ANALYTICAL
BIOCHEMISTRY
Enhanced
74, 636-637 (1976)
Visibility of Precipitin Disks in Radial lmmunodiffusion Measurements
We are reporting a simple one-step artifice for improving the visibility of precipitin disks in radial immunodiffusion measurements. Our routine method for determination of rat a-fetoprotein (AFP) is the familiar single radial immunodiffusion of Mancini et al. (1). Generally speaking, the concentration of this protein in samples with which we are concerned is high enough to allow incorporation of sufficient antiserum in the agarose plate that resulting precipitin disks are quite visible and readily recordable when photographed using indirect lighting on a dark background (Fig. 1A). Recent work has required AFP determinations at levels in the range of l-2 pg/rnl, amounts for which the precipitin disks on our plates of routine antibody concentration are unsuitably small. It is possible to effect a greater precipitin-disk diameter for a given antigen concentration by lowering the amount of antibody incorporated in the agarose, though disk intensity is thereby compromised (1). In our experience, when a useful diameter is achieved, the disks become too faint for the simple photographic method of recording normally used (Fig. 1B). Staining the plates with Coomassie blue, then measuring disk diameter directly from the stained plate has been an acceptable alternative recording procedure but unfortunately adds another day to an already long procedure. We have found that immersion of a fully developed plate in 95% ethanol for 1 hr results in an intensification of the precipitin disks, providing more contrast for improved photographs (Figs. 1C and D). It is of particular interest that this treatment makes staining of the lower-antiserum-level plates unnecessary. ACKNOWLEDGMENT Supported by Research Grant No. BC-158 from the American Cancer Society.
REFERENCE 1. Mancini,
G., Carbonara.
235-254.
A. O., and Heremans, J. F. (1965) Immunochemistry
2,
JOHN G.WIEJA CAROL J. SMITH Department of Medicine Given Building. C-244 University of Vermont College of Medicine Burlington, Vermont 05401 Received March 12. 1976; accepted April
20, 1976
636 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
637
FIG. 1. Radial immunodiffusion of rat sera using 0.75% (w/v) agarose in 0.05 M Tris-HCl buffer, pH 8.0; development time, 48hr. (A), Before ethanol treatment, antiserum concentration 0.5% (v/v); (B), before ethanol treatment, antiserum concentration 0.1%; (C). after ethanol treatment, antiserum concentration 0.5%; (D), after ethanol treatment, antiserum concentration 0.1%.
BIOCHEMISTRY
Enhanced
74, 636-637 (1976)
Visibility of Precipitin Disks in Radial lmmunodiffusion Measurements
We are reporting a simple one-step artifice for improving the visibility of precipitin disks in radial immunodiffusion measurements. Our routine method for determination of rat a-fetoprotein (AFP) is the familiar single radial immunodiffusion of Mancini et al. (1). Generally speaking, the concentration of this protein in samples with which we are concerned is high enough to allow incorporation of sufficient antiserum in the agarose plate that resulting precipitin disks are quite visible and readily recordable when photographed using indirect lighting on a dark background (Fig. 1A). Recent work has required AFP determinations at levels in the range of l-2 pg/rnl, amounts for which the precipitin disks on our plates of routine antibody concentration are unsuitably small. It is possible to effect a greater precipitin-disk diameter for a given antigen concentration by lowering the amount of antibody incorporated in the agarose, though disk intensity is thereby compromised (1). In our experience, when a useful diameter is achieved, the disks become too faint for the simple photographic method of recording normally used (Fig. 1B). Staining the plates with Coomassie blue, then measuring disk diameter directly from the stained plate has been an acceptable alternative recording procedure but unfortunately adds another day to an already long procedure. We have found that immersion of a fully developed plate in 95% ethanol for 1 hr results in an intensification of the precipitin disks, providing more contrast for improved photographs (Figs. 1C and D). It is of particular interest that this treatment makes staining of the lower-antiserum-level plates unnecessary. ACKNOWLEDGMENT Supported by Research Grant No. BC-158 from the American Cancer Society.
REFERENCE 1. Mancini,
G., Carbonara.
235-254.
A. O., and Heremans, J. F. (1965) Immunochemistry
2,
JOHN G.WIEJA CAROL J. SMITH Department of Medicine Given Building. C-244 University of Vermont College of Medicine Burlington, Vermont 05401 Received March 12. 1976; accepted April
20, 1976
636 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
637
FIG. 1. Radial immunodiffusion of rat sera using 0.75% (w/v) agarose in 0.05 M Tris-HCl buffer, pH 8.0; development time, 48hr. (A), Before ethanol treatment, antiserum concentration 0.5% (v/v); (B), before ethanol treatment, antiserum concentration 0.1%; (C). after ethanol treatment, antiserum concentration 0.5%; (D), after ethanol treatment, antiserum concentration 0.1%.
