CytoBead ANA 2 assay - a novel method for the detection of antinuclear antibodies

Sci Rep. 2025 Jul 1;15(1):22131. doi: 10.1038/s41598-025-04583-3.

Abstract

Detection of anti-nuclear autoantibodies (ANA) is based on a two-step algorithm including indirect immunofluorescence (IIF) on HEp2 cells and subsequent reflex/confirmatory testing for specific autoantibodies. Simultaneous cell- and microbead-based autoantibody detection by IIF may be utilized for the evaluation of systemic autoimmune rheumatic diseases (SARDs). In the present study, we assessed the performance of CytoBead ANA 2 in the detection of ANA and ANA-specific autoantibodies, compared to ANA IIF and BioPlex™ 2200. We also tested the ability of CytoBead ANA DFS-70 to identify dense-fine speckled (DFS) pattern associated with anti-DFS70 antibodies in non-SARDs patients. Hundred-twelve routine sera samples were assessed by manual CytoBead ANA 2 for the presence of ANA and specific autoantibodies. In parallel, these samples were analyzed by HEp2 ANA IIF test and a subsequent multiplexed assay BioPlex™ 2200 ANA. Twenty-nine ANA-positive samples obtained from non-SARDs patients and exhibiting DFS pattern by ANA IIF were further tested by CytoBead ANA DFS-70. A substantial agreement was observed between classical ANA IIF and manual CytoBead ANA 2 for the detection of ANA (k = 0.74). Discordant results were mainly associated with the presence of anti-SSA/Ro antibodies detected by CytoBead ANA 2 in ANA IIF negative patients. A good to almost perfect agreement was found between CytoBead ANA 2 and BioPlex™ 2200 for detection of specific antibodies with kappa values ranging from 0.70 to 0.90. Twenty samples (68.9%) obtained from 29 ANA IIF positive without SARDs patients exhibited DFS pattern in CytoBead ANA DFS-70, which confirmed the presence of anti-DFS70 antibodies. The diagnostic performance of manual CytoBead ANA 2 for ANA screening and detection of ANA specific antibodies is comparable to the diagnostic performance of ANA IIF followed by BioPlex™ 2200. This novel one-step assay enables simultaneous ANA screening and confirmation and represents a promising alternative approach to the time-consuming and costly two-tier ANA analysis.

Keywords: Anti-DFS70 antibodies; Anti-nuclear antibodies; CytoBead technology; Indirect immunofluorescence; Multiplexed assay.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Adult
  • Antibodies, Antinuclear* / blood
  • Antibodies, Antinuclear* / immunology
  • Autoimmune Diseases* / blood
  • Autoimmune Diseases* / diagnosis
  • Autoimmune Diseases* / immunology
  • Female
  • Fluorescent Antibody Technique, Indirect / methods
  • Humans
  • Male
  • Middle Aged
  • Rheumatic Diseases* / blood
  • Rheumatic Diseases* / diagnosis
  • Rheumatic Diseases* / immunology
  • Transcription Factors

Substances

  • Antibodies, Antinuclear
  • PSIP1 protein, human
  • Transcription Factors
  • Adaptor Proteins, Signal Transducing