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The Journal of Immunology, 1959, 82, 290 -297
Copyright © 1959 by The American Association of Immunologists, Inc.

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Immune Hemolysis at 0°

I. Introduction: The Role of Amboceptor and Complement1,2,

David L. McVickar and Gertrude R. Weinfeld

From the Department of Infectious Diseases, Division of Mycology, School of Medicine, University of California, Los Angeles

Abstract

1. Immune hemolysis at 0° takes place when the concentration of amboceptor used to sensitize erythrocytes is greater than optimal (optimal as determined by the usual amboceptor titration at 37°.)
2. In the presence of an excess of C', the following relationships between amboceptor concentration and maximal velocity of hemolysis hold: a) below optimal concentration the maximal velocity of hemolysis falls precipitously to zero; b) from optimal to 10 x optimal amboceptor concentration the maximal velocity of hemolysis is directly proportional to amboceptor concentration; c) the maximal velocity does not increase further when amboceptor concentration is increased beyond about 10 x optimal.
3. The curve relating time required for 50% hemolysis to amboceptor concentration is a hyperbolic one, with asymptotic values of approximately 40–50 min and 0.6–0.8 optimal amboceptor concentration, respectively.
4. A 100-fold increase in C' concentration (from 1:200 to 1:2) will not cause appreciable hemolysis of erythrocytes sensitized at amboceptor concentrations lower than 0.6–0.8 optimal. With erythrocytes sensitized at 10 times optimal amboceptor concentration, the following relationships between C' concentration and maximal velocity of hemolysis hold: a) as C' concentration is increased from zero to about 1:200, a gradual increase in the maximal velocity occurs, not proportional to the C' concentration; b) from C' concentrations of about 1:200 to 1:20, the maximal velocity of hemolysis is a linear function of the C' concentration; c) the maximal velocity does not increase further when C' concentration is increased beyond about 1:20.
5. The curve relating the time required for 50% hemolysis to C' concentration is hyperbolic, with an asymptotic value of about 40–50 min for the former, and close to zero for the latter.
6. The significance of these results is discussed with particular reference to a) the possibility that the requirement for increased amboceptor concentration reflects the fact that two (or more) amboceptor sites, each with its quota of C'1,4,2, must cooperate to initiate the terminal step through reaction with C'3; and b) the suggestion that the inability of increased concentrations of amboceptor and C' to increase the maximal velocity of hemolysis, or decrease the time required for 50% hemolysis, beyond a certain point, indicates that at this point the terminal step is limiting the hemolytic reaction.
7. The suggestion is made that much useful information may be obtained by further study of immune hemolysis at 0°, primarily because of the comparative slowness of the reaction, and the negligible amount of nonproductive deterioration of intermediate complexes of C' and sensitized erythrocytes.

Footnotes

1 Aided by a grant (E-341-C) from the National Institutes of Health, United States Public Health Service, Bethesda, Maryland.

2 A preliminary report of this work was presented to the American Association of Immunologists at the 1956 meeting of the Federated Societies for Experimental Biology in Atlantic City, N. J. (Federation Proc., 15: 604, 1956).







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