies and subclasses.

Scope of use

It is used for the identification of mouse monoclonal antibody Igs and subclasses and the research of related contents.

usage method

one First, restore the kit to room temperature (about 30 minutes), and then use pure water to prepare the cleaning solution to the working concentration (one part of concentrated cleaning solution plus 19 parts of pure water).

two take out Microplate 。 Each sample needs 6 holes, and the positive control is 6 holes. Store the surplus in a self sealing bag, and remember to add desiccant.

three Add 50 μ l of cell culture supernatant (or specific affinity purified antibody) to enzyme labeled microplate, add 6 holes for each sample, and add 50 μ l of each hole for positive control. Then add 50 μ l sample into each hole Diluent 。 Stick on the sealing film and incubate at 37 ℃ for 30 minutes.

four Discard the liquid in the board, and pat dry or machine wash the board on the absorbent material that can not remove the fiber for 5 times after washing the board for 5 times. Then add 100 μ l of each of the six enzyme labeled secondary antibodies to each of the six wells of each sample, as well as the six wells of the universal positive control. On the sample drawing or Microplate Mark it. Stick on the sealing film and incubate at 37 ℃ for 30 minutes.

five Absorb and discard the liquid in the board. After washing the board for 5 times, pat it dry or machine wash it for 5 times on the absorbent material that can not remove fibers. Each hole is added separately Chromogenic agent 50 μ l for A and 50 μ l for B respectively, and a new sealing film plate shall be used for color development at 37 ℃ for 20 minutes.

six The specificity of this kit is good. Generally, the results can be observed with the naked eye, and the Ig class or subclass of this sample can be known by looking at the enzyme labeled secondary antibody corresponding to the hole with blue color. In addition, the reaction termination solution (50 μ l per well) can be used to terminate the reaction and use the microplate analyzer to measure the wavelength at 450nm, and the dual wavelength measurement results at 630nm reference wavelength. See the secondary antibody of the enzyme labeled corresponding to the high value hole to know the Ig class or subclass of this sample. The positive control 0D is less than 0.5. The experiment is invalid and needs to be redone.

Saving conditions

The shelf life is one year at 2-8 ℃ in dark. Improper storage or frequent use may shorten the validity period.

matters needing attention

one Although this Kit It is still useful after expiration, but please use it within the validity period.

two When testing ascites monoclonal antibody, it should be diluted to 1/50000. Although it can be distinguished, it is not recommended. The composition of such samples is very complex, which may interfere with the results. In this case, you can select C030215 antibody identification reagent. It will bring you satisfactory results.

three When washing the board by hand, be sure to fill the hole, stay for 10 seconds, then discard it, and finally clean it. In particular, the enzyme must be sucked out rather than thrown out when washing the plate after the temperature resistance of enzyme label II. This is very important for the results when washing plates manually.

four The boards that are not used up at one time should be sealed in a self sealing bag with desiccant and stored together with the box.

five Do not shake the board violently when handling it to avoid wrong results due to cross contamination between holes.

The specificity of monoclonal antibody can be identified by various methods, such as immunization Fluorescence method ELISA, indirect hemagglutination and Immunoblotting Technology, and immune blocking test is also required.

Monoclonal titer The agglutination reaction, ELISA or radioimmunoassay can be used. Different determination methods have different potencies. The titer of culture supernatant is far lower than that of ascites. The titer of ascites can reach 5 × 10 by agglutination reaction four 。 By ELISA, the titer of ascites can reach 1.0 × 10 six 。 The titer of monoclonal antibody is used to cultivate the Dilution express.

If necessary, the affinity and recognition of the monoclonal antibody can also be measured Antigenic epitope Capacity measurement of.

Cloning method

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The positive hole after antibody determination can be expanded for culture and cloning to obtain the monoclonal antibody secreted by the offspring of a single cell. Generally speaking, the earlier the cloning time, the better. Because in this period Hybridoma cell At the same time, they grow vigorously, competing for nutrition and space, and the cells that produce the designated antibodies may be submerged and eliminated. However, the cloning time should not be too early. If it is too early, the cell traits are unstable, and the number is small and easy to lose. Cloned positive hybridoma cells, after a period of culture, will also be caused by cell mutation or specific chromosome Some cells lose the ability to produce antibodies due to the loss of. The number of cloning times is determined by the strength of secretory capacity and antigen Immunity It depends on the strength. Generally speaking, antigen with strong immunity can be cloned fewer times, but it needs at least 3-5 times to be stable. There are many cloning methods, including Finite dilution method , micromanipulation, soft agar plate method and fluorescence activation separation method.

