Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment

Abstract

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.

Development of Direct Competitive Enzyme-Linked Immunosorbent Assay for the Determination Cadmium Residue in Farm Produce

“…The cross-reactivity for several metal ions including Cd 2+ , Pb 2+ , Hg 2+ , Zn 2+ , Na + , Ca 2+ , Fe 3+ , Mg 2+ , Mn 2+ , Cu 2+ , Al 3+ , Co 2+ , Cr 2+ , Ni 2+ , Sn 2+ , or K + was tested using the DC-ELISA [15,16]. Metal ions with different concentrations which ranged from 0.01 to 10 5 μg/L were mixed with 5 mM EDTA overnight to form hapten metal chelate.…”

A Potent Anti-HB-EGF Monoclonal Antibody Inhibits Cancer Cell Proliferation and Multiple Angiogenic Activities of HB-EGF

“…2). In addition, the resulting Hill coefficient (n  =  1.68) was greater than 1 which also indicated positive cooperativity (n  =  1 signifies independent binding) [39].…”

“…This steeper slope can only be explained by use of a positive cooperativity model. In the positive cooperativity model, the binding of HB-EGF to one monoclonal antibody binding site causes the affinity of the second binding site of the antibody to increase [39]. Hence, a positive cooperativity equilibrium model was used to fit the dual-curve titration data which provided an improved fit to the dual curve data set, yielding an effective K D  =  1.50 pM (Fig.…”

Websites of note

“…A number of studies have shown that it is possible for IgG antibodies to display cooperativity in binding to antigens that enhances their interaction affinity 46–48. Monoclonal antibodies have also been shown to exhibit allosteric binding phenomena in the presence of appropriate binding partners and solution conditions 49…”