A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents

Brendan K. Reed, Laura B. Chopp, Courtney S. Malo, Danielle N. Renner, Virginia S. Van Keulen, Megan A. Girtman, Wendy N. Nevala, Kevin D. Pavelko, Diana Gil, Adam G. Schrum, Aaron J. Johnson, Larry R Pease

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 β2 M, 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.

Original languageEnglish (US)
Article numbere0137984
JournalPLoS One
Volume10
Issue number9
DOIs
StatePublished - Sep 21 2015

Fingerprint

T-cells
Structural integrity
Assays
T-Lymphocytes
Peptides
Molecular interactions
Flow cytometry
Antibodies
assays
T-lymphocytes
Quality Control
peptides
Quality control
Conformations
Epitopes
Flow Cytometry
antibodies
Monoclonal Antibodies
mice
Clinical Trials

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Reed, B. K., Chopp, L. B., Malo, C. S., Renner, D. N., Van Keulen, V. S., Girtman, M. A., ... Pease, L. R. (2015). A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents. PLoS One, 10(9), [e0137984]. https://doi.org/10.1371/journal.pone.0137984

A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents. / Reed, Brendan K.; Chopp, Laura B.; Malo, Courtney S.; Renner, Danielle N.; Van Keulen, Virginia S.; Girtman, Megan A.; Nevala, Wendy N.; Pavelko, Kevin D.; Gil, Diana; Schrum, Adam G.; Johnson, Aaron J.; Pease, Larry R.

In: PLoS One, Vol. 10, No. 9, e0137984, 21.09.2015.

Research output: Contribution to journalArticle

Reed, BK, Chopp, LB, Malo, CS, Renner, DN, Van Keulen, VS, Girtman, MA, Nevala, WN, Pavelko, KD, Gil, D, Schrum, AG, Johnson, AJ & Pease, LR 2015, 'A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents', PLoS One, vol. 10, no. 9, e0137984. https://doi.org/10.1371/journal.pone.0137984
Reed BK, Chopp LB, Malo CS, Renner DN, Van Keulen VS, Girtman MA et al. A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents. PLoS One. 2015 Sep 21;10(9). e0137984. https://doi.org/10.1371/journal.pone.0137984
Reed, Brendan K. ; Chopp, Laura B. ; Malo, Courtney S. ; Renner, Danielle N. ; Van Keulen, Virginia S. ; Girtman, Megan A. ; Nevala, Wendy N. ; Pavelko, Kevin D. ; Gil, Diana ; Schrum, Adam G. ; Johnson, Aaron J. ; Pease, Larry R. / A versatile simple capture assay for assessing the structural integrity of MHC multimer reagents. In: PLoS One. 2015 ; Vol. 10, No. 9.
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