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 w ith PBS; (B) inactivated at 56 degreesC, 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 im munofluorescence. In a parallel assay, the substrate, acetone-fixed human f ibroblasts, was preincubated with-sera prepared as if it were to identify A AA, but instead, a rhodamine-phalloidin was used to identify F-actin, by di rect immunofluorescence. All sera from patients were reactive to AAA when h eat-inactivated and/or calcium-chelated, and 60% of them when diluted with unmodified sera (P = 0.004). F-actin continued to be present after preincub ation with heat-inactivated or calcium-chelated sera from patients and heal thy controls, and in 41.5% of reactions with unmodified serum (P = 0.000000 1). The heat inactivation and the calcium chelation were both efficient pro cedures for maintaining the microfilament structure intact after serum incu bation and, therefore, for identifying AAA. (C) 2001 Academic Press.