Development of monoclonal antibodies to proteins separated by two-dimensional gel electrophoresis.

We have developed techniques for the production of monoclonal antibodies using Coomassie blue-stained protein spots cut from high resolution 2-dimensional polyacrylamide gels. The gel spots were homogenized with Freund's adjuvant and injected sub-cutaneously into a mouse, at several places along the flank. After boosting twice the spleen cells were hybridized by standard methods. Hybrids, clones and ascitic fluids were also screened with antigen prepared from 2-dimensional gel spots. The spots were cut from gels, homogenized in the presence of guanidinium chloride, and extracted by shaking overnight. The acrylamide was removed, the sample dialyzed to remove denaturant and the protein labeled with 125I. An alternative method for the production of screening antigen using column chromatography is described. These techniques allow the production of monoclonal antibodies to specific protein components of complex mixtures, even in the presence of other immunodominant proteins.