Temperature-dependent binding of monoclonal antibodies to C hordein.
The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.
Brett
Gary M
GM
of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
Mills
E N Clare
EN
Bacon
James
J
Wellner
Nikolaus
N
Husain
Rhonda D
RD
Tatham
Arthur S
AS
Shewry
Peter R
PR
Morgan
Michael R A
MR
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005983:Glutens; D001467:Hordeum; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D000911:Antibodies, Monoclonal; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D005983:Glutens