I. Development of Paper Electrophoresis Technique for Observation of Microgram Quantities of Protein. II. Electrophoretic and Ultra-Centrifugal Studies of Soluble Antigen-Antibody Complexes as a Method of Determining Antibody and Antigen Valences

Citation

Bradbury, James Thomas (1956) I. Development of Paper Electrophoresis Technique for Observation of Microgram Quantities of Protein. II. Electrophoretic and Ultra-Centrifugal Studies of Soluble Antigen-Antibody Complexes as a Method of Determining Antibody and Antigen Valences. Master's thesis, California Institute of Technology. doi:10.7907/9CZJ-ZS08. https://resolver.caltech.edu/CaltechETD:etd-03182004-155047

Abstract

Quantities of protein as small as five micrograms can be positively detected after electrophoresis on paper if strong adsorption to the paper, known as tailing, is prevented. Adding 0.1% gelatin to the buffer solution used to soak the paper is the simplest way to reduce tailing, and allows the conventional bromphenol blue stain to be used to locate the protein. A protein as nearly identical to the sample as possible eliminates tailing more completely than gelatin, and can be used if the sample can be located in a background of the other protein. Making the sample fluorescent under ultraviolet light by coupling 1-dimethylaminonaphthlalene-5-sulfonyl chloride to it before electrophoresis is an effective way to locate the sample in the presence of otherwise identical proteins. Oxidation of the paper with nitrogen dioxide to reduce tailing has been attempted, but the method has not been refined to a practical degree of effectiveness. Paper electrophoresis of BSA-rabbit anti-BSA complexes shows two zones attributed to the Ag2Ab and Ag3Ab2 complexes, in qualitative agreement with moving boundary electrophoresis and ultracentrifugation by Singer and Campbell. As a supplementary argument as to the identity of the complexes, a simple statistical theory is developed to show the relative proportions of different complexes to be expected from antigens and antibodies of given valences, or to show the valences of antigens and antibodies from the quantities of the different complexes formed. Using the ultracentrifugal data of Singer and Campbell in the theoretical equations limits the valence of the antibodies to a value close to two, and the valence of the BSA antigen to a value around five.

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