The preparation process for monoclonal antibodies.
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Last Update: 2020-10-19
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Source: Internet
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Author: User
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1.Screening and cloning of hybrid tumor-positive clones
hybrid tumor cells grown in HAT
culture
-based cells, only a few of which secrete predetermined specific monoclonal
antibodies
cells, therefore, must be screened and cloned. Limited dilution is usually used for cloning and culture of hybrid tumor cells. Using sensitive, fast and specific immunological methods, positive hybrid tumor cells that can produce the desired monoclonal antibodies are screened and cloned and amplified. After a comprehensive identification of the immunoglobulin type, subsexual, specificity, affinity, identification of the
antigen
of the table and its molecular weight, timely freeze deposit.
2.Large-quantity preparation of monoclonal antibodies
preparation of monoclonal antibodies is important using in vivo induction method and in vitro culture method.
(1) in vivo induction method to take Balb/c mice, the first abdominal cavity injection 0.5 ml of liquid shira or deplantation for pre-treatment. After 1-2 weeks, the interbreeding tumor cells are inoculated in the abdominal cavity. Hybrid tumor cells proliferate in the abdominal cavity of mice and produce and secrete monoclonal antibodies. At about 1-2 weeks, the mouse's abdomen was visible. A large number of monoclonal antibodies can be obtained by extracting asynal water with a syringe.
(2) in vitro culture method to place hybrid tumor cells in a culture bottle for culture. During the culture process, hybrid tumor cells produce and secrete monoclonal antibodies, collect cultured saliva, centrifuge the removal of cells and their fragments, you can obtain the required monoclonal antibodies. However, this method produces a limited amount of antibodies. In recent years, a variety of new culture technologies and devices have emerged, greatly improving the production of antibodies.
hybrid tumor cells are fused and screened before they can be used. Hybrid tumor cells are divided into two times: first, screening out hybrid tumor cells, and second, screening out hybrid tumor cells that produce specific antibodies in the primary hybrid tumor cells, the methods and principles of these two screenings are different.
3.The preparation process of asphalt preparation and single anti-purificationascension is relatively simple: the first step ofis to inject the abdominal cavity of mice with 0.5 ml of Fleury incomplete admission one week in advance;The second step, a week after the injection of admission will grow strong hybrid tumor cells centrifugal abandoned
culture base
, with PBS or incomplete culture base rehang after injection of the mouse abdominal cavity, generally 7-10 days or so can see the mouse abdominal cavity obviously swollen, at this time can be executed mice to collect abdominal water, can also be repeatedly pumped with syringe the next day. The yield of ascension obtained by repeated extraction method is higher than that obtained by one-time death (the maximum yield obtained by the station director by repeated extraction method is 30.5 ml per mouse).
addition to celiac injection hybrid tumor, can also be injected into the spleen, the method is somewhat similar to the spleen intra-immunity. The specific operation is: a week in advance of the injection of Fleury incomplete admission, after injection about a week with ether anaesthetized mice, cut open the back skin, you can see the spleen, with a syringe pierced the back skin, hybrid tumor cells injected into the spleen, and then with AB glue to the wound hair stick, coated with antibiotics can be, 7-10 days after the formation of abdominal water. The advantage of this method is that the number of hybrid tumor cells requires less, generally more than 1000 cells can successfully prepare ascertic water.
The newly extracted asphalt contains a large amount of grease, macrophages, hybrid tumor cells, endocysts in the abdominal cavity, cellular ingredients in the blood and other impurities, especially these cellular ingredients, if not removed may slowly rupture in the long-term preservation of abdominal water, the
protein
ingredients in the cell will be released To the abdominal water, increase impurities in the abdominal water, so that purification becomes difficult, so the abdominal water after collection, whether long-term preservation or immediate purification should be centrifugal removal of cells, but also remove grease (the density of grease is relatively small, generally floating on the surface of the abdominal water, with a gun to suck out can be thrown away).
purification of monoantigens is currently mainly several:
(1) salt/precipitation method;
(2) ion exchange method;
(3) gel
filtration
layering;
(4) protein A or protein G purification;
(5) antigen affinity and purification. Here are just a few brief introductions.
two main methods, one is ammonium sulfate two-step method, the other is the two-step precipitation method of ammonium acid-ammonium sulfate. The principle of the former method is that under high concentrations of salt ions (usually neutral salts), protein molecules in an aqueous solution, the hydration film on the molecular surface will be deprived, thus predisulate the solution, which is manifested as a decrease in solubility and predispulation.
