1、第四章-2,单克隆抗体 Monoclonal Antibodies,单克隆抗体,抗体的结构与功能,单克隆抗体的制备和生产,单克隆抗体的改造和应用,Human lymphoid organs,Central lymphoid organs? Secondary lymphoid organs? Thymus, bone marrow Tonsil, adenoid, lymphatic vessels, lymph nodes, spleen , peyers patches in small intestine, appendix,Innate immune response Adaptive
2、 immune response,Humoral immunity,Cell-mediated immunity,抗原：进入机体内对机体的免疫系统产生刺激作用的外源物质。包括：蛋白质、多糖、核酸、病毒、细菌、各种细胞等。特点：外源性、结构性（分子表面具有稳定的环状结构基团作为识别位点）、特异性（抗原与抗体的反应）。 Antigens are molecules that react with antibodies, whereas immunogens are molecules that induce an immune responses. Substances foreign to th
3、e body, such as disease-causing bacteria and viruses and other infectious agents, known as antigens, are recognized by the bodys immune system as invaders.,动物脾脏有上百万个B淋巴 细胞，一个细胞就是一个克隆，它针对抗原上的一个抗原决定簇产生的抗体是单克隆抗体。 antigen determinants immunodominant 一种抗原通常具有多个不同的抗原决定族，因此能刺激多个B淋巴细胞产生相应的单克隆抗体，因此血清中的抗体是针对不
4、同抗原决定族的单克隆抗体混合物，称为多克隆抗体。类别，表位，个体间比例，时间,Antibodies that arise in an animal in response to typical antigens are heterogenerous, because they are formed by several different clones of plasma cells-Polyclonal. Antibodies that arise from a single clone of cells, eg, in plasma cell tumor(myeloma) are homo
5、geneous, they are monoclonal.,Antibodies are globulin proteins that react specifically with the antigen that stimulated their production. There are five classes of antibodies based on differences on their heavy chains.,Functions Neutralize toxins and virus Opsonize microbes Activate complements Prev
6、ent attachment Catalytic capability,Monoclonal Antibody Technology - The Basics Substances foreign to the body, such as disease-causing bacteria and viruses and other infectious agents, known as antigens, are recognized by the bodys immune system as invaders. Our natural defenses against these infec
7、tious agents are antibodies, proteins that seek out the antigens and help destroy them. Antibodies have two very useful characteristics: 1) extremely specific- each antibody binds to and attacks one particular antigen. 2) some antibodies, once activated by the occurrence of a disease, continue to co
8、nfer resistance against that disease; classic examples are the antibodies to the childhood diseases chickenpox and measles. The second characteristic of antibodies makes it possible to develop vaccines. A vaccine is a preparation of killed or weakened bacteria or viruses that, when introduced into t
9、he body, stimulates the production of antibodies against the antigens it contains.,It is the first trait of antibodies, their specificity, that makes monoclonal antibody technology so valuable. Not only can antibodies be used therapeutically, to protect against disease; they can also help to diagnos
10、e a wide variety of illnesses, and can detect the presence of drugs, viral and bacterial products, and other unusual or abnormal substances in the blood. Given such a diversity of uses for these disease-fighting substances, their production in pure quantities has long been the focus of scientific in
11、vestigation. The conventional method was to inject a laboratory animal with an antigen and then, after antibodies had been formed, collect those antibodies from the blood serum (antibody-containing blood serum is called antiserum). There are two problems with this method: It yields antiserum that co
12、ntains undesired substances, and it provides a very small amount of usable antibody.,Structure and function of IgG,Amino terminal end,Carboxy terminal end,Variable regions.,Constant regions,Fab fratgments,Fc fragment,Antibody,( ) is not only the first class of antibody to appear on the surface of de
13、veloping B cell but also the major class secreted into blood in the earlt stages of primary antibody response, on first exposure to antigen ( ) is the only antibody that can pass from mothrt to fetus via the placenta. ( ) is the precipal class of antibody in secretions including saliva, tears, milk
