Two antipeptide monoclonal antibodies that recognize adhesive sequences in fibrinogen: identification of antigenic determinants and unrelated sequences using synthetic combinatorial libraries.
The fine specificity of two different monoclonal antibodies raised against synthetic peptides, each representing one of the two Arg-Gly-Asp (RGD) sequences in fibrinogen, was examined using synthetic combinatorial libraries (SCLs). The monoclonal antibodies (mAb), mAb LJ-134B/29 and mAb LJ-155B/16, recognize both the immunogenic peptide and native fibrinogen. The specificity of mAb LJ-134B29 was mapped using hexa- and decapeptide positional scanning SCLs (PS-SCLs) and competitive ELISA. The most active amino acids at each position of the two libraries were identified from a single screening. Individual hexa- and decapeptides were synthesized and assayed to determine their binding affinities. The 16 individual hexapeptides represented single and multiple substitutions of the antigenic determinant sequence, -GDSTFE-, eight of which had affinities less than 10nM. Four of the twelve individual decapeptides were found to have binding affinities of approximately 300nM, or nearly three-fold less than the peptide immunogen. A dual-defined hexapeptide library was screened against mAb LJ-155B/16, and individual peptides were obtained through an iterative selection and synthesis process. Surprisingly, one of the most active sequences was Ac-WWYESW-NH2 (IC50 = 40nM), which showed no similarity to the sequence of the immunizing peptide. Further mapping of the specificity of this antibody revealed that the antigenic determinant within the peptide immunogen was not completely linear. Recognition of this unrelated sequence by mAb LJ-155B/16 was confirmed in a direct binding assay using biotinylated peptide. The use of SCLs for the elucidation of high affinity peptides recognized by these two antibodies may provide additional information on the molecular mechanisms of fibrinogen binding to different integrin receptors.
Pinilla
C
C
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA.
Buencamino
J
J
Appel
J R
JR
Houghten
R A
RA
Brassard
J A
JA
Ruggeri
Z M
ZM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010449:Peptide Mapping
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D005340:Fibrinogen