Thermolabile and calcium-dependent serum factor interferes with polymerized actin, and impairs anti-actin antibody detection.
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.
Cançado
E L
EL
Department of Gastroenterology, University of Sao Paulo School of Medicine, Sao Paulo, Brazil. edulrc@uol.com.br
Abrantes-Lemos
C P
CP
Vilas-Boas
L S
LS
Novo
N F
NF
Carrilho
F J
FJ
Laudanna
A A
AA
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000097:blood / Q000276:immunology* / Q000378:metabolism*; D000328:Adult; D001323:Autoantibodies / Q000032:analysis / Q000097:blood*; D002118:Calcium / Q000097:blood / Q000502:physiology*; D051345:Destrin; D005455:Fluorescent Antibody Technique; D019693:Hepatitis, Autoimmune / Q000097:blood / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D008840:Microfilament Proteins / Q000097:blood / Q000502:physiology*; D011108:Polymers / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D001323:Autoantibodies; D051345:Destrin; D008840:Microfilament Proteins; D011108:Polymers; D002118:Calcium