14、and the respiratory and intestine secretions.,人体内的五种抗体,单克隆抗体(monoclonal antibody，McAb),定义：利用细胞融合技术，将能够产生抗体的B淋巴细胞（antigen stimulated B lymphoblast)和具有无限增殖能力的骨髓瘤细胞(myeloma)融合在一起，形成杂交瘤细胞(hybridoma cell)。杂交瘤细胞既能在体外无限增殖，又能持续分泌成分单一的特异性抗体。这种单一类型的只针对某一特定抗原决定簇的抗体分子，就是单克隆抗体。,单抗的制备过程,动物免疫与亲本细胞的选择 细胞融合：淋巴细胞杂交瘤的
15、制备 杂交瘤细胞的筛选：有限稀释法等 单克隆抗体的制备和冻存 单克隆抗体的纯化,Monoclonal antibody technology allows us to produce large amounts of pure antibodies in the following way: We can obtain cells that produce antibodies naturally; we also have available a class of cells that can grow continually in cell culture. If we form a hy
16、brid that combines the characteristic of “immortality“ with the ability to produce the desired substance, we would have, in effect, a factory to produce antibodies that worked around the clock. In monoclonal antibody technology, tumor cells that can replicate endlessly are fused with mammalian cells
17、 that produce an antibody. The result of this cell fusion is a “hybridoma,“ which will continually produce antibodies. These antibodies are called monoclonal because they come from only one type of cell, the hybridoma cell; antibodies produced by conventional methods, on the other hand, are derived
18、from preparations containing many kinds of cells, and hence are called polyclonal. An example of how monoclonal antibodies are derived is described below,A myeloma is a tumor of the bone marrow that can be adapted to grow permanently in cell culture. When myeloma cells were fused with antibody-produ
19、cing mammalian spleen cells, it was found that the resulting hybrid cells, or hybridomas, produced large amounts of monoclonal antibody. This product of cell fusion combined the desired qualities of the two different types of cells: the ability to grow continually, and the ability to produce large a
20、mounts of pure antibody. Because selected hybrid cells produce only one specific antibody, they are more pure than the polyclonal antibodies produced by conventional techniques. They are potentially more effective than conventional drugs in fighting disease, since drugs attack not only the foreign s
21、ubstance but the bodys own cells as well, sometimes producing undesirable side effects such as nausea and allergic reactions. Monoclonal antibodies attack the target molecule and only the target molecule, with no or greatly diminished side effects.,Generation of Hybridomas: Permanent Cell Lines Secr
22、eting Monoclonal Antibodiesanimal, immunized with the antigen of interest. sacrifice the mouse dissect the spleen and culture the lymphocytes from spleen. prepare the culture of myeloma cells, 2 attributes:growing indefinitely in culture and does not produce immunoglobulins. Fusion of 2 cells by PEG
23、 or electroporation HAT medium used to support the growth of fused, hybrid cells but not of the parental cells. The resulting clones of cells are screened for the production of antibody th the antigen of interest.,亲本细胞的选择 骨髓瘤细胞myeloma：一般不分泌抗体，能在体外无限繁殖和连续继代培养，且为HPRT（次黄嘌呤鸟嘌呤磷酰核糖转移酶）或TK（胸腺嘧啶核苷激酶）。多用BAL
24、B/C 小鼠的骨髓瘤细胞。 淋巴细胞lymphocytes：经过免疫处理的淋巴细胞，多用大鼠或小鼠。免疫方法：体内法或体外法。 体内法：对细胞或微生物抗原可直接注射如小鼠体内，可溶性蛋白抗原可与等量的福氏完全佐剂混合乳化后，注入到动物体内。34天后，在无菌条件下取出脾或淋巴结制成悬液，存活率在95以上的可以用于融合。 体外法：直接提取大、小鼠淋巴细胞，调整为107个/ml，加适当浓度抗原，34天后，收集被刺激的淋巴细胞。,Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies Producti
25、on of monoclonal antibodies involves in vivo or in vitro procedures or combinations thereof. Before production of antibodies by either method, hybrid cells that will produce the antibodies are generated. The steps in producing those cells are outlined below。The generation of mAb-producing cells requ