（1） Finite dilution method

1. Materials

（1） Microculture Disc, holes in disc Cloning The peritoneal cells (i.e. feeder cells) of mice were cultured for 20000 to 40000 per well the day before.

（2）HT culture medium

2. Operation method

(1) Take out antibody positive Pore cell Cell suspension was made with HT culture medium. The samples were stained with trypan blue and counted.

(2) The cells were diluted with HT culture medium into 200/ml, 40/ml, 20/ml and suspension.

(3) Use a straw to seed the cell suspension into the micro culture disk, 0.05ml for each hole, and the cell content is 10/hole, 2/hole, 1/hole and 0.5/hole respectively.

(4) 5% CO2 saturation humidity, 37 ℃ culture.

(5) Observe the growth of clone with inverted microscope every day, and select only one clone Colony Growing holes, discard more than two and none Cell growth Hole for.

(6) After the clone is propagated in large numbers, when it is covered with 1/3~1/2 of the hole bottom, the antibody in the culture medium is measured.

(7) Antibody positive pore cells, move to Feeder layer Organization of Culture flask In the second to fourth generation, it can be separated from feeder cells and cloned.

Cells

（2） Soft agar Cloning The specific operations are as follows:

1. Prepare 30ml of 2.5% agarose, dissolve it in water bath, and then transfer it to 45 ℃ water bath.

2. Mix 117ml of complete DMEM solution and 3ml of DMEM solution with 10 times concentration, and place them in a 45 ℃ water bath for preheating.

3. Mix agarose with DMEM solution, which is a complete DMEM solution containing 0.5% agarose, and add 75 × 108 spleen cells.

4. Add 10ml to each plate and solidify at room temperature.

5. The cells in DMEM are mixed with DMEM agar 1:1, and 2ml of the cell agar mixture is spread on the solidified plate to cover it completely.

6. Put it into the CO2 tank for saturation humidity, and incubate it at 37 ℃ for 10 days.

7. Use PBS to prepare 0.6% agarose, dissolve it in boiling water bath, set it at 45 ℃, take a test tube under heat preservation, quickly add 0.1ml 25% sheep red blood cell, 0.2ml guinea pig Complement, 2.7 ml 0.6% agarose.

8. 3ml agarose - Sheep red blood cell mixture was used to cover the clone. Incubate in 37 ℃ CO2 chamber for 1h~2h. The hemolysis range of sheep red blood cells dissolved from the upper part of the clone can be used to screen anti sheep red blood cell Ig.

（3） Microscopic operation

Monoclonal antibody preparation process

Add 1ml 1.0 × 108 cell suspension in a culture dish with a diameter of 6cm, place it in a 5% CO2 saturated humidity, place it in a 37 ℃ incubator for more than 30min, invert it under a microscope, look for those individual cells far away from the surrounding capillary Port (one end has a right angle elbow capillary, and the other end is connected with a foot long latex Put the tube horizontally on the liquid level, move it slightly from left to right until the tube mouth is seen, aim at the cell, inhale the capillary, move the cell in the tube into the 96 well plate with 2.0 × 104~5.0 × 104 feeding cells in advance, and after culture, obtain the kelon formed by a single cell. （4） Fluorescence activation separation method

Activating cells with a fluorescence classifier (Fluorescein Activafed Cell Sorter, FACS). The basic principle is: after the cells are dyed with fluorescent antibodies, they form linear droplets of single cells through the nozzle Lesser Under light excitation, fluorescein Emit fluorescence, this signal is received by the photomultiplier tube, and then combined with the cell shape and size to generate light scattering signal. After computer processing, the signal is generated and compared with the predetermined signal. According to the different cell fluorescence intensity and cell size, the cells are divided into different levels electric field And collected in different containers.

Cell selection

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（1） Splenocyte

1. Materials

(1) Immunized Serum antibody Balb/c mice with high titer.

Monoclonal antibody preparation process

(2) 1640 culture medium (3) 2.5% FCS-1640 nutrient solution

2. Operation method

(1) The mice were killed by neck pulling or CO2.

(2) Put the mice in 70% alcohol The spleen was taken out and fixed on the plate under sterile conditions.

(3) Put the spleen into 5ml of 1640 liquid containing 2.5% FCS, and gently wash off the red blood cells on the spleen under the ice bath.

(4) Gently squeeze the spleen with tweezers to make a suspension of spleen cells, and transfer the suspension into a small test tube with capillary tubes.

(5) Erect the small test tube for 3min, and make the large connective tissue Sink and transfer the cell suspension into the centrifuge tube.

