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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
Fleury Med & Hlth Lab, Res & Dev Div, Sao Paulo, Brazil.
Fleury Med & Hlth Lab, Res & Dev Div, Sao Paulo, Brazil.
Univ Florida, Dept Oral Biol, Gainesville, FL 32610 USA;Shanghai Jiao Tong Univ, Renji Hosp, Sch Med, Dept Lab Med, Shanghai, Peoples R China.
Univ Florida, Dept Oral Biol, Gainesville, FL 32610 USA.
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2019 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 57, no 11, p. 1754-1763Article in journal (Refereed) Published
Abstract [en]

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 mu L-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 antiDFS70 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.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH , 2019. Vol. 57, no 11, p. 1754-1763
Keywords [en]
antinuclear antibodies, autoantibodies, autoimmunity testing, DFS70, HEp-2 cells, reference standards
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-396699DOI: 10.1515/cclm-2019-0087ISI: 000489114100021PubMedID: 31005948OAI: oai:DiVA.org:uu-396699DiVA, id: diva2:1368894
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved

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