26、ires the use of animals, usually mice. The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumor from this “immortal“ cell line is called a hybridoma. No method of generating a hybridoma that avoids the use of animals has been found. Recent in vitr
27、o techniques allow the intracellular production of antigen-binding antibody fragments, but such techniques are still experimental and have an uncertain yield, efficacy, and antibody function (Frenken and others 1998). It has also been possible to genetically replace much of the mouse mAb producing g
28、enes with human sequences, reducing the immunogenicity of mAb destined for clinical use in humans. Before the advent of the hybridoma method, investigators could produce only polyclonal serum antibodies; this required large numbers of immunized animals and did not immortalize the antibody-producing
29、cells, so it required repeated animal use to obtain more antibodies. Development of the hybridoma technology has reduced the number of animals (mice, rabbits, and so on) required to produce a given antibody but with a decrease in animal welfare when the ascites method is used. Step 1: Immunization o
30、f Mice and Selection of Mouse Donors for Generation of Hybridoma Cells Mice are immunized with an antigen that is prepared for injection either by,Cell Fusion and Selection Solutions: IMDM complete + 15% MCM, -PBS-, PEG 50% ( w/v ), Aminopterin Prepare a T-150 flask with IMDM complete and keep it in
31、 the incubator for fused cells. Isolation of spleen cells Sacrifice mouse by cervical dislocation. Immerse mouse in 70% Ethanol. Remove spleen ( on the left side ) and transfer into a small petri dish which contains IMDM at room temp. Clean fat from the spleen and transfer the spleen into an empty d
32、ish. Using sharp tipped forceps, one end is punctured. A curved forceps is used to hold down the intact end, and the spleen is gently rubbed towards the opened end with another set of forceps. The cells from inside the spleen will ooze out with very little damage. Stop the process when you are left
33、with a nearly empty, transparent skin. Collect the cells by rinsing with IMDM. Transfer cell suspension to a 15ml conical tube and let the cell debris settle out (approx. 5 min.). Remove the cell suspension (without disturbing the settled cell debris) and transfer to a 50ml conical tube. Add an addi
34、tional 30ml of IMDM and pellet the cells at 1200 rpm for 5 - 10 min. Aspirate the medium, resuspend pellet and wash again with 30ml of IMDM. One immunized spleen has approx. 108 cells. After this wash the cells are ready for the fusion. Myeloma cell preparation: It is essential that the myelomas be
35、free of debris, rounded and refractive under phase contrast, and that they are harvested in log or late log phase growth (between 3.5 and 9 x105 cells/ml). Thaw cells 7 days before scheduled fusion. Myeloma do not grow well after being in culture for more than a few weeks. Make sure you refreeze cel
36、ls for future use. We have been using a ratio of 2 spleen cells : 1 myeloma cell. However, workers have been using a ratio from 1 to 10 spleen cells per myeloma successfully. For one spleen we harvest 5x107 cells. It is advisable to do this spin at the same time that the second wash of the spleen ce
37、lls is done.,The fusion The washed myeloma and spleen cells are pooled in 30ml of PBS (room temp.) and spun gently at 1000 rpm for 10 min. Aspirate the PBS and resuspend pellet gently by tapping the tube. Volume should be approx. 0.8 ml. Set a timer. Add an equal volume of PEG solution, slowly, drop
38、wise, with gentle tapping, over 1.5 min. at room temp. Then gently wiggle the tube for 1.5 min.at 37C. Some cell clumping will be evident. The suspension is spun at 1000 rpm for 3-4 min. (at this point you should see the different layers of cells with PEG on top). Slowly add 37C IMDM to 10 ml, witho
