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.
University of Copenhagen, Denmark.
C
C
Sylvester-Hvid
N
N
Kristensen
T
T
Blicher
H
H
Ferré
S L
SL
Lauemøller
X A
XA
Wolf
K
K
Lamberth
M H
MH
Nissen
L Ø
LØ
Pedersen
S
S
Buus
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