[8] Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein

doi:10.1016/S0076-6879(99)09010-2

Methods in Enzymology

Volume 309, 1999, Pages 106-122

https://doi.org/10.1016/S0076-6879(99)09010-2 Get rights and content

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Prion diseases are transmissible neurodegenerative diseases in humans and other mammals. The infectious agent, the prion, is thought to comprise a conformational isoform—PrP^Sc—of a membrane-anchored ubiquitous host protein PrP^C of unknown function. During the replication of prions it is hypothesized that disease-specific PrP^Sc converts host-resident PrP^C, possibly with the help of other cofactors, to new PrP^Sc molecules. The two isoforms of the prion protein—PrP^C and PrP^Sc have the same amino acid sequence, but are folded differently. On conversion to the disease-specific isoform, PrP^Sc aquires characteristic features such as partial protease resistance increase in β-sheet structure, and insolubility as opposed to PrP^C, which is protease-sensitive, consists primarily of an α-helical secondary structure, and is soluble. This chapter describes in detail how a monoclonal antibody (MAb) has been raised that specifically recognizes only native, disease-associated PrP^sc but not normal PrP^C. The protocol involved immunization of knockout mice with recombinant bovine PrP produced in Escherichia coli and thorough screening of hybridoma for the production of monoclonal antibodies against recombinant bovine PrP as well as disease-associated PrP^BSE (i.e., native, bovine PrP^Sc).

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Cited by (39)

