Autoimmunity Profiling

National facility

PPA_for_web

The Autoimmunity Profiling facility provides technologies and infrastructure for autoantibody profiling and antibody validation using large scale and customisable protein arrays. The protein array analysis is based on the resource of more than 42 000 human protein fragments generated within the Human Protein Atlas, representing more than 19 000 human protein coding genes. Two array formats are utilized, planar microarrays with either 384 protein fragments in 20 subarrays or one array with 42 100 protein fragments, secondly are also suspension bead arrays utilized for targeted antigen arrays and validation.  The facility also provides access to in situ synthesized ultra-high-density peptide microarrays, including a proteome-wide microarray with up to three million designed peptides covering all human protein encoding genes. Instrumentation is available for the measurement and analysis of commercially available protein microarrays and also for generating various customized spotted protein microarrays such as reverse phase arrays. The facility recently printed the world's largest protein array.

SERVICES

  • Autoantibody profiling
  • Epitope mapping
  • Antibody validation
  • Infrastructure for commercial protein arrays

 

Proposed projects are assessed and prioritized according to the following model.

1) Initial assessment by facility:

  • Technical feasibility and suitability
  • Required capacity and resources for the project

 

2) Project prioritization by committee:

  • Scientific potential
  • Supporting preliminary data
  • Significance of facility specific technique for project
  • Supports facility development (competence and techniques)

EQUIPMENT

  • Access to affinity reagents and high density arrays.
    • 42 100 mass spectrometry validated antigens (protein fragments), the Human Protein Atlas project.
    • High density antigen array containing 42 100 protein fragments from the Human Protein Atlas
    • Ultra-high-density peptide microarrays from Roche NimbleGen, specifically designed and containing 2.9M peptides in one array or 173 000 peptides in 12 replicate subarrays.
  • Liquid-handling robots. Two TECAN EVO150 (shared with Biobank Profiling and Cell Profiling Facilities)
  • Microarray printer. Arrayjet Marathon
  • Microarray scanners. Agilent G256BA, CapitalBio LuxScan HT24 and Roche NimbleGen Inc. MS200

APPLICATIONS

  • Profiling the autoimmunity repertoire in plasma, serum or CSF by screening on microarrays of randomly selected antigens. Usually done in the range of the lower hundreds of samples and lower thousands of antigens.
  • Verification of autoantibody profiles on suspension bead arrays with selected antigens. Usually in the range of lower hundreds of antigens and higher hundreds of samples.
  • Epitope mapping and off-target analysis of affinity reagents as well as identified autoantibodies on high density antigen or peptide microarrays.
  • Infrastructure and know-how to analyze commercial protein arrays such as ProtoArray (ThermoFisher), HuProt (Cambridge Protein Arrays), Immunome (Sengenics), and peptide arrays from Nimblegen Roche, Pepperprint and JPT.

 

RECENT PROJECTS

  • Autoantibody screening using planar protein arrays based on Human Protein Atlas protein fragments, with subsequent verification using suspension bead arrays in a larger patient sample material.
  • Epitope mapping of a selection of antibodies using peptide arrays containing 175 000 peptides covering hundreds of proteins.
  • Using assay infrastructure for autoantibody profiling with commercially available protein arrays
  • Verification of monoclonal antibody target interaction using protein arrays containing Human Protein Atlas protein fragments

 

Selected and recent publications using the facility: