TY - JOUR
T1 - Enhancement of ELISAs for screening peptides in epitope phage display libraries
AU - Valadon, Philippe
AU - Scharff, Matthew D.
N1 - Funding Information:
We thank Drs. David Beenhouwer and Nancy Green for editorial assistance.T his study was supported by grants from the National Institutes of Health (CA39838 and POlAI33184 (M.D.S.)). and the Harry Eagle Chair for Cancer Researchf rom the National Women’s Division of the Albert Einstein College of Medicine (M.D.S.). P.V. was partially supportedb y the Philippe Foundation.
PY - 1996/10/16
Y1 - 1996/10/16
N2 - The complete analysis of epitope phage display libraries requires sensitive assays capable of detecting peptides expressed on phage that have a wide range of affinities for antibody. We have compared two ELISAs, a 'direct' assay where the phage is captured by an anti-phage antibody and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibody. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 μM, above which phage clones are negative by the direct ELISA. We describe an enhancement of the direct assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-light chain antibody. The resulting polymerization of 2H1 induces an increase in antibody avidity that is responsible for the enhancement. The enhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELISA is not. The enhanced ELISA has also been used successfully for immunological screening of intermediate libraries, allowing detection of rare positive clones that would otherwise be lost. The combination of the three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synthesis and testing of many peptides.
AB - The complete analysis of epitope phage display libraries requires sensitive assays capable of detecting peptides expressed on phage that have a wide range of affinities for antibody. We have compared two ELISAs, a 'direct' assay where the phage is captured by an anti-phage antibody and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibody. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 μM, above which phage clones are negative by the direct ELISA. We describe an enhancement of the direct assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-light chain antibody. The resulting polymerization of 2H1 induces an increase in antibody avidity that is responsible for the enhancement. The enhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELISA is not. The enhanced ELISA has also been used successfully for immunological screening of intermediate libraries, allowing detection of rare positive clones that would otherwise be lost. The combination of the three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synthesis and testing of many peptides.
KW - ELISA
KW - immunological screening
KW - peptide library
KW - phage display
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U2 - 10.1016/0022-1759(96)00133-0
DO - 10.1016/0022-1759(96)00133-0
M3 - Article
C2 - 8890904
AN - SCOPUS:0030590643
SN - 0022-1759
VL - 197
SP - 171
EP - 179
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -