Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: a study using the hydatid vaccine EG95.
The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.
The University of Melbourne, Veterinary Clinical Centre, Werribee, Victoria, Australia. andrew.j.read@dpi.nsw.gov.au
A J
AJ
Read
C G
CG
Gauci
M W
MW
Lightowlers
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D048209:Echinococcus granulosus / Q000235:genetics / Q000276:immunology; D005849:Gerbillinae; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D014612:Vaccines / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D000947:Antigens, Helminth; C446034:EG95 protein, Echinococcus granulosus; D015801:Helminth Proteins; D019151:Peptide Library; D014612:Vaccines