Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies.
Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267, USA. William.Ball@uc.edu
W J
WJ
Ball
Z
Z
Wang
B
B
Malik
R
R
Kasturi
P
P
Dey
M K
MK
Short
M N
MN
Margolies
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001435:Bacteriophages / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D036022:Capsid Proteins; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004077:Digoxin / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; D010455:Peptides; D014760:Viral Fusion Proteins; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase