(T1530-01-07) Singlicate Analysis of Anti-Antigen IgA in Human Serum with Ligand-Binding Assay

Purpose: Duplicate measurements have traditionally been the industry standard for ligand-binding assays (LBAs) in clinical sample testing, ensuring data reliability and reducing variability. However, technological advancements, particularly the development of platforms like Meso Scale Discovery (MSD), which uses electrochemiluminescence detection, have significantly improved assay precision and reproducibility. These improvements enable the potential transition toward single-well (singlicate) measurements, providing significant benefits such as reduced reagent and labor costs, increased throughput, and faster assay turnaround times. This study assesses the feasibility of implementing singlicate analysis in vaccine immunogenicity testing by directly comparing singlicate and duplicate measurement formats in MSD-based binding assays. Here, the study focuses on quantifying immunoglobulin A (IgA) responses against antigen. Results will inform the suitability of singlicate formats in high-throughput immunogenicity testing environments.

Methods: The case study outlines a fit-for-purpose strategy for the development and qualification of an anti-Antigen immunoglobulin A (IgA) assay utilizing the MSD platform (Figure 1). Briefly, an MSD 96-Well 1-Spot SECTOR plate is coated with antigen proteins. Antibodies targeting the antigen are captured on the plate and subsequently bind to a SULFO-TAG conjugated anti-human IgA antibody. A tripropylamine solution (Read Buffer) is then added, generating electrochemiluminescence signals that are quantified by the plate reader. The approach emphasizes evaluating the feasibility of singlicate analysis for vaccine immunogenicity assessment. In the assay qualification, we assessed precision, accuracy, linearity, parallelism, specificity, and stability. Precision was evaluated using 32 serum samples spanning the full range of the assay. Each sample was analyzed in six independent runs, with up to nine replicate measurements generated per sample. This allowed for robust assessment of intra- and inter-run variability. Relative accuracy was determined through linearity experiments, where measured concentrations were compared to mean concentrations across different dilutions. These data were used to assess the consistency of quantitation across a range of expected IgA concentrations. For the comparison of singlicate and duplicate analysis, the singlicate format utilized only the first replicate from each run, whereas the duplicate format incorporated both replicates. Additionally, the study evaluated the influence of calibrator format, singlicate versus duplicates on assay’s output and performance metrics. The results demonstrate the potential for adopting singlicate analysis in regulated immunogenicity testing, with implications for assay efficiency, cost, and throughput without compromising data quality.

Results: To assess the feasibility of singlicate measurements in clinical testing, singlicate analysis was evaluated alongside the traditional duplicate format to compare assay accuracy, precision, and linearity. Precision assessment was first evaluated through Intra-assay precision, expressed as coefficient of variation (%CV), was 3.8% for duplicate analysis and 10.3% for singlicate analysis, indicating acceptable repeatability under both formats. Inter-assay precision was similarly robust, with %CV values of 9.2% (duplicate) and 10.4% (singlicate) for the anti-Antigen IgA assay. These data demonstrate consistent performance across runs and are summarized in Figure 2A, which presents inter-assay precision results for the full precision panel. Next, Linearity was tested using a panel of six human serum samples diluted in series up to a 1024-fold pre-dilution as shown in Table 1. All six samples exhibited strong linearity across the tested range, with slopes ranging from 0.85 to 1.15 and R² values > 0.95, in both duplicate and singlicate formats. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were eventually established based on precision and linearity criteria. For duplicate analysis, the LLOQ is 44.5 AU/mL and the upper limit of quantification (ULOQ) is 523,000 AU/mL. For singlicate analysis, the LLOQ is 43.6 AU/mL and the ULOQ is 469,000 AU/mL. These results indicate that both formats support a broad and comparable dynamic range suitable for clinical applications. Furthermore, singlicate analysis demonstrated performance comparable to duplicate analysis, yielding a strong concordance with a slope of 0.99, as shown in Figure 2B. Additional key parameters including parallelism, specificity, and stability were evaluated and qualified using the duplicate format prior to deployment in clinical sample testing, ensuring assay robustness under regulatory expectations.

Conclusion: The anti-Antigen IgA assay was successfully developed and qualified in both singlicate and duplicate formats. Each format met all predefined acceptance criteria for accuracy, precision, and linearity. Additional parameters, including parallelism, specificity, and stability, were evaluated using the duplicate format, thereby completing the qualification process and confirming the assay’s fitness for clinical sample analysis. The strong concordance observed between singlicate and duplicate results underscores the reliability and applicability of the singlicate format for vaccine immunogenicity assessments.

Acknowledgements: We thank Naila Hunter from Antigen Design and Selection department for providing antigen proteins and Huiling Chen from Clinical Biomarkers, Infectious Disease department for helpful discussions. This work is funded by Moderna, Inc. All authors are Moderna’s current employees and may hold stock/stock option in the company.

Figure1. MSD anti-Antigen IgA Assay mechanism

Figure 2 :Comparison of Singlicate and duplicate analysis
(A) Precision (inter-assay precision) Profile of anti-Antigen IgA with singlicate and duplicate analysis
(B) Concordance between singlicate analysis and duplicate analysis for anti-Antigen IgA


Table 1: Summary of Linearity of anti-Antigen IgA with duplicate analysis and singlicate analysis