I am currently using a syringe and a syringe filter (0.45um) to filter the ascites for antibody purification. The filter easily clogs up and I am only able to collect significantly low amount of filtered ascites from my initial volume.
If anyone can suggest or recommend a different technique please let me know.
Hi Leo,
You are dealing with a well known problem unfortunately. The first step is of course to spin down the ascites to pellet cells and other insolubles. There should be a considerable amount of fat floating above the clear supernatent and this is what intereferes with filtration. Collect all sup and transfer to a fresh tube. Add some Cab-O-Sil (M-5 scinitllation grade) Sigma cat# 7631-86-9. This is a substance that would selectively adsorb all the fat rather quickly. It is very "fluffy" and takes a lot of room in a tube. Don't over do it, you need about 50-100 ul volume of this stuff per 1ml ascites to absorb all of the fat. Takes about 10-20 mins at RT to get it done with gentle agitation. Spin again hard (14kxg) to pellet both Cabosil and fat, recover the sup that now can be filtered 0.45 or 0.22mc. Proceed with downstream MAb purification on PA or PG or other method of your liking
Hope this helps
Andrew
Hi Andrew,
Thank you! I will definitely look into this method.
Have you worked with Cab-O-Sil before? Does this have any effect on the yield of purified antibody? Does it have any disadvantages?
Leo,
The yield of antibodies will not be affected by treating ascites with Cabosil but only by your hybridoma and time. Cabosil is a very fine silica preparation, which is generally inert but has strong affinity to bind fat. Silica was used decades back to study/quantify fat in serum and other bio liquids and was well characterized then. Just be careful working with it not to inhale- stays permanently in your lungs- this is a disadvantage but easy to prevent.
Andrew
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