KINETICS AND LOCUS OF FAILURE OF RECEPTOR-LIGAND-MEDIATED ADHESION BETWEEN LATEX SPHERES .1. PROTEIN-CARBOHYDRATE BOND

We previously described the use of a counter-rotating cone and plate r heoscope to measure the time and force dependence of break-up of doubl ets of sphered, swollen, and fixed red cells (SSRC) cross-linked by mo noclonal IgM antibody. It has been shown that doublet break-up can occ ur by extraction of receptors from the membrane, rather than by antibo dy-antigen bond break-up, and is a stochastic process. We therefore pr epared 4.62-mu m carboxyl modified latex spheres with a covalently cou pled synthetic blood group B antigen trisaccharide. Using a two-step c arbodiimide process, ethylene diamine was covalently linked to the car boxyl modified latex spheres, and the trisaccharide, having an eight c arbon spacer modified to bear a terminal carboxyl group, was linked to the ethylene diamine, Using these antigen spheres we carried out stud ies in Couette flow, in a transparent cone and plate rheoscope, of the shear-induced break-up of doublets cross-linked by monoclonal IgM ant i-B antibody in 19% and 15% Dextran 40. As previously found with SSRC, over a range of normal force from 55 to 175 pN, there was a distribut ion in times to break-up. However, the fraction of antigen sphere doub lets broken up, which increased from 0.08 to 0.43 at 75 pM IgM, and fr om 0.06 to 0.20 at 150 pM IgM, was significantly lower than that for t he SSRC, where the fraction broken up at 150 pM IgM increased from 0.1 0 to 0.47. Thus, significantly higher forces were required to achieve the same degree of break-up for doublets of antigen-linked spheres tha n for SSRC. Computer simulation using a stochastic model of break-up s howed that the differences between antigen sphere and SSRC doublet bre ak-up were due to a change in bond character (the range and depth of t he bond energy minimum) rather than to an increase in the number of bo nds linking antigen-sphere doublets. This supports the notion that ant ibody-antigen bonds are ruptured in the case of antigen spheres, where as antigen is able to be extracted from the membrane of SSRC, although changes of receptor substrate from cell to latex and the possibility of latex strand extraction from the microspheres are potential complic ating factors.