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Published on: March 25, 2014
A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents
Brendan K Reed1, Laura B Chopp1, Courtney S Malo1
1Department of Immunology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States of America.
Insights
Researchers developed a new method to check the structural integrity of MHC:peptide multimers. This quality control assay ensures accurate visualization of T cell responses, especially with limited or irreplaceable samples.
Area of Science:
- Immunology and Molecular Biology
- Biotechnology and Reagent Development
Background:
- Major Histocompatibility Complex (MHC):peptide multimers are essential tools for visualizing antigen-specific T cell responses.
- Assessing the structural integrity of MHC multimer reagents is crucial before use, particularly with limited or irreplaceable samples, but robust controls are often unavailable.
Purpose of the Study:
- To develop and validate a method for probing the structural integrity of MHC multimers.
- To enable quality control of MHC multimer reagents prior to their use in critical immunological studies.
Main Methods:
- A capture assay was designed using beads coated with anti-mouse immunoglobulin (Ig).
- These beads were incubated with conformation-specific mouse monoclonal antibodies, followed by fluorescently tagged MHC multimers.
- The capture efficiency of labeled multimers by the beads was measured semi-quantitatively using flow cytometry.
Main Results:
- The developed method allows for the visualization of correct MHC multimer folding.
- It enables semi-quantitative comparison of different batches of MHC multimers for quality control.
- The assay can assess the fidelity of MHC multimer structure, including interactions between heavy chain, peptide, and β2M, depending on antibody availability.
Conclusions:
- This novel capture assay provides a reliable method to assess MHC multimer structural integrity.
- It is particularly valuable for studies involving precious or irreplaceable samples, such as patient samples in clinical trials.
- The approach enhances the reliability of T cell response visualization using MHC:peptide multimers.
Abstract:
Antigen-specific T cell responses can be visualized using MHC:peptide multimers. In cases where robust T cell controls are not readily available to assess the integrity of multimer reagents prior to analyzing limited sample, the ability to assess the structural integrity of MHC multimers before their use in critical experiments would be useful. We present a method to probe the structural integrity of MHC multimers using antibodies specific for conformational determinants. Beads coated with anti-mouse Ig are incubated with conformation-specific mouse monoclonal antibody and then with fluorescently tagged MHC multimer. The ability of the bead to capture the labeled multimer can be measured semi-quantitatively by flow cytometry. In this manner, the correct folding of MHC multimers can be visualized and batches of multimer can be compared for quality control. Because there are multiple conformational epitopes formed by various molecular interactions among heavy chain, peptide, and β2M, this capture assay can assess the fidelity of each aspect of multimer structure, depending on the availability of antibodies. The described approach could be particularly useful for studies using irreplaceable samples, including patient samples collected in clinical trials.
