sepharose and Amyloidosis

Protein aggregation is a hallmark of several degenerative diseases, including Alzheimer's disease, Parkinson's disease and familial amyloidosis (Finnish type) (FAF). A method to isolate and detect amyloids is desired for the diagnosis of amyloid diseases. Here, we report the synthesis of pentameric thiophene amyloid ligand (p-FTAA) linked to agarose resin for selective purification of amyloid aggregates produced in vitro and in vivo. Using amyloid fibrils produced in vitro from α-synuclein, gelsolin, and Aβ

Topics: Acetates; alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidosis; Animals; Corneal Dystrophies, Hereditary; Gelsolin; Humans; Mice; Peptide Fragments; Protein Aggregates; Sepharose; Thiophenes

Dialysis-related amyloidosis (DRA) is a devastating and costly condition that affects patients with end stage kidney disease. A key feature of DRA is the formation of amyloid fibrils, consisting primarily of beta2-microglobulin. Except for kidney transplantation, conventional kidney replacement therapies, which are based on nonspecific mechanisms, do not adequately address beta2-microglobulin removal. An antihuman beta2-microglobulin single-chain variable region antibody fragment (scFv) was developed to confer specificity to beta2-microglobulin removal during hemodialysis.. The scFv was immobilized onto agarose and characterized for beta2m binding capacity, thermal stability at 37 degrees C, regeneration capacity, storage conditions, and sterility.. The beta2-microglobulin binding capacity was 1.3 mg/ml scFv gel. The immunoadsorbent is thermally stable, can be regenerated, stored short-term in 20% ethanol, lyophilized for long-term storage, and withstand process conditions similar to that of a patient's hemodialysis therapy.. The results support further investigation of immobilized scFvs as a novel tool to remove beta2-microglobulin from blood.

Topics: Amyloidosis; beta 2-Microglobulin; Drug Stability; Extracorporeal Circulation; Humans; Immunoglobulin Variable Region; Immunosorbent Techniques; Kidney Failure, Chronic; Renal Dialysis; Sepharose; Sterilization

Amyloid fibrils were isolated from kidney tissue of a dog that presented with renal failure due to spontaneous amyloidosis. This fibril material was reduced, alkylated and chromatographed on a column of Sepharose CL6B. A major retarded fraction, when subjected to amino acid sequencing, demonstrated a blocked amino terminus. The isolated protein was then degraded with cyanogen bromide, and the resultant three peptides were isolated by high-pressure liquid chromatography. The amino acid sequence of one peptide corresponded to the sequence of human amyloid protein AA from position 17 to 23. A second peptide gave an amino acid sequence homologous to the published human protein AA sequence starting with position 24. Although a high degree of homology between canine and human AA is seen, the blocked amino terminus is similar to the AA protein of mink. These data show that spontaneous canine amyloid is analogous to human reactive (secondary) amyloid and, therefore, may aid in defining mechanisms of human amyloid pathogenesis.

Topics: Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Chromatography, Ion Exchange; Dog Diseases; Dogs; Kidney; Sepharose; Serum Amyloid A Protein

Clearing of turbid amyloid A fibril containing agarose gels by human serum has been ascribed to 'amyloid degrading activity'. We report here that this optical phenomenon is not due to an enzymatic reaction, does not involve proteolysis of the fibril subunits and is not inhibited by sera of patients with AA amyloidosis. The extent of clearing correlates closely with the serum albumin concentration and, as previously reported by others, serum albumin itself causes clearing comparable to that of whole serum. Furthermore addition of albumin solutions to turbid aqueous suspensions of AA amyloid fibrils causes immediate clearing. Serum albumin is known to clarify turbid non-amyloid fibril containing gels and is used commercially to improve the optical properties of radial immunodiffusion plates. We therefore propose that this property of albumin, the mechanism of which is not yet understood, underlies the so called 'amyloid degrading activity' of human serum and the latter is not therefore likely to be of in vivo biological or clinical significance.

Topics: Amyloid; Amyloidosis; Blood; Humans; In Vitro Techniques; Pronase; Sepharose; Serum Albumin; Serum Amyloid A Protein; Temperature; Trypsin