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Micromethods for the Study of Proteins and Antibodies

IV. Factors Involved in the Preparation and Use of a Stable Preparation of Formalinized, Tannic Acid-Treated, Protein-Sensitized Erythrocytes for Detection of Antigen and Antibody¹

From the Department of Microbiology, School of Medicine, Western Reserve University, Cleveland, Ohio

Abstract

The instability of fresh erythrocytes sensitized for use in hemagglutination reactions prompted the use of formalinized erythrocytes. In the course of attempts to avoid nonspecific agglutination, the variables affecting sensitization of formalinized erythrocytes were explored extensively. Uptake of antigen by cells increased as the concentration of tannic acid and the time of exposure to tannic acid were increased. Increasing the concentration of antigen to which tanned cells were exposed as well as increasing the temperature and time of reaction resulted in greater uptake of antigen by the cells. The sensitization reaction was pH dependent, with pH optima varying for different antigens. Optimal sensitization of cells for use in the hemagglutination reaction did not correspond with maximal uptake of antigen by the cells. The optimal conditions for sensitizing cells for use in hemagglutination were determined for a number of protein antigens. Under these conditions cell preparations of great stability were produced which were comparable in sensitivity to those made with fresh erythrocytes. The mechanism by which antigens are tightly bound to the erythrocyte surface remains unknown.

Footnotes

¹ This investigation was supported by a grant from the Institute of Allergy and Infectious Diseases, United States Public Health Service, E-1865.

² United States Public Health Service Research Fellow in Medicine, Departments of Medicine and Microbiology, Western Reserve University School of Medicine (United States Public Health Service Grant 2A5293 (C1)).

³ United States Public Health Service Medical Student Summer Research Fellow, Department of Microbiology, Western Reserve University School of Medicine (United States Public Health Service Medical Student Research Training Program Grant 2R-3 (C₂S₂) 1).