Mouse Anti-PRNP Recombinant Antibody (clone POM6) (CAT#: PABC-254)

Figure 1 C-terminal POMs do not bind to linear 25-meric PrP-peptides.

C-terminal-specific POMs were tested for binding on ELISA plates containing immobilized 25-meric PrP-peptides or rmPrP for control. While all antibodies bound strongly to rmPrP23-231, only POM4, 10 – and POM19, not shown – interacted with a 25-mer, spanning amino acids 200–225 of mouse PrP. As positive control, binding of POM11 on the octarepeat region – peptide 65–89 – was used. As expected POM11 did not interact with N-terminally truncated rmPrP121-231, while all other antibodies tested here did. When all peptides were mixed together and immobilized on the well (ALL P-MIX) no binding was observed, probably because of the inefficient amounts of the respective peptides. BLK: Blank.

Polymenidou, M., Moos, R., Scott, M., Sigurdson, C., Shi, Y. Z., Yajima, B., ... & Bellon, A. (2008). The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes. PLoS One, 3(12), e3872.

Figure 2 Cross-species specificities and binding idiosyncrasies of C-terminal specific POMs.

Western blot analysis on various wild type and mutated recombinant PrP proteins identified C-terminal POMs with particular binding patterns indicative for their epitopes (i.e POM4, 5, 6, 7 and 10). Blots were loaded with equal amounts and 6H4 serves as a loading control, since its epitope is conserved among the species tested and is not affected by the point mutations. (B) PrP sequence alignment of residues 165–182 demonstrating the position of POM5 epitope. (C) Western blot analysis on PrP variants, with various amino acids substituted by cysteine as indicated. Blots were loaded with equal protein amounts and a silver stained gel serves as a loading control (first panel). Only mild divergences in the binding patterns of the various POMs were found here. The most striking one is that 6H4, POM1 and POM17 are binding weaker to rmPrP with cysteine substitutions at positions 140 and 145.

Polymenidou, M., Moos, R., Scott, M., Sigurdson, C., Shi, Y. Z., Yajima, B., ... & Bellon, A. (2008). The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes. PLoS One, 3(12), e3872.

Figure 3 Immunoprecipitation, peptide competition and specific elution of PrP^C and PrP^Sc with POMs.

Brain homogenates from healthy or terminally scrapie sick mice were incubated with POM1–7, covalently coupled to paramagnetic beads. All antibodies tested immunoprecipitated PrP^C very efficiently and POM2, 5 and 6 showed competent immunoprecipitation of PK-digested PrP^Sc. Plasminogen was used as a positive and non-specific IgG as a negative control. (B) Brain homogenates from healthy or terminally scrapie sick mice were mixed with POM2-, POM3- or POM6-coupled beads, which were pre-incubated with a peptide corresponding to the epitopes of POM2 (QPHGGGW) or POM3 (HNQWNK) or without any peptide for comparison. Pre-incubation of POM2-coupled beads with its epitope blocked immunoprecipitation of both PrPC and PrPSc, whereas same amounts of an unrelated peptide (POM3-epitope) had no effect on the binding. Similarly, immunoprecipitation of PrP^C by POM3 was only blocked by its corresponding peptide-epitope and not by the epitope of POM2. As expected none of the peptides interfered with binding of POM6 which interacts with the C-terminal part of PrP. SCR: scrapie.

Polymenidou, M., Moos, R., Scott, M., Sigurdson, C., Shi, Y. Z., Yajima, B., ... & Bellon, A. (2008). The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes. PLoS One, 3(12), e3872.