Monoclonal Antibodies - Ascytes Fluid

Monoclonal Antibodies: Production via Ascytes Fluid

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The usual immune serum contains many different kinds of antibodies of which only a few are those of use to the scientist (why are all the other antibodies there?) Wouldn't it be wonderful to be able to produce solutions that contain much, much higher concentrations (titers) of the desired antibody with no more of the unwanted ones! There are two ways to do this. One is a very tedious method that is prone to contamination by other microbes that waft in. That method we might term as the "axenic method" as the only cells involved are those that are pumping out the desired antibodies. This is done purely in cell cultures that require lots of expensive equipment and has mixed success due to its proneness for contamination.

Wendell Zollinger (Walter Reed Army Institute of Research in Washington D.C.) reasoned that there must be a better way that takes much less proficiency and expense. He developed the following method in which antibody producing cells are fused with cancer cells to make "hybridoma" cells. Being cancer cells, they lost a lot of their inhibitions: they grow endlessly, readily invade other tissues, and go about their business at full throttle. In this case, the cells flood out fluid that contains up to thousands of times the normal concentration of the desired antibody. Yes, of course, the fluid still contains the background, low-level concentrations of the mouse's other antibodies, but the one you want is overwhelming and just what you want. What is especially nice is that the mouse's own immune system patrols your experiment and prevents contamination. Of course, not everything is totally rosy: the poor mouse eventually dies of cancer.

Production of Ascytes Fluid
Containing High Titer (nearly) Mono-Clonal Antibody

Step Picture Comments
1 The first step in producing ascytes fluid is much that same with any immunization. The antigen is injected into the mammal, and booster shots are given. Occasionally drops of blood are tested for their ability to agglutinate the original antigen soluution. Only if good agglutination occurs is it worth proceeding. (By the way, "ascytes" is a word that means "without cells." Squishing around among your intestines, ascytes fluid is much like serum.)
2 Instead of blood, the mouse's spleen is removed. The spleen is one of the several organs in a mammal that is loaded with B-lymphocytes, which produce IgG. The thing to remember is that each B-lymphocyte produces a different kind of antibody. Your problem is to find the one or very few that are producing the antibodies that you want to use. Once you have isolated that one out of the millions of unwanted ones, you want to clone it - make it proliferate into billions of cells - all making the antibody you want.
3 Above you have placed the mouse's spleen on the frosted end of a microscope slide. Now you lay another frosted end of a slide over the spleen and gently rub it within the sandwich. Have your partner slowly dribble normal saline (0.15M NaCl) onto your work and you will notice that the spleen very easily disintegrates into separated cells (spleenocytes), which drip away into a sterile petri plate.
4 From another lab down the hall, you obtain a suspension of mouse polyoma cells. Polyoma is a strange cancer in that causes a wide variety of cancers within the same mouse. (Mice, by the way, have very poor "anti-onco-genes", and so contract cancer much, much more easily than do people. But what the hey, mice don't live long enough for most cancers to kill them; cats get at the mice first; or snakes, or owls, or...) So now you mix the two cell suspensions: your spleenocytes and their polyoma cells.
5 After doing a few laboratory tricks like rapidly changing concentrations of calcium and gentle centrifuging, this is what happens. (1) The two different types of cells look at each other. (2) They snuggle up together. (3) Their cytoplasms fuse to form cells with two nuclei (some cancer cells are very acquisitive!). (4) Now the nuclei fuse. So, in your centrifuge tube you have a lot of cell-fusing going on. You are hoping that at least one of your few desired antibody-producing spleenocytes has fused so that it can divide like respectible hydridoma cells do, and soon that one cell produces many, many cells - a "clone" - and, hence, mono-clonal antibodies.
6 Now comes the tedious part that will take you several weeks. Dilute your hybridoma cells and distribute them among a thousand or so tubes. (Actually what are used are plastic plates that contain a hundred or so little 1 milliliter wells.) In each you will have maybe a hundred different hybridoma cells, which grow and produce antibody. After a few days, you test all the hundreds or thousands of wells for any that are producing the antibody you want. There are a number of ways of doing this, so the description will not be given here. Let's suppose that you found a well that contained the antibody. That means that there was one clone of the desired cells in that well - alas, along with several other clones of unwanted cells. But you have gone from maybe one cell in a million (step 5) to one out of every hundred cells. So you dilute those cells and distribute them into new wells, and so forth until finally you have a well that contains only the hybridoma cells you desire.
7 These hybridoma cells are merely injected into the tummy of the mouse (intraperineal) - into the space of the belly's sack that holds the intestines. Those hybridoma cells slosh around inside and eventually settle down and colonize various places on the lining of the belly (perineum).
8 There they multiply and together begin to produce large amounts of fluid. The mouse inflates incredibly into a small blimp that is filled with this fluid that is very rich in exactly the antibodies you want.
9 So you grab the nearly spherical mouse and stick a hypodermic needle into its belly. The mouse deflates yielding an amount of fluid that is nearly the volume of a normal mouse! That fluid is essentially cell-free (ascytes, remember?), and contains a very high titer of the antibody you need to use in your experiments.
10 You put the mouse back into its cage, where it acts like a mother who has just given birth: it flops down and dozes in exhaustion and relief.
11 But the hybridoma cells are still busy at work! The mouse can be deflated three or four times before the cancer finally kills it. Of course, smart researchers have frozen some hybridoma cells so that if they need more ascytes fluid at a later date, they need only thaw some and inject more mice. In three days or so, they can deflate more mice.

