Case Studies

Preclinical Case Studies

Real-world study data, kinetic screening workflows, and immunogenicity evaluation reports from preclinics' bioanalytical and in-vivo research programs.

Bio-Layer Interferometry (BLI)
NanobodiesBLI / KineticsAssay DevelopmentAntibody DiscoveryHigh-Throughput Screening

Developing a Biolayer-Interferometry Screening Workflow for Nanobody Clones in Crude Periplasmic Extracts

Phage-display campaigns produce dozens of ELISA-positive VHH clones, but purifying every candidate before kinetic screening is slow and costly. This case study documents how preclinics developed and qualified a BLI workflow to rank VHH candidates directly from crude periplasmic extract — testing and rejecting two configurations before arriving at a robust, screening-ready format.

Key Findings & Study Highlights

  • Three configurations evaluated: single-cycle kinetics (rejected), antigen-on-streptavidin/PE-as-analyte (rejected), VHH-captured-on-Ni-NTA/purified-antigen-as-analyte (selected)
  • Anti-GFP VHH positive-control pair: specific, fittable binding curves from crude periplasmic extract
  • Ni-NTA biosensors reusable up to ~10×
  • Tag-free antigen + Ni-NTA biosensors identified as hard prerequisites
  • His-tagged screening antigen artefact caught before screening — would have produced false positives for every clone
Bio-Layer Interferometry (BLI)
NanobodiesBLI / KineticsAffinity CharacterizationAntibody Discovery

Affinity and Kinetic Characterization of Nanobodies (VHHs) by Bio-Layer Interferometry

Two anti-PfTrx single-domain antibodies (A2 and G5) were characterized by BLI to determine binding affinity and kinetics against their antigen. VHH G5 yielded a robust, reproducible low-nanomolar affinity, while VHH A2 displayed complex non-1:1 kinetics and was deliberately not assigned a KD — illustrating preclinics' commitment to reporting only what the data support.

Key Findings & Study Highlights

  • VHH G5: KD ≈ 23–29 nM across two independent runs
  • VHH G5: Kon 7.9×10⁴–1.3×10⁵ M⁻¹s⁻¹; Koff 2.4×10⁻³–3.0×10⁻³ s⁻¹; R² > 0.99; 1:1 binding model fits
  • VHH A2: fast on-rate and off-rate, unstable association plateau, bi-modal dissociation — KD deliberately not reported
  • Ni-NTA biosensors adopted over anti-HisTag capture due to superior loading performance
IFN-γ ELISpot
ImmunologyVaccine DevelopmentT-Cell AnalysisELISpotSingle-Cell Analysis

Single-Cell Immunological Memory Assessment: IFN-γ ELISpot Protocol Applied to SARS-CoV-2 Spike Vaccine Immunogenicity Evaluation

A preclinical immunogenicity assessment of a SARS-CoV-2 spike vaccine in Balb/c mice using a two-dose vaccination protocol (d0 prime, d28 boost) with terminal splenocyte analysis at d42. IFN-γ-secreting T cells were quantified at single-cell resolution, revealing robust Th1-polarized responses in three of four vaccinated animals.

Key Findings & Study Highlights

  • SFU: 795 ± 45 (ID01), 725 ± 38 (ID02), 615 ± 32 (ID03) vs 15 ± 3 (control)
  • Fold-change over naive control: 53×, 48×, 41× for top three responders (mean 47.3×)
  • Cell seeding: 2×10⁵ splenocytes/well; linear dose-response confirmed
  • Detection sensitivity: 1 per 10⁵–10⁶ cells
ddPCR + ECLIA
Molecular AnalysisCytokine ProfilingmRNA TherapeuticsInnate ImmunityFormulation DevelopmentddPCRECLIA

Cytokine Response After Intramuscular Injection of mRNA Formulations

Two lipid-formulated mRNA candidates (mRNA-A and mRNA-B) were administered by intramuscular injection in Balb/c mice to assess their inflammatory cytokine profile. A dual-platform strategy combining ddPCR (tissue-level transcripts) and ECLIA (circulating serum cytokines) revealed a spatially organized response, with the draining lymph nodes as the most discriminating compartment and mRNA-A eliciting a consistently higher inflammatory signature.

Key Findings & Study Highlights

  • Tissue panel (ddPCR): Tnf, Cxcl10, Mx1, Ccl2 up to ~15,000 copies/ng RNA
  • Serum panel (ECLIA): 8 cytokines in pg/mL — mRNA-A higher than mRNA-B for 7 of 8 analytes
  • Tissues: injection-site muscles, popliteal lymph nodes, spleen, liver
  • 24 h post-injection, 1 µg/muscle; cytokine induction confined to injection sites + draining lymph nodes

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