Establishment of a quantitative ELISA capable of determining peptide - MHC class I interaction.
Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.
Sylvester-Hvid
C
C
University of Copenhagen, Denmark.
Kristensen
N
N
Blicher
T
T
Ferré
H
H
Lauemøller
S L
SL
Wolf
X A
XA
Lamberth
K
K
Nissen
M H
MH
Pedersen
L Ø
LØ
Buus
S
S
D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D020673:Protein Renaturation; D012680:Sensitivity and Specificity; D001613:beta 2-Microglobulin
D002021:Buffers; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D001613:beta 2-Microglobulin