A few thoughts. Is your affinity-purified codling moth GST enzymatically
active? It might have been denatured by the elution step and Abs to an
"unnatural" epitope generated. Alternatively, it's possible though a
little unusual for a polyclonal Ab ("PAb"?) to recognize native Ag but not
the denatured form on a Western. Also, sometimes perfectly good PAbs can't
see a protein in a crude prep on Western because of the large no. of other
proteins present. Does your PAb recognize purified GST in ELISA? Try
biotinylating a portion of the PAb and do a sandwich; PAb on plate, then
cell extract, then biotinylated PAb and streptavidin-HRP or Alk Phos.
Finally, even though GST is big enough to theoretically induce an immune
response itself, you might enhance development of the B cell response by
coupling it to a carrier such as KLH and immunizing with this.
Hope something here helps.
- Peter Blier