Step	Picture	Comments
1		The first step in producing ascytes fluid is much that same with any immunization. The antigen is injected into the mammal, and booster shots are given. Occasionally drops of blood are tested for their ability to agglutinate the original antigen soluution. Only if good agglutination occurs is it worth proceeding. (By the way, "ascytes" is a word that means "without cells." Squishing around among your intestines, ascytes fluid is much like serum.)
2		Instead of blood, the mouse's spleen is removed. The spleen is one of the several organs in a mammal that is loaded with B-lymphocytes, which produce IgG. The thing to remember is that each B-lymphocyte produces a different kind of antibody. Your problem is to find the one or very few that are producing the antibodies that you want to use. Once you have isolated that one out of the millions of unwanted ones, you want to clone it - make it proliferate into billions of cells - all making the antibody you want.
3		Above you have placed the mouse's spleen on the frosted end of a microscope slide. Now you lay another frosted end of a slide over the spleen and gently rub it within the sandwich. Have your partner slowly dribble normal saline (0.15M NaCl) onto your work and you will notice that the spleen very easily disintegrates into separated cells (spleenocytes), which drip away into a sterile petri plate.
4		From another lab down the hall, you obtain a suspension of mouse polyoma cells. Polyoma is a strange cancer in that causes a wide variety of cancers within the same mouse. (Mice, by the way, have very poor "anti-onco-genes", and so contract cancer much, much more easily than do people. But what the hey, mice don't live long enough for most cancers to kill them; cats get at the mice first; or snakes, or owls, or...) So now you mix the two cell suspensions: your spleenocytes and their polyoma cells.
5		After doing a few laboratory tricks like rapidly changing concentrations of calcium and gentle centrifuging, this is what happens. (1) The two different types of cells look at each other. (2) They snuggle up together. (3) Their cytoplasms fuse to form cells with two nuclei (some cancer cells are very acquisitive!). (4) Now the nuclei fuse. So, in your centrifuge tube you have a lot of cell-fusing going on. You are hoping that at least one of your few desired antibody-producing spleenocytes has fused so that it can divide like respectible hydridoma cells do, and soon that one cell produces many, many cells - a "clone" - and, hence, mono-clonal antibodies.
6		Now comes the tedious part that will take you several weeks. Dilute your hybridoma cells and distribute them among a thousand or so tubes. (Actually what are used are plastic plates that contain a hundred or so little 1 milliliter wells.) In each you will have maybe a hundred different hybridoma cells, which grow and produce antibody. After a few days, you test all the hundreds or thousands of wells for any that are producing the antibody you want. There are a number of ways of doing this, so the description will not be given here. Let's suppose that you found a well that contained the antibody. That means that there was one clone of the desired cells in that well - alas, along with several other clones of unwanted cells. But you have gone from maybe one cell in a million (step 5) to one out of every hundred cells. So you dilute those cells and distribute them into new wells, and so forth until finally you have a well that contains only the hybridoma cells you desire.
7		These hybridoma cells are merely injected into the tummy of the mouse (intraperineal) - into the space of the belly's sack that holds the intestines. Those hybridoma cells slosh around inside and eventually settle down and colonize various places on the lining of the belly (perineum).
8		There they multiply and together begin to produce large amounts of fluid. The mouse inflates incredibly into a small blimp that is filled with this fluid that is very rich in exactly the antibodies you want.
9		So you grab the nearly spherical mouse and stick a hypodermic needle into its belly. The mouse deflates yielding an amount of fluid that is nearly the volume of a normal mouse! That fluid is essentially cell-free (ascytes, remember?), and contains a very high titer of the antibody you need to use in your experiments.
10		You put the mouse back into its cage, where it acts like a mother who has just given birth: it flops down and dozes in exhaustion and relief.
11		But the hybridoma cells are still busy at work! The mouse can be deflated three or four times before the cancer finally kills it. Of course, smart researchers have frozen some hybridoma cells so that if they need more ascytes fluid at a later date, they need only thaw some and inject more mice. In three days or so, they can deflate more mice.

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