Analysis of antibody populations to oligo‐alanine determinants. I. An antibody assay by one‐hit sensitization of red cells

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Abstract
A sensitive method for the quantitation of mouse antibodies to oligo‐D‐alanine determinants is described. Receptor molecules carrying clusters of determinants are attached to the surface of sheep red blood cells. The receptor molecules are poly‐alanylated rabbit antibodies (IgG) directed to SRBC. The formation of a complex between cell‐bound receptor molecules and antibodies to oligo‐D‐alanine can act as a center of sensitization, leading to cell lysis by complement.The kinetics of sensitization of receptor‐equipped SRBC by antibodies to oligo‐D‐alanine was studied. Antibodies of the IgM class sensitized SRBC according to one‐hit kinetics. Single‐hit sensitization by antibodies of the IgG (7 S) class also occurred, as shown by dose‐response curves with receptor as well as with antibody, provided that antibodies are complexed with anti‐allotype antibodies subsequent to their attachment (facilitation). Facilitation thus not only serves to detect sensitively the attachment of 7 S antibodies, but also reveals the phenotype (allotype) of the constant portions of their H‐chains.The velocity of sensitization was proportional both to the antibody concentration and to the receptor density on cells. A velocity constant was defined, and compared to the probability of collision between antibodies and receptors, derived from the diffusion constant of IgG molecules. It appears that, with some antisera, as much as every second collision may lead to a successful union between antibody and receptor molecules.