Developing novel luminescence methods for measuring antibodies

  • Matthew J Randell

Student thesis: Master's ThesisMaster of Science by Research (MScR)


Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by destruction of the insulin-producing pancreatic β-cells. Islet autoantibodies to antigens such as the protein tyrosinephosphate (PTP)-like region of Islet Antigen 2 (IA-2) predict progression to T1D. SARS-CoV-2 is a betacoronovirus which causes COVID-19. Antibodies to the spike protein of the virus provide important information regarding exposure and the magnitude and waning of the immune response, either to natural infection or vaccination. This project aimed to develop plate-based ‘bridging’ luciferase assays as alternative testing methods with improved performance for large-scale screening for antibodies to SARS-CoV-2 spike and IA-2.
NanoLuciferase-tagged IA-2 or SARS-CoV-2 receptor binding domain were diluted and used to label plates of sera. This mixture was then incubated in high-binding optiplates coated with unlabelled PTP-IA-2 or unlabelled spike protein. The specific antibodies in the sera formed a bridge between the plate-bound protein and the free luciferase-tagged conjugate antigens, trapping the reporter for detection.
Results were expressed as units/ml using a standard curve of serially diluted positive sera. Receiver operator characteristic (ROC) curve analysis for the IA-2A bridging assay was carried out using sera from 265 schoolchildren (aged 9-13 years) and 139 people with type 1 diabetes (aged 1-21, samples taken within 3 months of diagnosis) from the Bart’s-Oxford Family Study (BOX). The assay was then compared with the PTP-IA-2 radiobinding assay in a blinded set of 150 samples from the 2020 Islet Autoantibody Standardisation Program Workshop. ROC curve analysis for the SARS-CoV-2 spike antibody bridging assay was performed using sera from 401 known negative samples and 46 individuals with PCR-confirmed SARS-CoV-2 infection. The assay was then compared with the Roche anti-spike antibody test in a blinded set of 182 samples.
The optimised bridging assays required only 2-3µl of serum. The IA-2A bridging assay had 70% sensitivity at 99% specificity, and the area under the ROC curve was 0.858 (95% CI 0.811-0.905). There was good correlation with the PTP-IA-2 results measured by radioimmunoassay (Spearman’s r=0.84, pWe established novel, non-radioactive, high-performance tests for autoantibodies to IA-2 and antibodies to SARS-CoV-2’s spike protein. This plate-based format facilitates automation and therefore high-throughput, large-scale screening for pre-type 1 diabetes or humoral immunity in COVID-19.
Date of Award27 Sept 2022
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorAnna E Long (Supervisor) & Kathleen M Gillespie (Supervisor)

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