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Utility of the HEp-2000 antinuclear antibody substrate
  1. Adrian Y S Lee1,
  2. Dimitra Beroukas2,
  3. Peter J Roberts-Thomson1
  1. 1 Clinical Immunology and Allergy, Flinders Medical Centre, Bedford Park, South Australia, Australia
  2. 2 Immunology, SA Pathology, Bedford Park, South Australia, Australia
  1. Correspondence to Dr Adrian Y S Lee, Clinical Immunology and Allergy, Flinders Medical Centre, Bedford Park, South Australia, Australia; adrian.lee2{at}

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We write with regards to the recently published consensus paper on antinuclear antibody (ANA) staining patterns by Damoiseaux et al which was most informative.1 We were surprised, however, that there was no mention or discussion of the HEp-2000 substrate (Immunoconcepts, Sacremento, California, USA) despite the acknowledgement that anti-SS-A/Ro60 is frequently missed on indirect immunofluorescence assay (IIFA).1 The HEp-2000 substrate is a modified HEp-2 substrate that is transfected with Ro60 cDNA and hence, over-expresses this antigen.2 It produces a bright nuclear/nucleolar staining pattern under IIFA when anti-SS-A/Ro60 antibodies are present (figure 1), and is one of the most commonly detected extractable nuclear antigen antibodies.3

Figure 1

Typical anti-SSA/Ro60 nuclear/nucleolar staining on the HEp-2000 substrate using serum specific for anti-Ro60 at (A) 1:80 dilution and (B) 1:2560 dilution. Even with a 32-fold dilution, the staining is still quite distinct at 1:2560 (B). Insets represent enlarged versions of representative areas on the micrograph.

The HEp-2000 substrate is a sensitive way to detect anti-SS-A/Ro60 antibodies that may be otherwise missed on conventional immunoassays, such as immunoblotting and the standard HEp-2 substrate.4 5 However, it is not a perfect screen as the HEp-2000 substrate may still miss detection of anti-SS-A/Ro60 sera6 7 indicating that at least a second method for anti-SS-A/Ro60 and other antibody detection is still required.8 For other ANA specificities, HEp-2000 performs similarly to the standard HEp-2 substrate with no significant impact on the detection of other ANA fluorescent patterns9 10 and in fact, improved sensitivities.5

It is important to maximise the chances of identifying anti-SS-A/Ro60, for the antibody, as an example, has significant implications for fertile women for the development of neonatal lupus erythematosus which may have devastating sequelae.11 We therefore recommend others to consider the HEp-2000 substrate if employing the IIFA method for ANA detection.



  • Contributors AYSL drafted the manuscript. DB contributed the figure, provided intellectual content and contributed to the manuscript. PJR-T contributed to the manuscript.

  • Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; internally peer reviewed.

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