Yet again the reply to this question demonstrates what a helpful group of people are on the list. I got a number of requests to pass on information so instead of replying individually I'll put a summary down for those interested. But first it may be of interest to explain the problem for those who have not encountered it before. Most monoclonal antibodies are either IgG or IgM isotypes (including the sub-isotypes of IgG), the IgG antibodies are usually easily purified from tissue culture snate or ascites by Protein G affinity columns, and most are stable on purification. Some IgM isotype antibodies will separate on Protein G, so its often worth a try, however the majority will not or only some low level binding is seen. The added complication is that many of the IgM antibodies are unstable on purification. IgM isotype antibodies can be separated by traditional gel filtration (thanks Deb), however my antibodies are in tissue culture snates and I cannot concentrate them up enough to run on G200 or DEAE. The method of choice would FPLC of HPLC but neither is available to me. A number of people suggested making an anti-mouse Ig affinity column and this is the way I have decided to go. I intend making a caprylic acid cut of some goat anti-mouse sera, clean up the gamma globulin fraction and couple this to Sepharose via CNBR. I should be able to elute antibody bound to the anti-mouse by gradual drop in pH. Stability of the product will still be a problem but that will vary among individual monoclonals. There were several comments on the commercial columns, mainly favourable but not all. There was complete agreement on the high cost of them. Many thanks to all. Peter Peter J Macardle PhD Dept of Immunology, Allergy and Arthritis Flinders Medical Centre Bedford Park SA5051 South Australia Phone: 618) 8204 4535 Fax: 618) 8277 1397
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