Distribution of “Blocking” Antibody in Human Serum Proteins Fractionated by Electrophoresis-Convection
Authors: Loveless Mary Hewitt , Cann John R. 
Published: January 2023

Summary and Discussion
Sera procured from a pollen-immunized and from an insulin-refractory donor have been fractionated extensively by EC and examined for blocking activity as well as for electrophoretic composition. EC, which segregates molecules chiefly according to their electric charge, provided fractions which were comprised largely of proteins of low, intermediate or high mobility. One preparation, for example, consisted 96% of slow-moving or γ2-globulin, whereas another contained 75% of fast-moving γ1-globulin, and a third possessed 58% of β-globulin, while still another was comprised 90% of α-globulins. The remainder of the serum could be salted out so as to provide a final fraction which contained as much as 92% of albumin (see Table II). The foregoing components differed from each other in that they exhibited graduated mobility rates when examined at pH 8.1, the γ2-globulins migrating most slowly while the albumin moved most rapidly.
When such fractions were mixed in vitro with their respective allergens, a slight excess of the latter could be detected by testing the mixture in sensitized, normal skin. The amount that had to be added to one fraction to be slightly in excess of its blocking power could be compared with the amount required for other fractions, thus indicating their relative antibody content. When blocking power was expressed in terms of the total protein content of the preparation, it was found that activity of successive fractions fluctuated roughly with their γ-globulin concentration whereas no positive correlation with immunologic properties could be demonstrated for other serum constituents. In the first blood taken from the insulin-refractory donor, the γ-globulin of lowest (Top I) and highest mobility (Tops Va, Vb, and VI) were the chief participants in the blocking function, whereas all γ-globulins appeared to be similarly involved in a later specimen. The activity curve of fractions prepared from the pollen-immune donor, K. U., resembled that of the early insulin-resistant sample.
The blocking activity of the 10 K. U. fractions did not duplicate relationships found among these preparations when their sensitizing antibodies were titrated by the technique of serum dilution (3). Although there was admittedly an area of overlap in the region of Tops Va and Vb, where maximal blocking and sensitizing activity coexisted, there was distinct blocking activity to the left of this area on the activity curve (notably in the Top Fraction, I), whereas important reaginic qualities were encountered to the right of this peak (Tops VI and VII). At the same time it was noted, in examining the electrophoretic data, that over 90% of the area of fractions to the left was made up of slow-moving γ2-globulin, while the β-globulin-enriched preparations lay in the central and right-hand regions of the curve (Tops Va, Vb, VI and VII). Fast-moving γ1-globulins, on the other hand, were concentrated largely in Tops Va and Vb and to a lesser extent in VI. This concentration of both the β-globulins and the γ1-globulins in Fractions Va, Vb and VI made it difficult, at first glance, to decide whether the two types of antibody were related to one or both of these serum proteins. Investigation of the area and activity data for the remaining fractions, however, made it clear that the β-globulins were concerned with sensitizing antibody whereas γ-globulins carried blocking qualities. It was not surprising, therefore, that fractions rich in both these components should also be efficient in both of these antibody functions.
As might have been expected from the fact that Fractions Va, Vb and VI had been immobilized and isolated at pH 6 and 5.5, the electrophoretic mobilities of their β-globulin and γ1-globulin molecules differed from each other only slightly, so that the complete separation of these two types of globulin poses a difficult technical problem. We have recently attempted to accomplish this by rerunning Fraction Vb of a reaginic serum at a slightly different pH level and were rewarded by the finding that sensitizing antibody and β-globulin content had both been concentrated in Top Vc (4). Similar efforts with blocking serum have not as yet been made.
The generally lower mobility of the blocking antibody justifies the conclusion that it is an entity distinct from the sensitizing antibody, the latter being associated chiefly with molecules of greater mobility. That some overlapping of the 2 activities occurred in fractions of intermediate mobility, such as Tops Va and Vb, is not surprising. A similar situation was encountered by Kuhns when he subjected human antitoxic sera to zone electrophoresis on a starch column. Whereas the precipitating antibodies migrated in association with a slow-moving component, the skin-sensitizing bodies moved in general more rapidly, with some overlapping at the interface.
The clinical behavior of our insulin-resistant patient, as well as the outcome of mouse-protection tests, made it apparent that she possessed some means of interfering with the hypoglycemic effect of insulin. Blocking studies showed that her serum carried antibodies which prevented insulin from reacting with sensitized skin. Whether one and the same mechanism accounted for both the hormonal refractoriness and the antiallergenic power of her serum can be only surmised. Although such antibodies might well have engendered the original refractoriness, it is also theoretically possible that some unrelated mechanism was initially involved and that antibody production was only secondary to the unusual amounts of insulin given in an effort to overcome the refractoriness. In favor of the former hypothesis is the fact that the patient's blocking power per ml of serum was more than adequate to account for the large doses she was required to take daily. Whether, in this case, the interference with sugar metabolism was due to a direct union of antibody with the hormonally active groupings of the insulin molecule or was dependent upon complexing with other, nearby groupings which led to steric hindrance was not subject to experimental test. In the rabbit, the latter situation appears to be productive of insulin resistance according to Lowell (11).
The tendency for B. E. R.-resistant serum to delay rather than prevent hypoglycemic death in the mouse suggests steric hindrance, although the formation of unstable antigen:antibody complexes might be an alternate explanation. The intermittent hypoglycemic shock experienced by the patient, at times many hours after her latest dose of insulin, might rest on instability of the hypothetical antigen:antibody complex, or on the release of insulin from some postulated reservoir.
The observation that blocking end-points with B. E. R. fractions were the same for unheated as for heated samples rules out the hypothesis that the blocking phenomenon is dependent upon heat-denatured reagin rather than on a separate, blocking antibody.
The use of serum:antigen mixtures for direct blocking tests in sensitized skin provided, in our experience, a more sensitive and practical means of measuring anti-insulin factor than the reagin-neutralization test with heated serum or the Lowell mouse-protection test—assuming that the same factor is being measured by all three procedures.