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Development and validation of a novel counter-immunoelectrophoresis assay for the detection of antibodies against extractable nuclear antigens

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posted on 2021-11-15, 15:12 authored by Wallis, Reuben

The identification of autoantibodies is a primary diagnostic marker for the diagnosis of some autoimmune diseases. The sera of patients with connective tissue diseases commonly contain autoantibodies that target nuclear antigens. As such these antibodies are called antinuclear antibodies (ANAs). Furthermore, the clinical identification of ANAs in patient sera to specific nuclear antigens is a primary tool for the diagnosis of connective tissue diseases. For this purpose specific extractable nuclear antigens (ENAs) are used. The most commonly employed laboratory technique for the detection of ENA antibodies is the enzyme linked immunoabsorbant assay (ELISA). ELISA is a simple technique that can be automated and provides high diagnostic sensitivity for the detection of ENA antibodies comparatively to a counter immunoelectrophoresis (CIE) assay. However, compared to CIE ELISA lacks diagnostic specificity. Therefore there is a demand for an assay with high diagnostic specificity as a secondary diagnostic test for the detection of ENA antibodies. The central aim of this project was to develop and validate a novel CIE assay that used fluorescently labelled ENAs to detect ENA antibodies in patient serum.   Development of the assay began with comparative testing of fluorescently labelled and unlabelled antigens to confirm that the immunochemical properties of the antigen remained intact. The CIE assay conditions were optimised for the detection of four ANAs SSA, SSB, RNP/Sm, and Sm using their corresponding ENAs. Assay conditions optimised were flurophore type, gel composition, buffer composition, antigen concentration, the running time, and analytical specificity. Evaluation of 281 clinical sera samples known to have previously tested positive test by ANA indirect immunofluorescence were performed using the optimised CIE assay and ELISA. The inter-rater agreement between the CIE assay and ELISA results was determined. Clinical details were used to retrospectively classify the clinical sera samples into clinical categories and case groups. This data was used for the diagnostic comparison of the CIE assay and ELISA results.  There was strong inter-rater agreement between the SSA CIE assay and ELISA results. The diagnostic specificity and positive likelihood ratio values of the SSA CIE assay were superior to those of the ELISA, while diagnostic sensitivity and the negative likelihood ratio values were approximately the same. There was strong inter-rater agreement between the RNP/Sm CIE assay and ELISA. However, it was observed that the statistical measures of assay accuracy (diagnostic specificity and sensitivity, positive likelihood ratio, and negative likelihood ratio) for the RNP/Sm ELISA were superior to those of CIE assay. The diagnostic specificity of the SSB and Sm CIE assays were higher than that of their respective ELISAs. This was also true for the positive likelihood ratio values of the SSB and Sm CIE assays when compared to their respect ELISAs.  The SSA CIE assay results suggest that this assay maybe a suitable replacement for ELISA as a diagnostic tool for the identification of anti-SSA antibodies and Sjogren’s syndrome. The SSB and Sm CIE assay results suggest that these assays would be suitable as secondary confirmatory diagnostic assays for the identification of their respective ENA antibodies. However, further performance evaluation of the assays within a routine diagnostic laboratory is required.

History

Copyright Date

2012-01-01

Date of Award

2012-01-01

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Biomedical Science

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Molecular Bioscience

ANZSRC Type Of Activity code

970111 Expanding Knowledge in the Medical and Health Sciences

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Jordan, Bill; Cook, Neil