Epitope mapping by phage display: random versus gene-fragment libraries.
We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.
Fack
F
F
Institute of Molecular Genetics, University Heidelberg, Germany.
Hügle-Dörr
B
B
Song
D
D
Queitsch
I
I
Petersen
G
G
Bautz
E K
EK
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D018063:Hantaan virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D017912:Inovirus / Q000235:genetics*; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology; D019151:Peptide Library; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D019151:Peptide Library; D014759:Viral Envelope Proteins; C090197:glycoprotein G2, Hantavirus