P12.06 GLP-compliant validation of a FluoroSpot assay to monitor tumor neoantigen vaccinations in preclinical mouse models

April, 04, 2024 | Select Oncology Journal Articles


While immunomonitoring as secondary endpoint gets increasingly attractive in neoantigen clinical trials for personalized tumor vaccines, the preclinical testing of neoantigen candidates in animal models may help in neoantigen selection, optimization of formulation or adjuvant development to obtain optimized individual immune responses. The FluoroSpot assay allows to quantify antigen-specific immune cells on a single-cell level. Due to its biologically dynamic setup using living immune cells, a standardization is necessary to comply with international regulatory requirements for GLP-compliant (pre-)clinical sample analysis.1–3

We performed a generic mouse FluoroSpot validation with IFN- as model cytokine for potential multiplexing using splenocytes derived from naive C57Bl/6 mice vaccinated with an immunogenic peptide (SIINFEKL/OVA257-264) as a potent model antigen for neo- or cancer vaccine antigens.

Materials and Methods

Spleens were derived from a preclinical adjuvant development project conducted by Synovo GmbH (Tübingen, Germany). Per treatment group, 5 naive C57Bl6 mice were vaccinated s.c. with 100 nM OVA257-264 peptide (± adjuvant in development) on days 7 and 14 with termination at day 15 or 21. Control animals received adjuvant only. After splenocytes isolation, FluoroSpot analysis was done to quantify antigen-specific IFN- secreting cells. Splenocytes were plated in triplicates including negative control (NC, medium), mock control (actin peptide pool), positive control (PC, Pokeweed) and OVA257-264 peptide to assess inter-/intra-assay precision in three runs performed by three operators. Moreover, assay linearity, specificity, minimum positive control response, LOD, LLOQ and ULOQ were determined (table 1).


Abstract P12.06 Table 1 Parameter Acceptance criteria Acceptance criteria met (Y/N/NA) Splenocyte viability 15 of 15 samples ≥ 50% upon overnight resting Y Minimum PC response 50 spots (based on study results and empirical determination). NA LOD 6 spots (negative control), 7 spots (mock control). NA Intra-assay precision 5 of 5 samples ≥ LLOQ showed a CV ≤ 30%. Y Inter-assay precision 6 of 6 samples ≥ LLOQ showed a CV ≤ 30%. Y Linearity Analyzed samples showed a coefficient of determination of R2 ≥ 0.9 for spot counts ≥ LLOQ (linear range 300,000 – 100,000 cells per well). Y LLOQ 7 spots. NA ULOQ 384 spots. NA Specificity a. All positive controls >> negative controls. b. 17 of 18 mock peptide controls ≤LOD (negative control). c. Adjuvant-only group: 5 of 5 samples < LOD d. OVA257-264 + adjuvant-treated group: 5 of 5 samples >LOD/LLOQ and SI≥3 Y


All predefined acceptance criteria for IFN- FluoroSpot validation could be met. Conclusively, the validated IFN- FluoroSpot is suitable for the detection of antigen-specific immune responses in a preclinical mouse model fulfilling regulatory requirements for bioanalytical assays. Next, a partial assay cross-validation with a neoantigen peptide pool for an established mouse tumor cell line will be done.


  • FDA Bioanalytical Method Validation Guidance for Industry. Version May 2018.

  • ICH guideline M10 on bioanalytical method validation and study sample analysis. Version 25 July 2022.

  • Corsaro B, et al. Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation Bioanalysis. 2021 Mar;13(6):415-463. PMID: 33533276.

  • A. Mauthe: A. Employment (full or part-time); Modest; Immune Analytics, GenID GmbH. R. Villar-Hernández: A. Employment (full or part-time); Modest; Immune Analytics, GenID GmbH. M. Springer: A. Employment (full or part-time); Modest; Immune Analytics, GenID GmbH. R. Preyer: A. Employment (full or part-time); Modest; Immune Analytics, GenID GmbH. M. Gutekunst: A. Employment (full or part-time); Modest; Immune Analytics, GenID GmbH.

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