1. Dilution of pooled plasma with more than an equal volume of saline solution destroys its ability to produce conglutination of red cells sensitized by univalent antibody. This can be correlated with Pedersen's work showing that X-protein is readily dissociated by dilution. The observation explains the discrepancy between the reports of British and American workers regarding the incidence of Rh "agglutinins" in the serum of Rh-negative mothers of erythroblastotic babies.
2. Plasma has a higher conglutinating activity than serum as shown by the finding that plasma gives titers on the average more than twice as high as those obtained with serum. The greater activity of plasma would seem due to the presence of fibrinogen which is apparently an important component of the colloidal complex of plasma proteins making up conglutinin.
3. Aside from its action in precipitating fibrinogen, heating at 56°C. for onehalf hour has no harmful effect on conglutinin.
4. Fetal plasma and serum yield much lower conglutination titers than adult plasma and serum, indicating that fetal blood is deficient in conglutinin. After birth, there is generally a marked increase in the conglutinin content of the blood. There is little or no variation in the conglutinin activity of sera from different normal adult individuals.
5. The use of whole citrated blood in exchange transfusion to an erythroblastotic baby caused an appreciable rise in the total plasma proteins after the transfusion and a corresponding increase in the conglutinating activity. When however, in another instance, two-fifths of the plasma was removed from the donor's blood and replaced with saline, there was no appreciable change in the protein concentration or conglutinin activity of the infant's plasma after the transfusion.
6. The fortification of pooled plasma by mixing 4 parts of it with 1 part of 25 per cent human albumin solution markedly increased its conglutinin content as shown by a fourfold increase in the conglutination titers obtained. Addition of less or more than this optimal amount of albumin resulted in lower titers. The 25 per cent human albumin solution itself yielded titers only half as high as did unmodified pooled plasma and was difficult to work with because of its high viscosity. Similar results were obtained in experiments with immune globulin solutions and pooled plasma.
7. Albumin solutions of less than 12.5 per cent concentration had little or no conglutinin activity; similarly, immune globulin solutions of less than 4.6 per cent concentration gave only relatively low titers when used as conglutinin. Yet, mixtures of these dilute solutions in certain optimal proportions yielded solutions with conglutinin activities considerably higher than that of pooled plasma. The albumin-globulin ratio in the mixtures giving the best results proved to be approximately the same as the albumin-globulin ratio of normal human serum or plasma.
8. Suitable mixtures of albumin and globulin solutions with a total protein concentration equal to that of normal plasma gave conglutination titers about four times as high as those obtained with unmodified pooled plasma. This suggests that there may be substances in normal plasma which tend to maintain the albumin and globulin in molecular dispersion. Another possibility is that in the fractionation process the albumin and globulin are rendered less hydrophilic, thus increasing their tendency to form colloidal aggregates.
9. The experiments described support the theory that clumping of cells by univalent antibodies in plasma media occurs in two stages, namely, (1) specific adsorption of univalent antibodies, and (2) non-specific adsorption of conglutinin by the sensitized cells causing them to stick together. The experiments further support the concept of conglutinin or X-protein as a colloidal aggregate of plasma proteins. Finally, they demonstrate that the intensity of the clumping (conglutination—not agglutination) depends on the quantity and quality of conglutinin and not merely on the total protein content of the medium of suspension.