Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. II. Protein-protein bond

Abstract

In an extension of the previous paper, we describe the force dependence of break-up of doublets of latex spheres cross-linked by protein G-IgG bonds via the Fc region of the antibody. The receptor, the monoclonal Bear-1 antibody, was either covalently linked to 4.75-microns aldehyde/sulfate (A/S) latex spheres in a one-step reaction, or physically adsorbed to the 4.63-microns carboxyl-modified latex spheres used in Part I of this paper. The spheres were suspended in 19% buffered Dextran 40 containing the ligand, the bivalent recombinant protein G (Gamma-Bind G), and observed in the counter-rotating cone and plate Rheoscope. Break-up of doublets, tracked individually under the microscope, as well as in populations of 50-150 particles, was studied over a range of normal force from 20 to 260 pN. In individual particle studies, the fraction of doublets of spheres with covalently linked IgG breaking up in the first 10 rotations, increased from 16% in the low-force to 63% in the high-force range. In population studies, the fraction broken up increased with duration and magnitude of the applied force, and decreased with increasing ligand concentration. Moreover, doublets of physically adsorbed IgG spheres required significantly lower force than doublets of covalently linked IgG spheres for the same degree of break-up, possibly because of surface detachment of IgG molecules rather than rupture of receptor-ligand bonds. Computer simulation, using the Bell stochastic model of break-up and a Poisson distribution for the number of bonds, described in Part I, showed that the parameters of the protein-protein bond differed significantly from those of the carbohydrate-protein bond studied in Part I of this paper, the former being much more responsive to force than the latter.

Dissecting Individual Ligand–receptor Bonds With a Laminar Flow Chamber

“…Ligand-receptor couple [38] Clearly, zero-force dissociation rates displayed a wide range (a thousandfold) of values, and there was no obvious correlation between k of f and k B t/d. Now, while Bell's model was considered to account satisfactorily for experimental results found in many studies [39], additional complexity was soon disclosed.…”

Modeling of Cell Aggregation Dynamics Governed by Receptor–Ligand Binding Under Shear Flow

“…For experimental measurements of shear-induced cell aggregation and disaggregation performed in a cone-plate viscometer, the time course of percentage of cell aggregation was determined either from microscopic observations directly 21,48,49 or by a twocolor flow cytometry technique 10,17,18,24,28,31,32,47,51 : % aggregation = (number of cells/beads in aggregates)/ (total number of cells/beads in suspension) 9 100. The resulted data were compared with the adhesion fraction P a t ð Þ Â 100 ¼ 1 À p 0 t ð Þ ½ Â100 predicted from the above model and the intrinsic kinetic parameters of interacting molecules are able to be determined from the best-fit of the data.…”

“…21,48,49 The limitations of the previous model lie in: (1) doublet breakage at low shear rate and the subsequent doublet formation at high shear rate were neglected; (2) contact duration between two singlets might be over-estimated by keeping the singlets in contact all the time; and (3) the model, even simplified, was still complicated to the exact process described above (i.e., low shear induces doublet formation first and then high shear enforces doublet breakage). Here we first validated the current model by fitting it to the same data set of latex beads cross-linked by protein G-IgG bonds 21 and then compared the predictions with those previously described. 27 …”

“…2b was in excellent agreement with the measurement that it takes~30 min to reach a~20% aggregation percentage. 21 …”

“…In previous studies of doublet break-up under shear flow, 21,48,49 bond number at the end moment of low shear was assumed to follow a Poisson distribution. 4 Noting that the kinetic theory employed was initially developed for a cell centrifugation assay and further observed in a micropipette adhesion frequency assay where cells were kept in contact all the time, 5 this should be a distinct case for many published results derived from a cone-plate viscometer assay, in which the two cells/beads are brought into transient contact to allow bond formation only during the interval of two-body collision.…”