Immunization procedures forE. coli proteins

Abstract

Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins. An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization. Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d. This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure. The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used. Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund. Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.

Quantitation of DNA and protein impurities in biopharmaceuticals

“…This may result in spuriously low detection of such proteins when they are in a state of antigen excess; this so-called prozone effect may be minimized by dilution of the sample. The same group evaluated two immunization procedures designed to improve the antibody response to weakly immunogenic components in complex antigen mixtures (43). The general strategy was to eliminate the most immunogenic proteins from the boosting doses given to the recipient animal.…”