Using a phage display technique to identify conformation-specific antibodies in vitro, researchers from the lab of Marcus Fandrich at the Fritz Lipmann Institute in Jena, Germany, have produced a novel amyloid-binding antibody. Their reagent recognizes a surface epitope present on Aβ fibrils and protofibrils, and other protein amyloids. Unlike some anti-Aβ antibodies, this one does not dissolve amyloid. However, it does prevent protofibrils from forming mature fibrils. Thus, the antibody may be useful for detection of amyloid protofibrils or modulation of fibril formation.

It is too early to know if the actions of this particular antibody might be beneficial in a therapeutic sense (it may enhance protofibril degradation) or detrimental (it may stabilize toxic Aβ aggregates), but nonetheless the technique described for antibody discovery should be useful, and readily applicable, to probe other conformations of Aβ and additional proteins as well.

With researchers chasing Aβ in all its different shapes to understand its toxicity and how to quell the same, conformation-specific antibodies are in high demand. They could help sort out the structures and activities of Aβ monomers, dimers, oligomers, protofibrils, and mature fibrils. In the new work, which appears in this week’s online edition of PNAS, first author Gernot Habicht and colleagues took an in-vitro approach to antibody production, using Aβ40 fibrils immobilized on magnetic beads to pan a phage display library of synthetic camelidae immunoglobulins (yes, that’s the animal family that includes camels, dromedaries, and llamas). The advantage of camelid proteins lies in their single-chain structure, whereas the antigen recognition portions of other mammalian antibodies are made up of two polypeptide chains. By screening with mature amyloid fibrils in the presence of an excess of monomeric Aβ40, Habicht and colleagues identified an immunoglobulin that bound fibrillar, but not free, peptide. Making the binding site multivalent by fusing it to a dimerizing alkaline phosphatase protein led to a higher affinity binder (Kd = 7 nM), and yielded a reagent that could be used to detect Aβ fibrils immunochemically. The resulting antibody, B10AP, labeled Aβ40 but not isolated peptide in surface plasmon resonance assays and Western blots, and it detected amyloid plaques in AD brain tissue.

The antibody was truly conformation-specific, as indicated by its ability to recognize the amyloid forms of several other disease-causing proteins, including Aβ42, amyloid A and IgG light chains. It bound to fibrils formed in vivo or in vitro, and recognized a common epitope on the long surfaces of the amyloid fibers, as revealed by electron microscopy. In contrast to the fibrils, Aβ oligomers, which the scientists visualized as 10 to 60 nm diameter particles with EM, were not well recognized by B10AP. Further characterization of the oligomers by circular dichroism differentiated them from fibrils, and they also displayed a relative lack of staining with amyloid dyes compared to fibrils.

To determine if the antibody could affect fibril folding or stability, the researchers added it to soluble Aβ and followed amyloid formation by both thioflavin T binding and electron microscopy. Their analysis showed that the antibody prevented formation of mature fibrils by attaching to and stabilizing protofibrillar structures. The protofibrils, which are a precursor to mature fibrils, expressed the B10 epitope but did not bind thioflavin T, suggesting that the protofibrils and fibrils have the same surface structure, but are in other ways distinct. The investigators do show that the antibody-stabilized protofibrils are more sensitive to proteolytic destruction in vitro than amyloid fibers, which suggest that the antibody has the potential to promote clearance. The antibody was not a plaque buster—it showed no ability to disaggregate mature fibrils.

The antibody may be useful for research and diagnosis, but measuring up the therapeutic possibilities of Aβ antibodies may depend for now on clarification of which aggregate forms are toxic, and which may be protective. (For the latest on this topic, see the recent coverage from the SfN meeting.) The technique Fandrich and colleagues describe for identifying conformation-specific antibodies in vitro could help sort out that issue, if it can be used to generate antibodies specific for other aggregates.—Pat McCaffrey

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References

News Citations

  1. San Diego: Oligomers Live Up to Bad Reputation, Part 1

Further Reading

Primary Papers

  1. . Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils. Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19232-7. PubMed.