Usually used salt is mainly ammonium sulfate, in the case of 50% saturation, the abdominal water in addition to albumin hemoglobin will be precipitated, but at the same time immunoglobulin will also be precipitated, in 45% saturation conditions, immunoglobulin solubility is minimal and precipitated. When operating first to the abdominal water to add the same volume of PBS, and then add ammonium sulfate solid to 50% saturation, how much can be added to the reference given ammonium sulfate solid solubility table, "Guide to Protein Purification" book has a detailed solubility table, sit for about two hours, 10000 g centrifugal half an hour later, discard the upper clear, with PBS resoluble precipitation, and then Add ammonium sulfate solids to 45% saturation again, sit for about two hours about 10000 g centrifugation half an hour, discard the upper clear, with a small amount of PBS dissolved precipitation, that is, to obtain a purer antibody, because this antibody still has a higher concentration of ammonium sulfate, so the need for PBS dialysis, general dialysis one day and one night more appropriate, dialysis process replacement dialysis two to three times.
if the purity requirements are not very high, can also not do the first precipitation, directly with 45% saturated ammonium sulfate precipitation can also be. Compared with the two-step precipitation method of ammonium sulfate, the antibody obtained by the two-step precipitation method of ammonium sulphate is purer. The principle of the two-step precipitation method of ammonium sulphate is: acid is an organic acid, under acidic conditions (pH 4.5), can make large molecule protein precipitation, especially pi value in the vicinity of 4.5 albumin, these hemoglobins with posit acid precipitation, and then use antibody precipitation can be.
: This is . (1) The asphalt with 0.06 M sodium acetate (pH4.0) by 1:3 dilution, stirring at 4 degrees C to add positive acid, generally according to each milliliter of undiluted asphalt to add 40 ul positive bitter acid, and then 4 degrees C to set aside 2 h; (2) 4 degrees C10000 g centrifugation 20min, it can be seen that the bitterness due to low temperature and crystallization from the system floating in the abdominal water layer, discarding this crystallization and bottom precipitation, adding 1/1 in the upper clear 10×PBS (0.1M, pH 7.4) of 0 volume, with a concentration of 0.277 g/ml of ammonium sulfate added to it at a temperature of 4 degrees C and more than 1h; (3) 10000 g centrifugation 20min; discarded, will be precipitated in the appropriate amount of PBS, and then put it into a dialysis bag to PBS dialysis (dialysis liquid should be more than 100 times the volume of antibodies, preferably stirred), 4 degrees C overnight; (4) collection of dialysis-good antibodies, directly used or added protective agents and
antiseptics
long-term preservation. The save condition is similar to the multi-anti-save condition. With these two purified antibody purity can reach 80-90%, less loss, less loss of activity of the antibody,
reagents
low cost, but the operation is more time-time-saving. the principle of ion exchange method is that the antibody itself carries an electric charge, when it passes through the filler of a suitable ion exchange column, it can exchange the same charged ions on the filler, thus separating from other impurities with different charges in the abdominal water, and finally washed away with a buffer with high ion strength. This method is similar to the principle of affinity antigen affinity and layering, the advantage is low cost, the filler used can be used repeatedly, the disadvantage is that it is difficult to obtain high purity antibodies through a single exchange, while direct elusion of the product concentration is low, often need to be concentrated. About its specific operation here is no longer detailed, interested friends can refer to "a rapid purification of monoclonal antibody method - yin and yang dual ion exchange layering method" (Schwei, etc.). The principle of gel filtration analysis is that the abdominal water through a column equipped with suitable aperture microbeads, small molecules can enter the small hole of microbeads, but the large molecular size is larger than the aperture of microbeads, so will not pass through the microbeads inside. Driven by semen, large molecules flow out of the column first, while small molecules flow out of the column. The most important application of filter filtration layering is the purification of IgM single resistance, IgM is a ligand, molecular weight of more than 900kD, compared to other molecules in the abdominal water, it is definitely a "huge" molecule, so it is particularly suitable for purification with gel filtration. See the literature for specific procedures. . Protein A and Protein G are the mainstream directions of single anti-purification in recent years. Here's a brief look at how they purify and how. Protein A is a protein on the cell wall of Staphylococcus auspicious staphylococcus aegypti with a molecular weight of 42kD and six IgG binding points in the sequence, five of which have a strong specific affinity for IgG's Fc fragments, and when saturation is reached, a Protein A molecule can bind to an average of two IgG molecules.
