Cell-based ELISA using intact HEp-2 cells: an alternative approach for antinuclear antibody detection
Immunologic Research, cilt.74, sa.1, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 74 Sayı: 1
- Basım Tarihi: 2026
- Doi Numarası: 10.1007/s12026-026-09816-9
- Dergi Adı: Immunologic Research
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, EMBASE, MEDLINE, Natural Science Collection (ProQuest), Biological Science Database (ProQuest), Biomedical Reference Collection: Corporate Edition (EBSCO), Health Research Premium Collection (ProQuest), Pharma Collection (ProQuest)
- Anahtar Kelimeler: ANA, Antinuclear antibodies, Cell-based ELISA, HEp-2 cells
- İnönü Üniversitesi Adresli: Evet
Özet
Background: 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). Materials and methods: 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. Results: 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%. Conclusions: 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.