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Thermolabile and calcium-dependent serum factor interferes with polymerized actin, and impairs anti-actin antibody detection.

Abstract：

:The detection of anti-actin (AAA) by immunofluorescence is hindered by the presence of a serum factor. To better understand how it interferes with AAA detection, we tested sera from 20 patients with autoimmune hepatitis, and from 21 healthy adults, diluted 1:10 and prepared as follows: (A) diluted with PBS; (B) inactivated at 56 degrees C, and diluted with PBS; (C) diluted with 34 mM EDTA/PBS; (D) heated and diluted with EDTA/PBS. To reveal AAA, a fluorescein-labelled anti-human IgG was used in the process of indirect immunofluorescence. In a parallel assay, the substrate, acetone-fixed human fibroblasts, was preincubated with sera prepared as if it were to identify AAA, but instead, a rhodamine-phalloidin was used to identify F-actin, by direct immunofluorescence. All sera from patients were reactive to AAA when heat-inactivated and/or calcium-chelated, and 60% of them when diluted with unmodified sera (P=0.004). F-actin continued to be present after preincubation with heat-inactivated or calcium-chelated sera from patients and healthy controls, and in 41.5% of reactions with unmodified serum (P=0.0000001). The heat inactivation and the calcium chelation were both efficient procedures for maintaining the microfilament structure intact after serum incubation and, therefore, for identifying AAA.

journal_name

J Autoimmun

journal_title

Journal of autoimmunity

authors

Cançado EL,Abrantes-Lemos CP,Vilas-Boas LS,Novo NF,Carrilho FJ,Laudanna AA

doi

10.1006/jaut.2001.0540

subject

Has Abstract

pub_date

2001-11-01 00:00:00

pages

223-8

issue

3

eissn

0896-8411

issn

1095-9157

pii

S0896-8411(01)90540-2

journal_volume

17

pub_type

杂志文章

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