Cell-based ELISA using intact HEp-2 cells: an alternative approach for antinuclear antibody detection.

Authors

Faruk Dişli, Sedat Yıldız

Published in

Immunologic research. Volume 74. Issue 1. Aug 06, 2026. Epub Aug 06, 2026.

Abstract

Antinuclear antibodies (ANAs) are serological markers of clinical importance in the diagnosis and monitoring of autoimmune diseases. This study aimed to develop a HEp-2 cell-based ELISA method as a quantitative and standardized alternative to traditional ANA detection techniques such as indirect immunofluorescence (IFA).
HEp-2 cells were seeded in ELISA plates at ~ 3.0 × 10⁴ cells per well and incubated. After fixation with formaldehyde and acetone, cells were quenched with 1% H₂O₂, permeabilized using Triton X-100, acetone, and methanol, and blocked with Well Champion or 1% BSA. Sixteen sera (8 ANA-positive, 8 ANA-negative), confirmed by Chorus ANA-8 and DRG ANA-8-Screen ELISA, were applied. ANA levels were measured by indirect ELISA with OD readings at 450 nm. Three HRP-conjugated secondary antibodies (anti-IgG gamma chain, Fc-specific, whole IgG) were tested.
Fixation and permeabilization agents did not interfere with the distinction between ANA-positive and ANA-negative samples. In the absence of quenching and when 1% BSA was used for blocking, background levels increased. Among the secondary antibodies, the anti-IgG gamma chain showed the least cross-reactivity, while Fc-specific and whole IgG antibodies had higher nonspecific binding. The assay showed an average intra- and inter-assay variation of 13.13%, with a sensitivity of 87.5% and a specificity of 81.25%.
This study demonstrates that the HEp-2 cell-based ELISA is a feasible, promising method for detecting and quantifying ANA positivity. While it reliably distinguishes between positive and negative samples, it may serve as a practical alternative to conventional assays in research and diagnostics.

PMID:
42560627
Bibliographic data and abstract were imported from PubMed on 06 Aug 2026.

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