title: Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.
abstract: Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.
authors.affiliation: Chemistry Department, Oakland University, Rochester, Michigan 48309, USA.
authors.forename: Yuqin
authors.initials: Y
authors.lastname: Shang
authors.forename: Ray
authors.initials: R
authors.lastname: Mernaugh
authors.forename: Xiangqun
authors.initials: X
authors.lastname: Zeng
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis*; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D019151:Peptide Library; D058525:Quartz Crystal Microbalance Techniques; D011994:Recombinant Proteins / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D000068878:Trastuzumab