Pattern recognition with a fibril-specific antibody fragment reveals the surface variability of natural amyloid fibrils.

Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's ß amyloid (Aß) peptide, while disaggregated Aß peptide or Aß oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high ß-sheet content or ß-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.

PTAA and B10: new approaches to amyloid detection in tissue-evaluation of amyloid detection in tissue with a conjugated polyelectrolyte and a fibril-specific antibody fragment.

AIMS: We analysed the suitability of two little known substances for the detection of amyloid in surgical pathology specimens, that is the conjugated polyelectrolyte polythiophene acetic acid (PTAA) and the camelid antibody domain B10. METHODS: We compared the amyloid detection of PTAA and B10 to Congo red in 106 amyloid-containing tissue biopsies of diverse anatomical and precursor origin by evaluating the accordance in four grades (grade 0: no staining, grade 1: staining of <33% of the amyloid deposits, grade 2: 33-66% and grade 3:>66%). RESULTS: PTAA showed grade 2-3 staining in 57 (54%) cases, while B10 presented this accordance in only 25 (24%) tissue biopsies. Grade 1 staining was found in 11 (10%) samples with PTAA and in 62 (58%) cases with B10. No staining at all (grade 0) occurred in 38 (36%) biopsies when using PTAA and in 19 (18%) cases when using B10. CONCLUSION: Although conformation-sensitive detection seemed promising, PTAA and B10 stain only a fraction of the examined amyloid samples when using routine surgical pathology settings. This study emphasises the necessity of having optimised pre-analytical protocols for recovery, storage and handling of samples if these novel amyloid ligands are to be used in routine diagnosis of amyloid.

Amyloid in endomyocardial biopsies.

The prognosis of cardiac amyloidosis depends on the nature and origin of the amyloid protein deposited. However, little is known about the prevalence and origin of amyloid in heart muscle biopsies. We therefore examined retrospectively the distribution and origin of amyloid in a consecutive series of endomyocardial biopsies. Endomyocardial biopsies with verified presence of amyloid from 101 patients were included. Amyloid was classified immunohistochemically in each of them. Our collective comprised 63 men and 38 women, with a mean age of 66 years (range 37-85 years). Cardiac amyloidosis was the most common of the AL (54 patients) or ATTR type (42 patients). In five individuals, amyloid remained unclassified. AL amyloidosis was subdivided into ALlambda (45 patients) and ALkappa amyloid (nine patients). AA amyloid was not found in any individual. The amount of amyloid was higher in AL than in ATTR amyloidosis. Genomic DNA was extracted and examined by DNA sequencing in 19 patients with ATTR amyloidosis. Five (26%) individuals carried TTR mutations (p.Val20Ile, p.Val30Met (twice), p.Asp39Val and p.Glu54Asp) and were classified as suffering from hereditary ATTR amyloidosis. Amyloid in endomyocardial biopsies is most commonly of immunoglobulin light chain origin, followed by non-hereditary and hereditary-type ATTR amyloid.