Enhancement of binding avidity by bivalent binding enables PrPSc-specific detection by anti-PrP monoclonal antibody 132

Abstract

Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119–127, enables us to reliably detect abnormal isoform prion protein (PrP Sc ) in cells or frozen tissue sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrP Sc -specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrP C ) are close enough to allow bivalent binding, mAb 132 binds to PrP C . These results indicate that weak monovalent binding to monomeric PrP C diminishes PrP C signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrP Sc in a bivalent manner.

Kinetics of Blood–Brain Barrier Transport of Monoclonal Antibodies Targeting the Insulin Receptor and the Transferrin Receptor

“…With regard to the association rate constant of IRMAb binding to the IR, the effective k on in vivo is 10 5 M −1 sec −1 (simulation 15, Figure 7), as a k on value of 10 4 M −1 sec −1 (simulation 19, Figure 7) or a k on of 10 6 M −1 sec −1 (simulation 20, Figure 7) produces a predicted brain uptake that is either too low, or too high, respectively, relative to the experimentally observed brain uptake. These estimates of the k on parameter are made in vivo at 37 • C and reflect binding of the MAb to the endothelial membrane-bound receptor, which is expressed at a receptor density, 0.03 fmol/mm 2 (35), that is 100-fold lower than the receptor density used in in vitro SPR experiments [76,77].…”

“…However, at the brain endothelial luminal membrane in vivo, the TfRMAb binds to a hetero-tetrameric complex of two Tf molecules bound to a TfR disulfide-linked receptor dimer (Figure 1A), which is embedded in the plasma or endosomal membranes. Second, the TfR1 surface density at the brain capillary in vivo, 0.03 fmol/mm 2 [35], is >100-fold lower than the receptor surface density used in SPR experiments [76,77]. Unknown rate constants were estimated by fitting the mathematical model to experimentally observed brain uptake of the TfRMAb [35].…”

Involvement of N- and C-terminal region of recombinant cervid prion protein in its reactivity to CWD and atypical BSE prions in real-time quaking-induced conversion reaction in the presence of high concentrations of tissue homogenates

“…Genes encoding the N-terminal replaced chimera of rCerPrP and rMoPrP (rMo N –CerPrP, rCer N –MoPrP) were generated using assembly PCR [ 57 ] with primer sets described in the Supplementary Table 2. Genes encoding the C-terminal replaced chimeras (rCer–Mo C PrP, rMo–Cer C PrP) were generated through two consecutive PCRs with primers in Supplementary Table 2.…”

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE)

“…This new cell-based ELISA can discriminate prion-infected cells from prion-uninfected cells without the need for cell lysate preparation and PK treatment. Such ELISA is a practical and simple method for the primary screening of anti-prion compounds [ 111 , 112 ].…”