Immune Response and Biomarkers

The Immune Response and Biomarkers Facility provides advanced quantification of a wide range of biomarkers across diverse biospecimens, using technologies such as ELISA, xMAP Luminex and targeted proteomics with OLINK (Certified International Core Lab).

The biomarkers analysed are primarily proteins, including antibodies of different isotypes and subclasses against customised antigen panels, as well as other immune-related analytes such as cytokines, chemokines and growth factors. In addition, biomarkers associated with other physiological pathways, including metabolic, neurological and cardiovascular processes, can also be assessed.

Key strengths of the facility include:

• Multiplexing capacity, enabling multiparametric data generation from single samples (Luminex and OLINK).
• Miniaturisation, suitable for studies with limited sample volumes (e.g. paediatric cohorts).
• Versatility and adaptability (customized needs, kits can be commercial or produced in-house).
• High-throughput capacity through semiautomated liquid handling systems.
• Functional antibody assays (e.g. binding inhibition and surrogate neutralisation).
• Complementarity of technologies, allowing cross-validation of biomarkers (e.g. Luminex vs OLINK for cytokines and other biomarkers).

Custom panels are developed using recombinant proteins (commercial or produced in-house in prokaryotic or eukaryotic systems) and monoclonal antibodies (mAbs) for customised applications. These enable in-depth immune phenotyping, using spectral cytometry, and the analysis of T-cell specific responses through intracellular cytokine staining (ICS) and T-cell activation-induced marker (AIM) assays by flow cytometry and ELIspot/FluoroSpot assays.

The following specific knowledge and capacities are offered as added values:

• Extensive expertise and experience in immune monitoring.
• A long track record of the team.
• Built-in QA/QC and data preprocessing R pipelines.
• The possibility to perform statistical analyses, consulting, training, and capacity building for LMIC laboratory personnel.

Services

• ELISA and colorimetric assays using commercial kits or developed in-house to measure concentrations of different analytes.
• Multiplex cytokine Luminex assays using commercial kits to measure concentrations of different proteins like cytokines, chemokines, growth factors and other, or gene expression by Invitrogen™ QuantiGene™ Plex panels.
• Multiplex antibody Luminex assays developed by our group to measure levels and avidity of IgM, IgA, IgA1, IgA2, IgG, IgG1, IgG2, IgG3, IgG4, IgE against flexible panels of antigens from different microbes.
• Multiplex functional antibody Luminex assays.
• Olink assays using commercial kits to measure levels of up to 92 biomarkers in multiplex format (T96, T48 and 21-Flex) (Certified International CoreLab).
• Other cellular assays (T cells, B cells) upon request (flow cytometry, ELIspot/FluoroSpot) For these technologies we offer:
  • Advice on what markers, technologies (ELISA, Luminex, Olink) and kits to use depending on the objective and specificities of the study.
  • Design of experiments.
  • Supplier contacts and preparation of quotations.
  • Purchase of reagents and kits.
  • Performance of the assays.
  • QA/QC of the data generated.
  • Additionally: Flow cytometry and ELIspot/FluoroSpot (sample acquisition/reading using IDIBAPS platform instruments).

Equipment

• CytoFLEX (Beckman Coulter)
• Access to the PCB flow cytometry unit (Cytek Aurora; 4 lasers, spectral)
• Access to the PCB ELISA microplate reader (TECAN Infinite® M Plex microplate)
• Two automatic washers (BioTek)
• Luminex instruments: one Magpix, one Luminex 100/200 and two FlexMap3D
• Accessories for the Luminex assays:
  • Kingfi sher Flex 96 deepwell (Thermo Fisher) for automated antigen coupling
  • Integra Assist Plus and Integra Viafl o 384 (Integra) for liquid handling in 384 well plates
  • Two 96 well plate orbital shakers (Thermo Fisher)
  • Two 384 well plate orbital shakers (Heidolph Titramax)
• Guava easyCyte for automatic bead and cell counting
• OLINK Signature Q100
• Accessories for the OLINK assays:
  • Signature Q100 Interface plate: 96.96, 48.48 and 24.192
  • Centrifuge for plates (Eppendorf (5430/5430R)
  • Microcentrifuge for strips and tubes (IKA Mini G Centrifuge, Fisher Scientific)
  • Controlled plate vortex (MaxMate Eppendorf 230V Version)
  • Tube holder for controlled plate vortex (MixMate PCR 96 tube holder, Eppendorf)
  • Single and multichannel precision pipettes (Rainin, Mettler Toledo)
  • Freezing block (-20ºC) for enzyme handling
  • PCR instrument (SimpliAMP Thermal cycler, Applied Biosystems)
• Softwares for running ELISA (GraphPad Prism), Luminex (X-ponent 4.3), Olink (NPX Signature software), and fl ow cytometry (FlowJo)
• Ella™ automated ELISA (Bio-Techne)

This core facility is part of the CERCAGINYS platform.