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Hemagglutination Studies with Formalinized Erythrocytes

Effect of Bis-Diazo-Benzidine and Tannic Acid Treatment on Sensitization by Soluble Antigen¹

From the Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts

Abstract

Formalinized erythrocytes sensitized with diphtheria toxoid using bisdiazotized benzidine (BDB) as a coupling agent, will detect 0.00015 units/ml of standard diphtheria antitoxin. Therefore, such erythrocytes are at least as sensitive as fresh or formalinized erythrocytes sensitized by the tannic acid method. They can be stored frozen, and thawed at any time for use.

The concentration of BDB in the reaction mixture is crucial. In contrast, optimal sensitization occurs over a wide range of diphtheria toxoid concentration, and 100-fold less antigen is necessary than had previously been needed to sensitize nonformalinized erythrocytes by the BDB method. The coupling procedure is complete within 5 to 10 min at 37°C.

Other factors related to the preparation of formalinized erythrocytes, tannic acid treatment of such erythrocytes, preparation of the BDB-antigen-erythrocyte reaction mixture, and performance of the hemagglutination test are discussed.

Footnotes

¹ This work was done during the tenure of a National Science Foundation Fellowship, and with the support of a Public Health Service Research Grant, H-2255, from the National Heart Institute, USPHS.

² Present address: Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda 14, Maryland.

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