The Protein A on the currently commercialized Protein A resin (which is to cure the filler on Agarose or Sephardex microbeads) is mostly recombinant proteins, and some non-necessary areas are removed during the recombination process, some of which also reduce the number of IgG binding bits (generally only four binding points are retained) in order to facilitate the exodus of antibodies.
Protein A has specific bindings to IgG in many animals and is therefore used more in the purification of antibodies. Protein G is a protein on the cell wall of streptococcus cells, similar to Protein A, it can also be specifically combined with the Fc segment of IgG, the natural Protein G can bind to IgG, but also can bind to albumin, commercialized for purifying antibodies Proteo In G is also recombined to remov areas that bind to albumin during the recombination process, the main difference between Protein A and Protein G is that they exhibit different affinity for different subsypes of antibodies, and the commercialized Protein G resin is generally more expensive than Protein A. Since this page only describes the purification of single resistance, here is only a list of the two mouse subsype affinity characteristics:
from the above, taking into account economic factors and purification efficiency, you can know that in addition to IgG1 needs to use Protein G, other sub-types of antibodies generally choose Protein A affinity and purification, IgM purification methods need luck, if you can use Protein A for affinity and purification, only later good-looking methods. The rules are passing, here is a brief introduction to the use of Protein A or Protein G purified monoantitor from abdominal water operation method: (1) pre-treatment. Protein A or Protein G resin is generally soaked in 20% ethanol, some also have special protective agents, need to be removed before use, the simple way is to fill into the washing column, and then wash with about 10 times the volume of PBS one to three times, you can also use PBS washing in the centrifuge tube, wash once and then centrifuge to remove the washing liquid, and then the next round of washing. (2) on the abdominal water. The abdominal water from mice often contains grease as well as somatic cells and hybrid tumor cells, so the upper column needs to do a simple treatment, generally the abdominal water first centrifugation, centrifugation generally choose 5000 g centrifugation 20min or so, after centrifugation , oil and other impurities floating on the ascension, cells and other heavier parts are sunk in the bottom, take the middle of the clear ascension can be added to the installed column, and then seal the column placed on the shaker shake, room temperature 2 h or 4 spent the night. It can also be done in a centrifugal tube. Sometimes if the volume of astrium is relatively large, and in order to ensure that affinity fillers can be used repeatedly, you can use 0.45 micron membrane to filter the ascertin water once. In addition, it has been reported in the literature that it is better to dilute the ascerin with PBS and then incubate with resin. (3) wash and unwash. Drain the assalin from the affinity column. If it is used centrifugal tube, can be centrifuged out of the abdominal water, the same between. Wash the column 3 times with PBS with 10 times the volume of the column, and finally wash the antibody off with a lotion. The purification and collection monitoring process can be exactly the same as multi-resistance purification. (4) antibody preservation. This step can also be exactly the same as multi-resistance purification. The advantage of Protein A or Protein G purification is that the resulting monoantigen purity is relatively high, antibody activity preservation is also very good, easy to operate, in addition, because they and antibody binding area is located in the Fc segment of the antibody, so they are similar to the affinity of most antibodies, in the multiclonal antibody purification process can also be used to concentrate other methods of purification of the thinner antibodies. But these two fillers are relatively expensive, and the subsype of the antibody needs to be determined before the monoantigen can be purified.
market also has a recombined Protein A and G recombination protein-coupled filler, Protein A/G, which combines the advantages of both to make the purification process easier. Another problem is that the single resistance of the IgM subtype is not always purified with Protein A, and a better approach is to purify it with another affinity filler: Protein L resin, which has the same principle as Protein A and G, and Protein L can specifically bind to the Kappa chain of antibodies. In mice, at least 95% of the antibodies are molybric (the other 5% are molybic), so most monoantibodies can be purified with Protein L affinity fillers, including Ig.
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