(6) Filled with 2.5% FCS - 1640 Centrifugal tube And centrifuged at 400g for 7 min to 10 min (preparation shall be started at the same time marrow cells ）。

(7) Use about 10ml fresh culture medium for precipitation Resuspension 。

(8) Repeat steps (6) and (7).

(9) Counting cells, for trypan blue staining Phase contrast microscope The number of living cells should be higher than 80%.

(II) Myeloma cells

Myeloma cells can produce and secrete a large number of immunoglobulins, such tumors cell fusion After that, it may affect or reduce the titer of the antibody secreted, so it is necessary to select myeloma cells of non secretory immunoglobulin deficiency type.

Conditions for selecting myeloma cells:

① The source of the tumor cell line should be the same strain as that of the spleen cell preparation mouse, so that the histocompatibility antigens of the two are consistent;

② Myeloma cells must be Resting state , no γ Globulin Or not secreted to the outside of cells;

③ The growth of myeloma cells needs a higher cell density, preferably 106 cells/ml;

④ Fast growth and short reproduction time.

frequently-used Balb/c mice Myeloma cell lines produced include:

①S194/5XXO·BU·1；

② SP2/0-Ag14 (SP2 for short);

③ P2 - X - Ag 8 · 6 · 5 · 3 (653 for short);

④ FO is most commonly used in 653, which is composed of mineral oil 4 methyl 15 alkane (Pristane, Norphytane ）Induced Plasmacytoma (MinerolOilplasmacytoma, MOPC) and cultivated to form an 8-azaguanine Resistance The subline of. Myeloma cells are generally introduced directly by relevant units and stored in liquid nitrogen tanks at - 195 ℃. Resuscitation, proliferation and passage are sufficient during the test. Because myeloma cells are semi adherent and easy to fall off, there is no need for trypsin treatment. To prevent Atavism Before fusion, 15 μ g/ml of 8-nitrogen can be added to the culture medium Guanine 。 Take about 1 × 107~6 × 107 Logarithmic growth period Myeloma cells (cultured for 15-20 h) were centrifuged at room temperature (400g) for 10 min, and precipitated with 2.5% FCS 1640 solution Resuspension And count.

（3） Feeder cell

In vitro cell culture, single cells or a small number of cells are not easy to survive and reproduce, and other living cells must be added to make them grow and reproduce. The added cells are called feeder cells. stay cell fusion In the process of selecting and monoclone, it is on the basis of a small number or a single cell to make it grow and reproduce into a population, so feeder cells must be used in this process. Many kinds of Animal cell Can be used as feeder cells, such as normal spleen cells Thymocyte Peritoneal exudate cells are often used, mainly macrophage And lymphocytes.

The advantages of using peritoneal exudate cells are:

On the one hand, it is used as feeder cells. On the other hand, macrophages can engulf dead cells and cell fragments, creating a good environment for the growth of fusion cells. The source of peritoneal cells can be the same strain of mice as myeloma cells. It can also be other kinds of mice, such as C57 mice, Kunming mice, etc.

1. Materials

(1) Several mice (Balb/c or C57 mice).

(2) 11.6% sucrose solution (autoclaved for 10 pounds and 30 minutes).

2. Operation method

(1) The mice were killed by neck pulling.

(2) Soak in 70% alcohol for disinfection for 10 min.

(3) Cut the mouse abdomen with a surgical scissors, peel off the skin, and expose the abdominal cavity.

(4) Inject 4ml 11.6% sucrose solution into the abdominal cavity with a syringe, gently rub it with fingers for 1min, and then use the syringe to draw back the peritoneal fluid, and add it into the centrifuge tube.

(5) Centrifuge at 1000 r/min for 10 min.

(6) The supernatant was discarded and cells were made into suspension with HT medium. Trypan blue Dye and count the living cells to make every milliliter contain 400000 living cells.

(7) Use 1ml pipette to seed cells into micro Petri dish 0.05ml per well, containing 20000 cells, put into the CO2 incubator for culture cell fusion and Cloning Use

application

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Since the advent of monoclonal antibodies, because of their unique characteristics, they have been rapidly applied to many fields of medicine. It is mainly manifested in the following aspects.

1. Laboratory medicine Diagnostic reagent

As a diagnostic reagent in the laboratory of laboratory medicine, monoclonal antibodies are widely used in ELISA 、 Radioimmunoassay , immunohistochemistry and Flow cytometry And other technologies. And the application of monoclonal antibodies has greatly promoted the development of commercial kits.

