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Reactions Between Lipoids and Antibodies

II. The Isoelectric Point and Composition of Heterophile Lipoid-Antibody Aggregates

From the Department of Bacteriology, School of Medicine, University of Pennsylvania, Philadelphia

Abstract

The ether and acetone insoluble, alcohol soluble fraction of sheep red blood cells and of guinea pig kidney, fortified by the addition of cholesterol and corn germ sterol, forms a crystalline suspension on dilution with salt solution. The isoelectric point of the lipoid particles in such a suspension is approximately pH 1.9. After they are placed in normal amboceptor-free rabbit serum and washed free of excess protein, their isoelectric point is found to be changed to pH 2.8 to 3.5, presumably due to the adsorption of serum protein, incompletely removed by washing. When the particles are placed in serially increasing concentrations of an anti-sheep red blood cell serum and washed free of protein, their isoelectric point at first rises to a maximum of pH 4.2 to 4.9. Further increase in the antiserum: antigen ratio occasionally causes a cataphoretic prozone, with a shift in the isoelectric point of the washed particles back towards the acid zone, reaching as low as pH 2.2 and 2.6 in two experiments.

The isoelectric point of the antibody bound to heterophile lipoid is therefore at least pH 4.7 to 4.9, and may be even more alkaline.

When a suspension of the washed crystals is placed in antiserum, the aggregates which form are found to contain considerable quantities of a nitrogenous serum substance. Because the amount so bound depends primarily on the concentration of antiserum, even when the antiserum is diluted with normal serum, this nitrogenous substance is believed to represent largely the specific antibody. Assuming it to be a protein, it constitutes up to 9.3 per cent of the washed aggregates. This was the maximum value observed, and may represent maximally sensitized particles. It is interesting to note that spherical molecules 7 µµ in diameter deposited as a close-packed unimolecular layer around the antigen particles would constitute approximately 10 per cent of the aggregates.

The minimum amount of antibody necessary to cause the aggregation of 0.1 cc. of the lipoid suspension under the experimental conditions described in the text is estimated as 0.0015 mgm.; and an anti-sheep cell serum which can cause such aggregation up to 1:500 dilution is estimated to contain 75 mgm. of antibody to sheep cell or guinea pig kidney lipoid per 100 cc. It is estimated that only a small proportion, 1:140, of the surface of the antigen particles here studied need be covered with antibody protein in order to produce aggregation. It should be pointed out that, because of the assumptions discussed in the text, these figures represent orders of magnitude rather than precise values.