Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: a study using the hydatid vaccine EG95

Abstract

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.

Fig. 1

Schematic representation of full length recombinant EG95 and three overlapping truncated versions of EG95. Linear epitope regions identified by Woollard et al., are shaded.

Fig. 2

Schematic illustrating affinity purification technique. Clarified pooled antisera was diluted in binding buffer. The diluted serum was applied to the five depletion columns – GST, EG95_4–74, EG95_54–109, EG95_92–156 and E. coli lysate. The flow through was reapplied to the depletion columns three times. Washed columns were eluted of antibodies. Anti-GST antibodies were collected. The flow through from the five depletion columns was applied to an EG95–GST column. The flow through from this column was reapplied three times. Washed EG95–GST column was eluted of antibodies and the antibodies against conformational epitopes on EG95 were collected (anti-cEG95).

Fig. 3

Comparison of the anti-EG95 and anti-GST titres of affinity purified antibodies. The EG95 (black bars) and GST (grey bars) reactivity of two affinity purified antibody solutions (anti-cEG95 and anti-GST). Panel A shows IgG affinity purified to conformational epitopes of EG95 (Anti-cEG95). Panel B shows IgG affinity purified to GST (Anti-GST). Panel C shows pooled whole antisera from sheep following third immunisation with EG95–GST (day 59). Panel D shows pooled whole antisera following removal of EG95 and GST determinants (flow-through fraction – see Fig. 2).

Fig. 4

Dot-blot of recombinant protein antigens reacted with antibody pools from sheep immunised with EG95–GST. 1 μg of each protein was spotted onto the nitrocellulose membrane. Lane A is pooled serum from sheep immunised with EG95–GST. Lane B is the pre-immune pool. Lane C is reacted with IgG affinity-purified to conformational epitope/s of EG95 (anti-cEG95). Lane D is reacted with IgG affinity-purified to GST (anti-GST). Lane E is reacted with antibody affinity-purified to E100 peptide (anti-E100). Lane F is reacted with antibody affinity-purified to G1 peptide (anti-G1).

Fig. 5

Western Blot. Recombinant proteins EG95–MBP (lane 1), EG95_4–74 (lane 2), EG95_54–109 (lane 3), and EG95_92-156 (lane 4) probed with three antibody sources. Pooled sera from sheep vaccinated with EG95–GST (Panel A), polyclonal antibodies affinity purified to conformational epitopes of EG95 (anti-cEG95) (Panel B) and polyclonal antibodies affinity purified to peptide E100 (Panel C). A Coomasie stained SDS-PAGE displaying recombinant proteins.

Fig. 6

Inhibition of anti-E100 antibody binding to EG95 using E100 peptide. The concentration of anti-E100 antibody in each well was kept constant at 35 ng/ml and the concentration of free peptides E100 and R1 varied.