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Purification and Analysis of fluorescein-Labeled Antisera by Column Chromatography¹

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

Abstract

1. Fluorescent rabbit anti-bovine serum albumin antibody with bright specific and negligible nonspecific staining was prepared from the conjugated antiserum by a single elution from a diethylaminoethyl cellulose column.

2. The yield was 40 to 60% of the labeled precipitating antibody when conjugates with a molar fluorescein to protein ratio of 4 or less were used. The antibody eluted under the conditions employed had a molar fluorescein to protein ratio of approximately 1.0.

3. Absorption and emission studies of a variety of conjugates show that after conjugation to protein a) amorphous and crystalline fluorescein isothiocyanate preparations have the same emission intensity, and b) fluorescein isocyanate has approximately twice the emission intensity of the isothiocyanate.

Footnotes

¹ The work reported in this paper was supported in part by USPHS Grant H-2255.

² This work was performed during the tenure of a Postdoctoral Research Fellowship from the National Institute of Allergy and Infectious Diseases, USPHS.

³ Research Fellow, American Heart Association.

⁴ Career Investigator, American Heart Association.

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