Determination of amyloid type by ELISA using milligram amounts of tissue

Kaplan, Batia; German, G P; Ravid, Mordchai; Pras, Mordechai

doi:10.1016/0009-8981(94)90239-9

Cited by 17 publications

(9 citation statements)

References 11 publications

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“…For aggregate extraction, we used the Kaplan method (Kaplan et al. 1994); 100 mg of autopsy samples of human FAP and control kidney and stomach from transgenic mice ( n = 3) were homogenized in ice cold phosphate buffered solution (PBS; 1 ml), centrifuged at 19,000 g for 10 min and 1 ml of PBS added to the pellet.…”

Section: Methodsmentioning

confidence: 99%

αB‐crystallin (HspB5) in familial amyloidotic polyneuropathy

Magalhães

Santos²,

Saraiva

2010

Int J Experimental Path

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The small heat shock protein αB-crystallin (HspB5) is known to be overexpressed in several neurodegenerative disorders. In familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder characterized by extracellular deposition of mutated transthyretin (TTR), activation of heat shock factor 1 -HSF1- by extracellular TTR deposition has been shown as well as induction of the expression of heat shock proteins, HSP27 and HSP70. Here we investigate the expression of αB-crystallin in FAP. We first detected αB-crystallin in aggregates extracted from tissues of both FAP patients and transgenic mice for the human V30M mutant TTR; however, subsequent studies by confocal fluorescence microscopy did not confirm the association of αB-crystallin with TTR aggregates; thus the presence of αB-crystallin in aggregate extracts might derive from the extraction procedure. Increased levels of αB-crystallin were observed by immunohistochemistry in human FAP skin, as compared to normal skin. Furthermore, skin, stomach and dorsal root ganglia from V30M transgenic mice showed increased expression of αB-crystallin as compared to controls without deposition. A human neuroblastoma cell line incubated with TTR aggregates displayed increased expression of αB-crystallin. Overall, these results show that extracellular TTR deposits induce an intracellular response of αB-crystallin. This small heat shock protein (HSP), which is important for anti-apoptotic and chaperone properties, may have a protective role in FAP.

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Section: Methodsmentioning

confidence: 99%

αB‐crystallin (HspB5) in familial amyloidotic polyneuropathy

Magalhães

Santos²,

Saraiva

2010

Int J Experimental Path

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“…Enzyme linked inimunosorhent as.say (ELISA), The non-competitive ELISA was performed as described earlier [22], Specimens of control and amyloid-containing tissues were obtained from 2mg of wet spleen using the extraction procedure described above and subjected to elecrophoresis on 10% polyacrylamide gel. Proteins from 8 to about 14 KDa were electroeluted as described earlier [23], dialyzed against distilled water, lyophilized and dissolved in 30/*!…”

Section: Methodsmentioning

confidence: 99%

Direct Evidence for SAA Deposition in Tissues During Murine Amyloidogenesis

et al. 1994

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To study the mechanism of amyloid deposition, the nature of amyloid proteins formed in experimental murine amyloidosis, was examined. Spleen specimens, 15-60 mg, were homogenized and extracted using aqueous acidic acetonitrile, in a recently developed procedure, making it possible to obtain amyloid proteins from minute amounts of tissue. The extracted material, 1.5-4 mg, was analysed by Western blotting and ELISA using antibodies recognizing differentially proteins AA and SAA. Two immunoreactive proteins of 8 and 12 KDa were isolated and subjected to amino acid analysis and N-terminal sequence determination. The results of immunochemical and chemical examination showed that the 8 and 12 KDa proteins represented proteins AA and SAA, respectively. The data obtained provide new direct evidence for SAA in tissues during murine amyloidogenesis.

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“…Amyloid proteins were extracted using a modified procedure based on the method developed by Kaplan et al [27]. In the present study, an additional step was added to extract lipids and phospholipids with a chloroform/methanol mixture before amyloid protein extraction.…”

Section: Extraction and Characterization Of Amyloid Deposits From Fapmentioning

confidence: 99%

“…Amyloid proteins were extracted from samples of FAP patients and non-FAP subjects following a modified procedure based on the method developed by Kaplan et al [27]. Briefly, adipose tissue was dispersed in 5 ml of 0.154 M KCl and incubated for 1 h at room temperature (20 • C).…”

Section: Extraction Of Amyloid Proteins From Adipose Tissuementioning

confidence: 99%

Argpyrimidine, a methylglyoxal-derived advanced glycation end-product in familial amyloidotic polyneuropathy

Gomes

Silva

Quintas

et al. 2005

Biochemical Journal

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FAP (familial amyloidotic polyneuropathy) is a systemic amyloid disease characterized by the formation of extracellular deposits of transthyretin. More than 80 single point mutations are associated with amyloidogenic behaviour and the onset of this fatal disease. It is believed that mutant forms of transthyretin lead to a decreased stability of the tetramer, which dissociates into monomers that are prone to unfolding and aggregation, later forming beta-fibrils in amyloid deposits. This theory does not explain the formation of beta-fibrils nor why they are toxic to nearby cells. Age at disease onset may vary by decades for patients with the same mutation. Moreover, non-mutated transthyretin also forms the same deposits in SSA (senile systemic amyloidosis), suggesting that mutations may only accelerate this process, but are not the determinant factor in amyloid fibril formation and cell toxicity. We propose that glycation is involved in amyloidogenesis, since amyloid fibrils present several properties common to glycated proteins. It was shown recently that glycation causes the structural transition from the folded soluble form to beta-fibrils in serum albumin. We identified for the first time a methylglyoxal-derived advanced glycation end-product, argpyrimidine [N(delta)-(5-hydroxy-4,6-dimethylpyrimidin-2-yl)-L-ornithine] in amyloid fibrils from FAP patients. Unequivocal argpyrimidine identification was achieved chromatographically by amino acid analysis using dabsyl (4-dimethylaminoazobenzene-4'-sulphonyl) chloride. Argpyrimidine was found at a concentration of 162.40+/-9.05 pmol/mg of protein in FAP patients, and it was not detected in control subjects. The presence of argpyrimidine in amyloid deposits from FAP patients supports the view that protein glycation is an important factor in amyloid diseases.

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Determination of amyloid type by ELISA using milligram amounts of tissue

Cited by 17 publications

References 11 publications

αB‐crystallin (HspB5) in familial amyloidotic polyneuropathy

αB‐crystallin (HspB5) in familial amyloidotic polyneuropathy

Direct Evidence for SAA Deposition in Tissues During Murine Amyloidogenesis

Argpyrimidine, a methylglyoxal-derived advanced glycation end-product in familial amyloidotic polyneuropathy

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