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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

Western blot analysis of Aβ42 fibrils and prefibrillar oligomers. Aβ42 fibrils (F) and prefibrillar oligomers (O) were run on SDS polyacrylamide gels, transferred to nitrocellulose and probed with 6E10, 4G8, OC and A11 antibodies as indicated at the top of the panel. Both fibrillar and prefibrillar oligomer samples contain bands that react with 4G8 ranging from monomer up to the size of material that accumulates at the top of the gel. OC only stains the bands from fibrillar samples of approximately dimer and above. A11 only stains the prefibrillar oligomer samples. 6E10 does not stain prefibrillar Aβ oligomer samples formed at pH 7.4 as previously reported [22].
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Figure 2: Western blot analysis of Aβ42 fibrils and prefibrillar oligomers. Aβ42 fibrils (F) and prefibrillar oligomers (O) were run on SDS polyacrylamide gels, transferred to nitrocellulose and probed with 6E10, 4G8, OC and A11 antibodies as indicated at the top of the panel. Both fibrillar and prefibrillar oligomer samples contain bands that react with 4G8 ranging from monomer up to the size of material that accumulates at the top of the gel. OC only stains the bands from fibrillar samples of approximately dimer and above. A11 only stains the prefibrillar oligomer samples. 6E10 does not stain prefibrillar Aβ oligomer samples formed at pH 7.4 as previously reported [22].

Mentions: The specificity of OC antisera was examined by ELISA and dot blot analysis of monomeric, prefibrillar oligomeric and fibrillar Aβ and other types of amyloids (Fig. 1). ELISA analysis indicates that OC is conformation dependent and fibril specific because it recognizes Aβ fibrils, but not Aβ prefibrillar oligomers or Aβ monomer (Fig. 1A). OC recognizes a generic epitope that is associated with the fibrillar amyloid state regardless of the sequence because it reacts equally well with α-synuclein fibrils and islet amyloid polypeptide (IAPP) fibrils. Dot blot analysis confirms that the fibril epitope of both Aβ and poly Q (Q36) fibrils is recognized by OC and is complementary and mutually exclusive with the generic prefibrillar epitope of both Aβ and polyQ oligomers that is recognized by A11. A11 does not recognize the fibrillar samples that stain with OC, while OC does not stain the prefibrillar samples recognized by A11 (Fig. 1B). The size distribution of Aβ aggregates that are recognized by OC and A11 was determined by western blotting of fibrillar and prefibrillar oligomer Aβ samples (Fig. 2). Aβ42 fibrils contain 6E10 and 4G8 reactive bands at the positions of 4.5 kDa monomer, dimer, tetramer and a broad smear of aggregates up to the top of the gel. Prefibrillar oligomer Aβ42 samples contain A11 positive aggregates of the same approximate size range as fibrils that are stained by 4G8, but the prefibrillar oligomers are not stained by 6E10. We have previously reported that 6E10 does not stain prefibrillar oligomers prepared by dilution of a stock solution of Aβ42 in 100 mM NaOH in PBS, but it does recognize prefibrillar oligomers formed in water at pH 2.5 and 6E10 recognizes random coil monomer and amyloid fibrils [22]. These results indicate that prefibrillar Aβ oligomers are polymorphic at the 6E10 epitope and that the Aβ monomer in the prefibrillar oligomer preparation has a stable conformation that is not reactive with 6E10. OC anti-fibril antiserum stains the Aβ42 dimer band and higher aggregates from fibrillar samples, but does not stain Aβ monomer (Fig. 2). However, OC does not stain the prefibrillar oligomer aggregates that are stained by A11. A11 stains bands corresponding to tetramer and a broad distribution of aggregates centred on a molecular weight of approximately 60 kDa, indicating that fibrillar oligomers are conformationally distinct from prefibrillar oligomers even though their sizes are broadly overlapping. The size of the bands detected by OC and A11 depend on the time of aggregation with the size increasing with incubation time (data not shown).


