Abstract
The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109-112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106-110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.
Highlights
The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-LysHis-Met (MKHM) corresponding to human PrP-(109 –112)
We revealed that the immunoreactivity of 3F4 with proteinase K (PK)-treated elk PrPSc in the brain of chronic wasting disease (CWD)-infected cervidized transgenic mice (Tg) expressing elk PrP is virtually equal to that of two other antibodies without species specificity: 6H4 and 8H4
Using peptide arrays on the cellulose paper and competition experiments with Western blotting or ELISA, we demonstrated that the epitope residues minimally required for 3F4 binding are PrP-(106 –110) (KTNMK), not PrP-(109 –112) (MKHM), and that the effect of Met112 in human PrP or Val115 in animal PrP on the species preference of the 3F4 antibody is largely modulated by the adjacent amino acids including Ala115
Summary
PrPC, cellular prion protein; PrPSc, pathological, scrapie isoform of prion protein; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; PK, proteinase K; g5p, Fd gene 5 protein; PBS, phosphate-buffered saline; sCJD, sporadic CJD; SPR, surface plasmon resonance; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; Tg, transgenic. Using peptide arrays on the cellulose paper and competition experiments with Western blotting or ELISA, we demonstrated that the epitope residues minimally required for 3F4 binding are PrP-(106 –110) (KTNMK), not PrP-(109 –112) (MKHM), and that the effect of Met112 in human PrP or Val115 in animal PrP on the species preference of the 3F4 antibody is largely modulated by the adjacent amino acids including Ala115
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