Identification of mimotope peptides which bind to the mycotoxin deoxynivalenol-specific monoclonal antibody.
Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.
Yuan
Q
Q
Departments of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824, USA. hartL@pilot.msu.edu
Pestka
J J
JJ
Hespenheide
B M
BM
Kuhn
L A
LA
Linz
J E
JE
Hart
L P
LP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014255:Trichothecenes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D009842:Oligopeptides; D019151:Peptide Library; D014255:Trichothecenes; C007262:deoxynivalenol