Disrupted in Schizophrenia 1 regulates the processing of reelin in the perinatal cortex
2020, Schizophrenia Research
Disrupted in Schizophrenia 1 (DISC1) is a prominent gene in mental illness research, encoding a scaffold protein known to be of importance in the developing cerebral cortex. Reelin is a critical extracellular protein for development and lamination of the prenatal cortex and which has also been independently implicated in mental illness. Regulation of reelin activity occurs through processing by the metalloproteinases ADAMTS-4 and ADAMTS-5. Through cross-breeding of heterozygous transgenic DISC1 mice with heterozygous reeler mice, which have reduced reelin, pups heterozygous for both phenotypes were generated. From these, we determine that transgenic DISC1 leads to a reduction in the processing of reelin, with implications for its downstream signalling element Dab1. An effect of DISC1 on reelin processing was confirmed in vitro, and revealed that intracellular DISC1 affects ADAMTS-4 protein, which in turn is exported and affects processing of extracellular reelin. In transgenic rat cortical cultures, an effect of DISC1 on reelin processing could also be seen specifically in early, immature neurons, but was lost in calretinin and reelin-positive mature neurons, suggesting cell-type specificity. DISC1 therefore acts upstream of reelin in the perinatal cerebral cortex in a cell type/time specific manner, leading to regulation of its activity through altered proteolytic cleavage. Thus a functional link is demonstrated between two proteins, each of independent importance for both cortical development and associated cognitive functions leading to behavioural maladaptation and mental illness.
Novel assay with fluorescence-labelled PrP peptides for differentiating L-type atypical and classical BSEs, and scrapie
2012, FEBS Letters
Citation Excerpt :
Furthermore, prions consist of multiple types that are classified by incubation periods and lesion profiles in inbred mice [2]. Some prions may also differ in their PrPSc properties, e.g. different electrophoretic mobilities [3], relative glycoform ratios [4], N-terminal ends of proteinase K (PK) digested PrPSc [5], immunoreactivities against conformational-specific antibodies [6,7], and stabilities against chaotropes [8]. These differences suggest that prion identity is encoded by the conformation of PrPSc, but little research has been done to confirm this.
Characteristic differences of prions may account for the conformational diversity of the pathogenic isoform of prion protein (PrP^Sc). Here, we applied a protein detection procedure by using fluorescent-labelled peptides for detecting PrP^Sc. Five prion protein (PrP) related peptides were found to change significantly their fluorescent intensities with prion-affected animal samples. Their reactivity was different among atypical L-BSE, classical BSE and scrapie. The pull-down assay revealed that they precipitated PrP^Sc specifically. These findings suggest that fluorescent intensity changes depend on peptide–PrP^Sc binding. This novel approach may distinguish the fine structural differences in PrP^Sc, which were not detected by the pull-down assay.
PrP-peptides HPP01, 02, 03, 06, 11 physically interact with PrP^Sc of L-type atypical and classical BSEs, and scrapie by pull down
Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity
2009, Molecular Immunology
The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP^Sc, associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP^Sc that recognizes both PrP^C and PrP^Sc. This scFv fragment had a dissociation constant (K_D) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP^Sc but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP^Sc and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.
Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues
2008, Journal of Immunological Methods
Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP^Sc) of the prion protein (PrP^C) on neuronal cells. These methods depend on antibodies directed against PrP^C and capable of reacting with PrP^Sc in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP^Sc, including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrP^Sc as well as PrP^Sc of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP^Sc blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP^RES) in situ.
Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153–165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153–165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153–165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP^Sc in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4 reached the same level of reactivity as 1.5D7 and 1.6F4. This shows that 6H4 needs denatured PrP^RES to reach maximal reactivity, confirming earlier results. As an exception, human PrP^RES still reacted relatively poorly with 6H4 in PET-blotting, while 1.5D7 and 1.6F4 reacted well with PrP^RES from most human CJD types.
Taken together this implies that the binding epitope of 1.5D7 and 1.6F4 is accessible in the aggregates of undenatured PrP^Sc (IHC) while the binding site of 6H4 is at least partly inaccessible. In techniques incorporating a denaturing and/or disaggregating step 6H4 showed good binding indicating increased accessibility of the binding site. An exception to this is human samples in PET-blotting suggesting that huPrP^RES might not be as easily unfolded by denaturation as BSE and scrapie PrP^RES. Also of interest was the ability of 1.5D7 and 1.6F4 to discriminate between two allelic variants of PrP CJD^Sc (VV vs. MM) in immunohistochemistry as opposed to the normally used antibody 3F4.
Identification of an epitope on the recombinant bovine PrP that is able to elicit a prominent immune response in wild-type mice
2007, Immunology Letters
Citation Excerpt :
Instead, mice lacking a functional Prnp gene (Prnp0/0 mice) were used predominantly [13] and there are numerous reports of the successful production of mAbs after immunization with recombinant PrPs [14–18]. The well-known mAb 6H4 was generated by immunizing Prnp0/0 mice with a reduced form of recombinant bovine PrP (recBoPrP) [14,15]. In another approach, the immune system has been circumvented by the immunization of wild-type mice with chemically modified recombinant PrP proteins.
The main cause for the development of transmissible spongiform encephalopathies (TSE) is the conformational change of prion protein from the normal cellular isoform (PrP^C) into the abnormal isoform, named prion (PrP^Sc). The two isoforms have the same primary structure, and with PrP being highly conserved among different species, no immune response to PrP^Sc has been observed in infected humans or other mammals so far. The problem of inducing immune response was encountered when producing monoclonal antibodies against PrP, therefore mice lacking a functional Prnp gene were predominantly used for the immunization. In the present paper we report that by immunizing wild-type BALB/c mice with chemically unmodified recombinant bovine PrP a potent humoral immune response was achieved. Furthermore, we were able to isolate the monoclonal antibody (mAb) E12/2 and few other mAbs, all reacting specifically with bovine and human PrP, but not with PrP from several other mammals. The epitope of mAb E12/2 is located at the C-terminal end of helix 1, with His155 being crucial for binding. It has been proven that mAb E12/2 is useful for human and bovine TSE research as well as for diagnostics. Our results show that there are sufficient structural differences between mouse and bovine PrP to provoke a prominent humoral immune response.
Novel single chain antibodies to the prion protein identified by phage display
2007, Virology
A well defined structure is available for the carboxyl half of the cellular prion protein (PrP^c), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrP^c recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrP^c as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised 1 predominant and 3 additional scFv-Ps that contained different V_H and V_L sequences and that bound specifically to the PrP^c target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrP^c residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrP^c that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the amino-central region of PrP^c. In addition, the binding of MAbs to known linear epitopes within PrP^c depends strongly on the endpoints of the target PrP^c fragment used.

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[8] Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein

Publisher Summary

Cell

J. Immunol. Methods

Biochemistry

J. Immunol.

J. Virol.

J. Infect. Dis.

J. Virol.

J. Immunol.

Nature

Mol. Med.

J. Infect. Dis.

J. Gen. Virol.

Neurology