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Appl Environ Microbiol. 1964 July; 12(4): 343-348
Copyright © 1964 American Society for Microbiology. All Rights Reserved.

Technical Considerations in the Preparation of Fluorescent-Antibody Conjugates

Communicable Disease Center, U.S. Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia

ABSTRACT

A comparison was made of (NH₄)₂SO₄, HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.