Limits...
Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers.

Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL, Rasool S, Gurlo T, Butler P, Glabe CG - Mol Neurodegener (2007)

Bottom Line: The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers.The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots.Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. cglabe@uci.edu.

ABSTRACT

Background: Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Abeta and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.

Results: We immunized rabbits with a morphologically homogeneous population of Abeta42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.

Conclusion: Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

No MeSH data available.


Related in: MedlinePlus

Comparison of A11 and I11 antibody specificity. A. Dot blots. Aβ monomer, Aβ prefibrillar oligomers (prepared in HFIP-H20, pH 2.5), Aβ fibrils and α-synuclein and IAPP prefibrillar oligomers were spotted on nitrocellulose strips and probed with A11, I11 and 6E10 as a control. A11 and I11 antibodies demonstrate the same specificity for prefibrillar oligomers and do not react with monomer or fibrils. 6E10 stains the Aβ-containing samples, including prefibrillar oligomers formed at acid pH. B. Western blots. Prefibrillar oligomer samples of calcitonin (Lane 1), insulin (Lane 2) and prion peptide 106–126 (Lane 3) were probed with A11 and I11, which give the same staining pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2100048&req=5

Figure 4: Comparison of A11 and I11 antibody specificity. A. Dot blots. Aβ monomer, Aβ prefibrillar oligomers (prepared in HFIP-H20, pH 2.5), Aβ fibrils and α-synuclein and IAPP prefibrillar oligomers were spotted on nitrocellulose strips and probed with A11, I11 and 6E10 as a control. A11 and I11 antibodies demonstrate the same specificity for prefibrillar oligomers and do not react with monomer or fibrils. 6E10 stains the Aβ-containing samples, including prefibrillar oligomers formed at acid pH. B. Western blots. Prefibrillar oligomer samples of calcitonin (Lane 1), insulin (Lane 2) and prion peptide 106–126 (Lane 3) were probed with A11 and I11, which give the same staining pattern.

Mentions: Since both A11 and OC recognize generic epitopes that do not depend on a particular amino acid sequence, we tested whether the immune response to other types of prefibrillar oligomers and fibrils is also specific for generic conformation dependent epitopes. We synthesized an oligomer mimic of islet amyloid polypeptide (IAPP) by coupling IAPP carboxy-terminal thioester to colloidal gold particles as previously described [19]. We used IAPP oligomer mimics and IAPP fibrils to immunize rabbits and we characterized the specificity of the immune response by dot blot. Immunization of rabbits with IAPP oligomer mimics gives rise to a prefibrillar oligomer-specific immune response that is indistinguishable from that obtained with Aβ prefibrillar oligomer mimics (Fig. 4). We call this serum I11 to indicate its derivation from IAPP antigen and its similarity to A11. Like A11, I11 recognizes prefibrillar oligomers derived from Aβ, α-synuclein and IAPP, but not monomers or fibrils on dot blots (Fig. 4A). Western blots of prefibrillar oligomer samples probed with I11 give the same staining pattern as A11 (Fig. 4B). Similarly, rabbits vaccinated with IAPP fibrils give rise to a fibril specific immune response that recognizes Aβ fibrils and does not recognize monomer or prefibrillar oligomers (Fig. 5). The resulting serum is called LOC (Like OC). Taken together these results demonstrate that the immune response to oligomer mimics and fibrils is predominantly conformation dependent and sequence independent.


Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers.

Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL, Rasool S, Gurlo T, Butler P, Glabe CG - Mol Neurodegener (2007)

Comparison of A11 and I11 antibody specificity. A. Dot blots. Aβ monomer, Aβ prefibrillar oligomers (prepared in HFIP-H20, pH 2.5), Aβ fibrils and α-synuclein and IAPP prefibrillar oligomers were spotted on nitrocellulose strips and probed with A11, I11 and 6E10 as a control. A11 and I11 antibodies demonstrate the same specificity for prefibrillar oligomers and do not react with monomer or fibrils. 6E10 stains the Aβ-containing samples, including prefibrillar oligomers formed at acid pH. B. Western blots. Prefibrillar oligomer samples of calcitonin (Lane 1), insulin (Lane 2) and prion peptide 106–126 (Lane 3) were probed with A11 and I11, which give the same staining pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100048&req=5

Figure 4: Comparison of A11 and I11 antibody specificity. A. Dot blots. Aβ monomer, Aβ prefibrillar oligomers (prepared in HFIP-H20, pH 2.5), Aβ fibrils and α-synuclein and IAPP prefibrillar oligomers were spotted on nitrocellulose strips and probed with A11, I11 and 6E10 as a control. A11 and I11 antibodies demonstrate the same specificity for prefibrillar oligomers and do not react with monomer or fibrils. 6E10 stains the Aβ-containing samples, including prefibrillar oligomers formed at acid pH. B. Western blots. Prefibrillar oligomer samples of calcitonin (Lane 1), insulin (Lane 2) and prion peptide 106–126 (Lane 3) were probed with A11 and I11, which give the same staining pattern.
Mentions: Since both A11 and OC recognize generic epitopes that do not depend on a particular amino acid sequence, we tested whether the immune response to other types of prefibrillar oligomers and fibrils is also specific for generic conformation dependent epitopes. We synthesized an oligomer mimic of islet amyloid polypeptide (IAPP) by coupling IAPP carboxy-terminal thioester to colloidal gold particles as previously described [19]. We used IAPP oligomer mimics and IAPP fibrils to immunize rabbits and we characterized the specificity of the immune response by dot blot. Immunization of rabbits with IAPP oligomer mimics gives rise to a prefibrillar oligomer-specific immune response that is indistinguishable from that obtained with Aβ prefibrillar oligomer mimics (Fig. 4). We call this serum I11 to indicate its derivation from IAPP antigen and its similarity to A11. Like A11, I11 recognizes prefibrillar oligomers derived from Aβ, α-synuclein and IAPP, but not monomers or fibrils on dot blots (Fig. 4A). Western blots of prefibrillar oligomer samples probed with I11 give the same staining pattern as A11 (Fig. 4B). Similarly, rabbits vaccinated with IAPP fibrils give rise to a fibril specific immune response that recognizes Aβ fibrils and does not recognize monomer or prefibrillar oligomers (Fig. 5). The resulting serum is called LOC (Like OC). Taken together these results demonstrate that the immune response to oligomer mimics and fibrils is predominantly conformation dependent and sequence independent.

Bottom Line: The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers.The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots.Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. cglabe@uci.edu.

ABSTRACT

Background: Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Abeta and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.

Results: We immunized rabbits with a morphologically homogeneous population of Abeta42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.

Conclusion: Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

No MeSH data available.


Related in: MedlinePlus