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)

Western blot analysis of Aβ42 fibrils and prefibrillar oligomers. Aβ42 fibrils (F) and prefibrillar oligomers (O) were run on SDS polyacrylamide gels, transferred to nitrocellulose and probed with 6E10, 4G8, OC and A11 antibodies as indicated at the top of the panel. Both fibrillar and prefibrillar oligomer samples contain bands that react with 4G8 ranging from monomer up to the size of material that accumulates at the top of the gel. OC only stains the bands from fibrillar samples of approximately dimer and above. A11 only stains the prefibrillar oligomer samples. 6E10 does not stain prefibrillar Aβ oligomer samples formed at pH 7.4 as previously reported [22].
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Western blot analysis of Aβ42 fibrils and prefibrillar oligomers. Aβ42 fibrils (F) and prefibrillar oligomers (O) were run on SDS polyacrylamide gels, transferred to nitrocellulose and probed with 6E10, 4G8, OC and A11 antibodies as indicated at the top of the panel. Both fibrillar and prefibrillar oligomer samples contain bands that react with 4G8 ranging from monomer up to the size of material that accumulates at the top of the gel. OC only stains the bands from fibrillar samples of approximately dimer and above. A11 only stains the prefibrillar oligomer samples. 6E10 does not stain prefibrillar Aβ oligomer samples formed at pH 7.4 as previously reported [22].
Mentions: The specificity of OC antisera was examined by ELISA and dot blot analysis of monomeric, prefibrillar oligomeric and fibrillar Aβ and other types of amyloids (Fig. 1). ELISA analysis indicates that OC is conformation dependent and fibril specific because it recognizes Aβ fibrils, but not Aβ prefibrillar oligomers or Aβ monomer (Fig. 1A). OC recognizes a generic epitope that is associated with the fibrillar amyloid state regardless of the sequence because it reacts equally well with α-synuclein fibrils and islet amyloid polypeptide (IAPP) fibrils. Dot blot analysis confirms that the fibril epitope of both Aβ and poly Q (Q36) fibrils is recognized by OC and is complementary and mutually exclusive with the generic prefibrillar epitope of both Aβ and polyQ oligomers that is recognized by A11. A11 does not recognize the fibrillar samples that stain with OC, while OC does not stain the prefibrillar samples recognized by A11 (Fig. 1B). The size distribution of Aβ aggregates that are recognized by OC and A11 was determined by western blotting of fibrillar and prefibrillar oligomer Aβ samples (Fig. 2). Aβ42 fibrils contain 6E10 and 4G8 reactive bands at the positions of 4.5 kDa monomer, dimer, tetramer and a broad smear of aggregates up to the top of the gel. Prefibrillar oligomer Aβ42 samples contain A11 positive aggregates of the same approximate size range as fibrils that are stained by 4G8, but the prefibrillar oligomers are not stained by 6E10. We have previously reported that 6E10 does not stain prefibrillar oligomers prepared by dilution of a stock solution of Aβ42 in 100 mM NaOH in PBS, but it does recognize prefibrillar oligomers formed in water at pH 2.5 and 6E10 recognizes random coil monomer and amyloid fibrils [22]. These results indicate that prefibrillar Aβ oligomers are polymorphic at the 6E10 epitope and that the Aβ monomer in the prefibrillar oligomer preparation has a stable conformation that is not reactive with 6E10. OC anti-fibril antiserum stains the Aβ42 dimer band and higher aggregates from fibrillar samples, but does not stain Aβ monomer (Fig. 2). However, OC does not stain the prefibrillar oligomer aggregates that are stained by A11. A11 stains bands corresponding to tetramer and a broad distribution of aggregates centred on a molecular weight of approximately 60 kDa, indicating that fibrillar oligomers are conformationally distinct from prefibrillar oligomers even though their sizes are broadly overlapping. The size of the bands detected by OC and A11 depend on the time of aggregation with the size increasing with incubation time (data not shown).

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