Single radial immunodiffusion has been adapted to the quantitation of low molecular haptens, making use of the principle of hapten inhibition of precipitation. As model haptens, isomaltose oligosaccharides were quantitatively estimated in a precipitating antidextran protein-dextran conjugate system. The antidextrans had been raised in sheep by immunization with a bovine serum albumin-dextran conjugate. After preparation of the agar plates containing the antidextran, the wells were filled with the haptenic oligosaccharide solutions. The latter were allowed to diffuse into the agar and to bind to the antidextran for 30, 120, or 300 min, and then the hapten inhibition halos were visualized or “developed” by immersing the plates in a solution of an ovalbumin-dextran conjugate for 24 hr. Thereafter, the diameters of the transparent hapten inhibition halos, contrasting against the remaining opaque parts of the plate, were measured. A linear relationship between area of hapten inhibition and concentration of individual isomaltose oligosaccharides was obtained, when sufficient time for complete hapten diffusion was allowed before visualizing hapten inhibition zones. In the examples studied, the smallest amount of oligosaccharide hapten measurable was about 10 μg/ml. The potential application of this procedure for quantitation of other low molecular monovalent haptens is discussed.
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