Figure 2. Isolating p75^NTR antibodies by neuronal retrograde transport in vivo. (A) The in vivo selection procedure. Phage library enriched for binding to p75^NTR from one round of in vitro panning was applied to the rat sciatic nerve in vivo at 5 x 10¹⁰ cfu. Phage was recovered 18 h later and used to infect E.coli for titer. The phage was then amplified for further rounds of in vivo selections (optional) or for sequence analysis. (B) Nerve pre-injury increases recovery of anti-p75^NTR phage. Phage recovered from nerve ligation 18 h after applying either WT phage (blue columns) or phage enriched for anti-p75^NTR by one round of in vitro panning (red columns) to either uninjured sciatic nerve or sciatic nerve injured 7 d earlier by ligation. Phage recovery increased 13-fold by nerve pre-injury: ***p < 0.001 Student’s t-test; NS, not significant; n = 3; cfu, colony forming units. (C) In vivo-selected anti-p75^NTR phage are transported to the cell bodies in the spinal cord. Anti-p75^NTR phage isolated from first round in vivo screening was reapplied to the injured sciatic nerve without blocking ligation. Twenty-four hours later phage co-localized on the cell bodies of p75^NTR expressing neurons. Control phage applied to the contralateral side also with induced injury showed no visible accumulation. p75^NTR expression is detected in green, phage as red, co-localization is visualized as yellow. Scale bar 150µm. (D) Anti-p75^NTR scFv with high ELISA scores are not internalized in vivo. ELISA scores of a sample of 96 phage clones enriched for anti-p75^NTR by one round of in vitro panning, and after either a further in vitro panning round or one round of in vivo phage selection. Note the absence of clones with high ELISA scores (arrows) after a single round of in vivo phage selection.