Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils
Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purifica...
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| Format: | Online |
| Language: | English |
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Public Library of Science
2014
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140755/ |
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pubmed-41407552014-08-25 Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils Greiner, Erin R. Kelly, Jeffery W. Palhano, Fernando L. Research Article Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids. Public Library of Science 2014-08-21 /pmc/articles/PMC4140755/ /pubmed/25144803 http://dx.doi.org/10.1371/journal.pone.0105433 Text en © 2014 Greiner et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Greiner, Erin R. Kelly, Jeffery W. Palhano, Fernando L. |
| spellingShingle |
Greiner, Erin R. Kelly, Jeffery W. Palhano, Fernando L. Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| author_facet |
Greiner, Erin R. Kelly, Jeffery W. Palhano, Fernando L. |
| author_sort |
Greiner, Erin R. |
| title |
Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| title_short |
Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| title_full |
Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| title_fullStr |
Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| title_full_unstemmed |
Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils |
| title_sort |
immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils |
| description |
Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids. |
| publisher |
Public Library of Science |
| publishDate |
2014 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140755/ |
| _version_ |
1613126478413692928 |