Selective Precipitation of Antibody with Ligand‐Modified Phospholipids: Effect of Lipid Chain Length

Abstract

AbstractThe selective precipitation from aqueous solutions of goat polyclonal anti‐biotin antibody (pABA) by complexation with ligand‐modified phospholipids (LMPs) is described. In this study, the effect of varying the acyl chain length of the LMP from six to 18 carbon atoms on the rate and yield of precipitation is detailed. As the acyl chain length increases, the hydrophobic driving force for interaction of ligand‐bound antibody molecules also increases, resulting in a larger yield of precipitated antibody. The rate of selective precipitation, however, is observed to pass through a sharp maximum at an acyl chain length of 10–12 carbon atoms. In the range of target antibody and LMP concentrations studied (1–10 μM), the maximum rates of precipitation are observed for those LMPs in sufficiently low concentrations in aqueous solution to be below their critical micelle concentration (CMC) . The longer chain length LMPs (12–18 carbon atoms at concentrations of 5–10 μM) gave considerably slower rates of precipitation and were all observed to be micellar solutions. The yield of target antibody as a percentage of antibody precipitated was not observed to pass through a maximum, rather all LMPs with acyl chain lengths longer than 12 carbon atoms were observed to give the maximum yield. Thus the optimal structure of an LMP for precipitation of a target antibody corresponds to the maximum chain length (10 carbon atoms) at a concentration level (5–10 μM) which still falls below its CMC. The kinetics of precipitation, as monitored by measuring turbidity, are well modelled by a theory which combines the Mie theory of light scattering with the Smoluchowski theory for the kinetics of precipitation. The maximum rate constants corresponding to Smoluchowski kinetics for precipitating pABA were approximately 25 000–30 000 M−1 s−1, while the maximum yields were 65–70%. The molecular picture which emerges is one in which the optimal rate is obtained by maximizing hydrophobic driving force for interaction of LMP acyl chains while still maintaining a submicellar state of aggregation.