Standardization of the Immunofluorescence Test for Autoantibody to Nuclear Antigens (ANA): Use of Reference Sera of Defined Antibody Specificity

Abstract

Standardization of the indirect immunofluorescence antinuclear antibody (IF-ANA) test can be improved for a given substrate with use of reference ANA sera, uniform assay conditions, and standardization of optical systems. To accomplish this, reference sera from the Arthritis Foundation with defined antibody specificities for nDNA, SS-B, RNP, Sm, nucleoli, and "speckled pattern" were reacted with commonly used IF-ANA substrates, mouse kidney sections, KB and HEp-2 tissue culture cells. Reagents and assay conditions used were those provided with the substrates in commercially available IF-ANA kits. A microscope slide with graded intensities of fluorescent beads was used to standardize microscope fluorescence intensity readings. The authors' fluorescence pattern and intensity results should be directly comparable to results obtained in other laboratories for the six antibodies for which reference sera are available. Although no defined sera are widely available for SS-A, Scl-70, PM-1, and centromere antibodies, the ability of each substrate to detect these antinuclear antibodies as well as mitochondrial, smooth muscle, ribosomal and microsomal antibodies also was tested. HEp-2 cells and KB cells were found to be superior to mouse kidney sections for detection of SS-A, Scl-70, PM-1 and centromere antinuclear antibodies. Mouse kidney sections were superior for screening of sera for the absence of ANA as well as for detection of smooth muscle and liver-kidney microsomal antibodies. Other antibodies were detected with equal sensitivity with all substrates and each of the three ANA kits used in the study performed satisfactorily. Use of reference sera as well as optical standardization is recommended for IF-ANA testing.

Antibodies to non‐bilayer phospholipid arrangements induce a murine autoimmune disease resembling human lupus

“…A modification of kaolin-activated partial thromboplastin time was used to determine lupus anticoagulant antibodies [21,27]. Hep-2 cells incubated with mice sera at different dilutions were used to determine antinuclear antibodies and FITC-conjugated goat anti-mouse antibodies were used as secondary reagents [28]. Total antihistone antibodies were determined in mouse sera at 1:500 dilution [29].…”

Assay of Antinuclear Antibodies by ELISA Using Nuclei as Antigen

“…Despite being the established method for detecting and quantifying antibodies, FANA has certain limitations, e. g. the use of fluorescent microscopy, wide variation in reagent quality, and the lack of a standard substrate for detecting a variety of autoantibodies (11,12).…”

Fator antinúcleo na dermatologia

“…From then on, the specificity of the obtained FANA is investigated (Chart 1). 24,25,[28][29][30] In short, a positive FANA can signify auto-antibodies present in the nucleus, nucleolus and also cell cytoplasm, and must interpreted according to the last postive dilution observed, as to fluorescence location (nucleus, nucleolus, cytoplasm or mitotic machinery) and to deposit morphology, whether homogenous, diffuse, speckled etc. 1,2,16,26,[36][37][38] FANA must be presented as follows: a) positive or reactant, maximal dilution in multiples of 1/40, reaction location and observed deposit pattern -in this way, a correlation of its specificity can be made, 1,16,24 as shown in chart 1; b) Negative or nonreactant FANA.…”

“…These auto-antibodies, when circulating, deposit in different organs and tissues, trigger fixation of the complement system, thus leading to inflammation and dysfunction of the target organ. 2,16,17,22,24 The formation of the "antinuclear factor" complex can have a specific meaning, as it is a marker for various diseases. It can have a prognostic meaning or even no important meaning at all regarding the presence of auto-immune diseases.…”