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Griffin:Antibody Basics: Difference between revisions
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→Antibody purification
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#Paper1 pmid=1401962 | #Paper1 pmid=1401962 | ||
</biblio> | </biblio> | ||
===Ultrafiltration=== | |||
[Millipore http://www.millipore.com/techpublications/tech1/koticha1] | |||
ABSTRACT | |||
Monoclonal antibodies (mAB) continue to gain importance as therapeutic and diagnostic agents for many types of cancer. The process of screening hybridoma libraries for candidate mABs is both time consuming and labor intensive. Once a hybridoma cell line expressing a suitable mAB is established, a bench-scale purification methodology (e.g. 50-500 mL) must be developed to produce sufficient mAB for further characterization. A traditional method for purifying mABs involves clarification of the hybridoma supernatant by centrifugation, followed by an ammonium sulphate precipitation to concentrate the mABs. The precipitate is then recovered by centrifugation, resolubilized and desalted using dialysis. After these steps, the mAB is further purified using Protein A/G affinity chromatography. The purified antibody is desalted and exchanged into a biological buffer using dialysis. The entire process typically requires several days to complete and can be particularly onerous if multiple mABs are to be evaluated in parallel. | |||
We describe a new and simplified method that minimizes the processing time to less than a day to obtain pure mAB. The method involves clarification of the hybridoma supernatant by microfiltration, followed by concentration using ultrafiltration (Saha K, et al, 1992). The mAB is further purified on Protein A/G beads. The purified mAB is desalted and buffer-exchanged using ultrafiltration. This approach was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E10 (Evan G, et al., 1985). The mAB purified by this new method performed comparably to the commercially purified mAB in downstream applications such as western blotting and ELISA. | |||
The data demonstrate that the new protocol is robust and delivers mAB of a high purity and yield as compared to the traditionally purified mAB. The application of ultrafiltration to mAB purification will be of considerable value to any researcher interested in screening hybridoma libraries and accelerating the purification of mABs. | |||
==Ascites== | ==Ascites== | ||
