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In Vitro Production of Diphtheria Antitoxin by Tissues of Immunized Animals

II. Development of a Synthetic Medium Which Promotes Antibody Synthesis and the Incorporation of Radioactive Amino Acids into Antibody¹

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

Abstract

Addition to various basal synthetic media of dialyzed or undialyzed homologous or heterologous serum, serum ultrafiltrate, chick embryo extract, purines, pyrimidines, vitamins, coenzymes, lipids and carbohydrates did not consistently enhance in vitro antibody synthesis by lymphoid tissues from immunized rabbits. The failure of paraamino benzoic acid and thiamin to produce an effect on these short-term experiments was contrasted with their effects in longer-term cultures and an explanation of the different findings advanced. It was found that optimal concentrations of certain amino acids must be present in the medium for maximal in vitro antibody synthesis and incorporation of radioactive amino acids into antibody. These data provide further evidence for the importance of amino acids as precursors of antibodies. The complete function of the amino acids in the medium is unknown because the medium containing the smallest concentration of each amino acid still contains 5–10 times the amount of each acid required for antibody synthesis. The failure of previous investigators to observe an amino acid requirement for incorporation of radioactive amino acids into antibody was attributed to their use of much larger amounts of tissue than in the present study. As evidenced by the incorporation data, antibody synthesis in vitro is a rapid process, which is in accord with the findings of other studies. These observations also indicate that antibody is rapidly secreted into the medium. As a result of this study, a completely synthetic medium has been developed which promotes appreciable antibody synthesis and incorporation of radioactive amino acids into antibody.

Footnotes

This work was supported in part by a grant from the Life Insurance Medical Research Fund.

² Predoctoral Fellow of the U. S. Public Health Service.

³ Present address: Department of Microbiology, School of Veterinary Medicine, University of Pennsylvania.

⁴ This work was conducted during the tenure of an Established Investigatorship from the American Heart Association.

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