39、ut disturbing pellet. After adding , swirl the tube gently to mix and dilute the PEG. Do not disturb the cells. Spin at 1000 rpm for 5 min. Aspirate medium, resuspend cells by tapping. Slowly add 5 ml of 37C IMDM-m. Bring to 20 ml and add to the flask in the incubator. (If using feeder cells, add th
40、em at this point; 106 cells/ml). Add Aminopterin (2ml to 200 ml of medium). (Some workers will leave the cells at this point for 24 hours before adding Aminopterin. We add the drug immediately.) Seed the cells in 96 well microtiter dishes, 250 ul per well, 8 plates per fusion. First clones may be se
41、en in 7-10 days. First screen will usually start after 2 weeks, with a second and third, if necessary, a few days later. Screening ELISA Materials: -Plates - 96 well Dynatech Immulon, type 2. (Fisher 17-0221-199).-PBS, TBS-PBS + 0.1% Tween 20 or TBS + 0.1% Tween 20.-Blocking solution = 2% BSA (type
42、V ) in PBS. (Add 0.02% azide for longer storage.)-Elisa buffer = 2% BSA + 0.1% Tween 20 in PBS ( azide optional ).-Enzyme linked antibody = Horseradish peroxidase1-Substrate = ABTS - 100X (from Zymed 00-2001)1-HRP buffer: 100mM Na Citrate pH 4.2( 490 mg citric acid + 720 mg Na citrate dehydrate + 50
43、 ml H2O, pH 4.2 ).-Hydrogen peroxide 30% ( 1000X )Protocol: ABSORBTION OF ANTIGEN Dilute Ag to 10 ug/ml in PBS. Add 100 ul of Ag solution to each well. Leave O.N covered with saran wrap, at 4C. BLOCKING Wash unbound Ag by inverting the plates and flicking the wells dry. Rinse by adding PBS to each w
44、ell and inverting it again (use squirt bottle). Repeat the rinse twice. Add 100 ul of blocking solution to every well, leave 1 hr at room Temp or O.N at 4C. PRIMARY ANTIBODY Add the antibody to be tested: Sup of cells = 25 ul, mix well by pipetting up and down (10 times).serum, ascites = 1:100 and a
45、 series of 1/5 dilutions. Do dilutions in blocking solution. Leave 1 hr at room temp or O.N at 4C. SECONDARY ANTIBODY Wash unbound antibody 4 times with PBS + 0.1% Tween 20. Add 100 ul of enzyme linked antibody to all wells. Do the appropriate dilutions in the Elisa buffer. (ex: HRP is 2000X). Leave
46、 1 hr at room temp or O.N at 4C. SUBSTRATE Dissolve substrate in water. Wash plate 4 times with PBS + 0.1% Tween 20 (use TBS instead of PBS for AP). Add 100 ul of substrate to every well. Match color development. This could take from a few seconds to 20 min. If needed, stop the reaction by adding 50
47、 ul of 4M NaOH. Read absorption in Elisa reader at correct wavelength (for HRP system 416 nm, for AP - 405 nm).,The fusion The washed myeloma and spleen cells are pooled in 30ml of PBS (room temp.) and spun gently at 1000 rpm for 10 min. Aspirate the PBS and resuspend pellet gently by tapping the tu
48、be. Volume should be approx. 0.8 ml. Set a timer. Add an equal volume of PEG solution, slowly, dropwise, with gentle tapping, over 1.5 min at room temp. Then gently wiggle the tube for 1.5 min.at 37C. Some cell clumping will be evident. The suspension is spun at 1000 rpm for 3-4 min. (at this point
49、you should see the different layers of cells with PEG on top). Slowly add 37C IMDM to 10 ml, without disturbing pellet. After adding , swirl the tube gently to mix and dilute the PEG. Do not disturb the cells. Spin at 1000 rpm for 5 min. Aspirate medium, resuspend cells by tapping. Slowly add 5 ml o
50、f 37C IMDM-m. Bring to 20 ml and add to the flask in the incubator. (If using feeder cells, add them at this point; 106 cells/ml). Add Aminopterin (2ml to 200 ml of medium). (Some workers will leave the cells at this point for 24 hours before adding Aminopterin. We add the drug immediately.) Seed the cells in 96 well microtiter dishes, 250 ul per well, 8 plates per fusion. First clones may be seen in 7-10 days. First screen will usually start after 2 weeks, with a second and third, if necessary, a few days later.,
