Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera

Alessandra Dellavance; Danielle C. Baldo; Bing Zheng; Rodrigo A. Mora; Marvin J. Fritzler; Falk Hiepe; Johan Rönnelid; Minoru Satoh; Ignacio Garcia-De La Torre; Mark H. Wener; Edward K.L. Chan; Luis E.C. Andrade

doi:10.1515/cclm-2019-0087

Article

Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera

Alessandra Dellavance , Danielle C. Baldo , Bing Zheng , Rodrigo A. Mora , Marvin J. Fritzler , Falk Hiepe , Johan Rönnelid , Minoru Satoh , Ignacio Garcia-De La Torre , Mark H. Wener , Edward K.L. Chan and Luis E.C. Andrade

Published/Copyright: April 22, 2019

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 57 Issue 11

Abstract

Background

International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies.

Methods

Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization.

Results

A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 μL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation.

Conclusions

This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

Keywords: antinuclear antibodies; autoantibodies; autoimmunity testing; DFS70; HEp-2 cells; reference standards

Corresponding author: Luis E.C. Andrade, MD, PhD, Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 740 3° andar, 04023-062 São Paulo, Brazil; and Immunology Division, Fleury Medicine and Health Laboratory, São Paulo, Brazil

Acknowledgments

The authors are deeply indebted to Vitor Anselmo Sakihara and Paulo Ho, from Butantan Institute, São Paulo, Brazil for lyophilizing the preliminary serum pools; Inova Diagnostics, Inc, San Diego, CA, and Euroimmun Medical Laboratory Diagnostics AG, Lübeck, Germany, for providing the anti-DFS70 ELISA and CLIA kits, respectively; Trinity Biotech, Jamestown, NY, for providing the HEp-2 ELITE slides; Kathryn Kohl, at the Plasma Service Group, for preparing the lyophilized aliquots of the Megapool; Kari B. Basso at the University of Florida Mass Spectrometry Research and Education Center supported by NIH S10 OD021758-01A1; Tomoko Hasegawa and Shin Tanaka from the Department of Clinical Nursing, University of Occupational and Environmental Health, Kitakyushu, Japan.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Fleury Medicine and Health Laboratory provided the material support for the selection and testing of all samples (NP-084). Luis Eduardo C. Andrade receives a research grant (#232711/2014-3) from the Brazilian Research Agency CNPq.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. MJF is a consultant or received honoraria form Inova Diagnostics, Euroimmun GmbH and Alexion Canada.

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Received: 2019-01-25

Accepted: 2019-03-14

Published Online: 2019-04-22

Published in Print: 2019-10-25

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https://doi.org/10.1515/cclm-2019-0087

Keywords for this article

antinuclear antibodies; autoantibodies; autoimmunity testing; DFS70; HEp-2 cells; reference standards