Commercialized kits made from monoclonal antibodies are widely used in:

① Pathogenic Microbes Detection of antigens and antibodies;

② tumor antigen Testing;

③ Immune cell And its subgroups;

④ Hormone determination;

⑤ cell factor Determination of.

Recognition of antigen by monoclonal antibody, and Polyclonal antibody There is a big difference. Different test kits use different monoclonal antibodies and different antigen recognition sites, leading to certain differences in test results. Therefore, standardization needs further study.

2. Protein purification

Monoclonal antibodies are affinity chromatography Important ligands in the. Monoclonal antibody is adsorbed on an inert solid matrix (such as Speharose 2B, 4B, 6B, etc.) and prepared into Chromatography column 。 When the sample flows through the chromatographic column, the antigen to be separated can specifically bind to the solid phase monoclonal antibody, and other components cannot bind to it. After the chromatographic column is fully eluted, change the ionic strength or pH of the eluent, the antigen to be separated will be dissociated from the antibody, and the antigen to be purified can be obtained by collecting the eluant.

3. Tumor targeted therapy and radioimmunoimaging technology

Monoclonal antibody against a tumor antigen is combined with Chemotherapy drugs Or radiotherapy material connection, which uses the targeting effect of monoclonal antibodies to carry drugs or radiotherapy materials to target organs and directly kill target cells, is called tumor targeted therapy. In addition, radioactive Markers It can be connected with the monoclonal antibody and injected into the patient's body for radioimmunoimaging to assist in tumor diagnosis. Monoclonal antibodies are mainly mouse derived antibodies, and xenogeneic animal serum can cause human allergic reactions. Therefore, the preparation of human human monoclonal antibody or Humanized antibody More important, but no significant progress has been made in this regard.

Advantages and limitations

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1. Advantages of monoclonal antibodies

(1) Hybridoma can survive and pass on permanently in vitro. As long as there is no gene mutation in the cell line, it can continuously produce antibodies with high specificity and high homogeneity.

(2) A large number of highly specific and uniform antibodies can be obtained by using relatively impure antigens.

(3) Since it is possible to obtain "unlimited" amounts of homogeneous antibodies, it is applicable to Labelled antibody Characteristic immunological analysis methods, such as IRMA and ELISA.

(4) Because of its high specificity and single biological function, monoclonal antibodies can be used for radioimmunoimaging and immuno targeting therapy in vivo.

2. Limitations of monoclonal antibodies

(1) The inherent affinity and limitation of monoclonal antibodies biological activity It limits its application range. Because monoclonal antibodies can not carry out precipitation and agglutination reactions, many detection methods cannot be completed with monoclonal antibodies.

(2) The reaction intensity of monoclonal antibody is not as strong as that of polyclonal antibody.

(3) The preparation technology is complex and time-consuming, so the price of monoclonal antibody is also high^[2] 。

Research progress

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The development of monoclonal antibody drugs originated in 1975. The advent of hybridoma technology makes it possible to prepare a large number of uniform mouse monoclonal antibodies. In 1986, the first mouse derived monoclonal antibody muronab-CD3 (OKT3) against post transplant immune rejection was approved by the US Food and Drug Administration (FDA) for marketing. However, the antibody from mouse derived lymphocyte hybridoma was recognized by the human immune system, which would cause serious human anti mouse antibody (HAMA), It not only shortens the half-life of therapeutic monoclonal antibody, weakens its efficacy, but also sometimes causes serious adverse reactions.

The development of monoclonal antibody drugs fell into a low ebb between 1988 and 1993. With the development of recombinant DNA technology, various antibody humanization technologies have developed rapidly. Monoclonal antibody drugs have gone through the stages of human mouse chimeric monoclonal antibody and humanized monoclonal antibody. The subsequent phage display library technology and transgenic mice Technology, making it possible to produce the whole human monoclonal antibody. In 2002, the first whole human antibody Adalimumab Listing.

Monoclonal antibodies have a broad prospect in medical treatment, and are used to treat tumors, autoimmune diseases, infectious diseases, transplant rejection and other diseases. Adamu is used to alleviate moderate to severe structural damage that is ineffective in the treatment of anti rheumatic drugs (DMARD) rheumatoid arthritis Arthritis (RA).

Human Monogram Antibody

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The development of monoclonal antibodies has gone through four stages:

Mouse derived monoclonal antibody chimeric monoclonal antibody , humanized monoclonal antibody and Whole human monoclonal antibody 。

Whole human monoclonal antibody: the variable region and constant region of its antibody are both human origin, eliminating immunogenicity and toxic side effects.

