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Determination of Relative Affinity and Heterogeneity of Mouse Anti-DNP Antibodies by a Plaque-Inhibition Technique¹

From the Department of Pathology and Hygiene, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801

Abstract

A plaque-inhibition technique was used to study the response of mice to the dinitrophenyl (DNP) determinant at the cellular level. Sheep erythrocytes were sensitized for use in the plaque assay by exposing them to dinitrophenylated antisheep hemolysin of low hemolytic efficiency. DNP-lysine was added to the agar to inhibit specifically indirect plaques formed by mouse spleen cells producing anti-DNP antibodies. The average relative affinity of these antibodies was defined as the reciprocal of the molar concentration of DNP-lysine producing 50% plaque inhibition. The average relative affinity increased by a factor of 130 between 1 and 8 weeks after initial immunization. A highly significant correlation, apparently linear, was found between the average relative affinity and the average association constant of the serum antibody as measured by a modified Farr technique. The slopes of the plaque-inhibition curves in the vicinity of 50% plaque inhibition were assumed to be inversely proportional to the degree of heterogeneity of the antibodies with respect to affinity. There was a highly significant positive correlation between affinity and heterogeneity.

Footnotes

¹ This investigation was supported by Contract N00014-67-A-0305-0018 with the Office of Naval Research, United States Department of the Navy, and by Research Grant AI 09743 from the National Institutes of Health. A portion of this research was supported by funds from the Illinois Agricultural Experiment Station.

² Recipient of United States Public Health Service Training Grant 2E-178. Present address: Department of Pathology, New York University Medical Center, 550 First Avenue, New York, N. Y. 10016.

³ Requests for reprints should be addressed to Dr. Diego Segre, Department of Pathology and Hygiene, College of Veterinary Medicine, University of Illinois, Urbana, Illinois.

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