Antibody purification from soup or ascites is one of the most important steps in the process of monoclonal Antibody production. The best method is selected according to the class of antibodies. Several methods have been introduced in order to IgM purification, for example Gel filtration, Ion exchange, Hydrophobic Chromatography and affinity chromatography.
There are several problems in the purification of IgM antibody from soup or Ascites by Gel filteration, like loss of Antibody, sample dilution. etc. Morever this step is overall time-consuming and has a low efficiency too.
Then, new designing is necessary for IgM purification from soup or ascites. We decided to prepare an affinity chromatography column including Rabbit Anti Mouse IgM for purifying the IgM form ascites and soup.
Material and Methods: Methods as follow:
1) Antibody producing cells were cultured and injected to mice for ascites production. IgM antibodies were purified from ascites by Gel filtration.
2) IgM antibodies were injected to rabbit and antibodies were produced against Mouse IgM in rabbit.
3) Rabbit anti-Mouse IgM was purified by affinity chromatography.
4) In order to purifing Mouse IgM from soup or ascites, affinity column were made by Sepharose-4B- Rabbit Anti Mouse IgM.
Results: Hybridoma lines that produced IgM were cultured and injected into the mouse peritoneum. We collected ascites from peritoneum after 15 days. Finally 3.32 mg mouse IgM was gotten from ascites by gel filtration chromatography. The purified mouse IgM was injected into two rabbits and 41 mg Rabbit Anti Mouse IgM were gotten by passing rabbits’ sera from Sepharose-4B-Mouse IgM column. Purified Rabbit Anti Mouse IgM was linked to Sepharose-4B for preparing the Sepharose-4B-Rabbit Anti Mouse IgM column. This column was used for purifying mouse IgM (Monoclonal Antibody) from cell culture and ascites. The results of our study show that purified IgM from ascites and soup by Sepharose-4B-Rabbit Anti Mouse IgM column was absolutely pure.
Discussion: Since IgM purification protocols except affinity chromatography have several steps and also rapid IgM purification were usually recommended, we have designed extremely rapid and easy new method for the purification of IgM antibody.