Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: The effect of hapten density

Tinglu Yang, Olga K. Baryshnikova, Hanbin Mao, Matthew A. Holden, Paul S. Cremer

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Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, KDD1, was 2.46 × 10-5 M, while the second was KD2 = 1.37 × 10-8 mol/m2. This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 ± 2.7 J/(mol·K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.

Original languageEnglish (US)
Pages (from-to)4779-4784
Number of pages6
JournalJournal of the American Chemical Society
Issue number16
StatePublished - Apr 23 2003

All Science Journal Classification (ASJC) codes

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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