The technologies related to the preparation of whole human antibody mainly include:

Human hybridoma technology, EBV transforming B lymphocytes technology, phage display technology, transgenic mouse antibody preparation technology and single B cell antibody preparation technology, etc.

Humanized and all human antibody drugs prepared from humanized and all human antibodies have the characteristics of high affinity, high specificity and small side effects, overcome various shortcomings of animal antibodies and chimeric antibodies, and have become an inevitable trend in the development of therapeutic antibody drugs.

Adalimumab As a biological monoclonal antibody, it is difficult to challenge in terms of efficacy and technical threshold; In addition, more and more doctors prefer the dosage form of subcutaneous injection in the treatment of rheumatoid arthritis, without the injection process and cost. Although the clinical trial of comparison with etanercept has not yet come out, people generally believe that the efficacy of adalimumab is better.

Developments

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Sales and growth

In 2010, the global total sales of therapeutic monoclonal antibody drugs reached 44 billion US dollars. If we add 10 billion US dollars of monoclonal antibody diagnostic and research reagents, the total market of monoclonal antibody drugs reached 55 billion US dollars. In 2011, the total market volume of monoclonal antibody drugs has reached US $62.8 billion. In the future, global monoclonal antibody drugs will still maintain a high growth rate. By 2015, the global sales of monoclonal antibody drugs are expected to reach about $98 billion. Monoclonal antibody drugs have emerged from scratch in China, and are playing an increasingly important role in the pharmaceutical market in China. At present, the market scale of McAb in China is increasing by more than 50% every year. Monoclonal antibodies are still high-end products in the Chinese market, mainly relying on imports. Roche is the biggest winner in China's McAb market, and the varieties with the highest market share are rituximab and Trastuzumab All come from Roche Pharmaceuticals, and the share of all Roche products exceeds half of the domestic McAb products. In 2010, the sales volume of tumor drugs in China reached 16 billion yuan, up 24% year on year, ranking seventh in the world, far exceeding 4.4% in the United States and other mature or emerging markets; In 2011, the market sales reached 20.8 billion yuan. Year on year growth rate 30%。 One of the factors restricting the market penetration of biosimilars is legislation, which prevents the use of biosimilars to replace original research products. This means that the use of biosimilars is limited to new patients and short-term indications. However, the medical cost control opportunities created by the use of biosimilars are huge. In those markets (including the United States) where no regulatory path has been established for biosimilars, the key factors for the success of biosimilars are patent uniqueness, clinical trial requirements and interchangeability.

Policy support for R&D

During the "Twelfth Five Year Plan" period, China's "Major New Drug Development" project will receive 10 billion yuan from the central government and 30 billion yuan of supporting funds. Special strategic priorities include innovative drug research and development, technological transformation of large drug varieties, etc. Malignant tumor Cardiovascular and cerebrovascular diseases The R&D of 10 major disease drugs, including neurodegenerative diseases, diabetes, and tuberculosis, is the key point of innovation funding support.

During the "Twelfth Five Year Plan" period, the development goal of the pharmaceutical industry is to increase the gross output value by 20% annually and reach 3.1 trillion yuan by 2015.

In 2011, monoclonal antibody drugs continued to lead the global drug market, with significant year-on-year growth.

From 2000 to 2010, the global market of monoclonal antibody drugs Compound growth rate Up to 32%.

In 2011, six of the top 20 prescription drugs in the world were monoclonal antibody drugs, and five of them had sales of more than US $5 billion. In 2009 and 2008, the figure was 40 billion US dollars and 37 billion US dollars respectively. At present, almost all large pharmaceutical companies have monoclonal antibody research and development projects.

Among the 20 most popular drugs in the world in 2011, Pfizer's Atorvastatin calcium It ranked first with US $13.3 billion. In 2011, the global market size of biopharmaceutical products exceeded US $155 billion, accounting for 25% of pharmaceutical/biopharmaceutical products. However, some research data show that the proportion of biopharmaceutical products in the global market will increase significantly in the next few years.

It is predicted that by 2014, the top three products in global sales will be Biotechnology drugs (Avastin, Humira and Enbrel), their sales will reach US $25.4 billion.

Monoclonal antibody drugs have become the most important category in biopharmaceuticals. In 2011, the sales of the seven largest antibody drugs reached 38.82 billion US dollars, accounting for nearly 50% of the whole biopharmaceuticals market share; Monoclonal antibodies are still the focus of research and development, and will also be an important driving force for the development of biopharmaceutical industry in the future^[2] 。

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