Methodology article

Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence

Vagia Tsiakalou^1* , Elena Tsangaridou^1* , Hara Polioudaki¹ , Artemissia-Phoebe Nifli² , Meri Koulentaki³ , Tonia Akoumianaki¹ , Elias Kouroumalis³ , Elias Castanas² and Panayiotis A Theodoropoulos¹

¹  Biochemistry, University of Crete, School of Medicine, P.O. Box 2208, Heraklion 71003, Greece

²  Experimental Endocrinology, University of Crete, School of Medicine, P.O. Box 2208, Heraklion 71003, Greece

³  Gastroenterology, University of Crete, School of Medicine, P.O. Box 2208, Heraklion 71003, Greece

author email corresponding author email* Contributed equally

BMC Immunology 2006, 7:20doi:10.1186/1471-2172-7-20

Published:	6 September 2006

Abstract

Background

Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera.

Methods

We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde.

Results

We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells.

Conclusion

Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.