Flow injection analysis of binding reaction between fluorescent lectin and cells.
A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.
Oda
Y
Y
Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka, 577-8502, Japan.
Kinoshita
M
M
Nakayama
K
K
Ikeda
S
S
Kakehi
K
K
D000372:Agglutination Tests; D000818:Animals; D001360:Avidin; D001419:Bacteria; D001710:Biotin; D003208:Concanavalin A; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D037102:Lectins; D008297:Male; D051379:Mice; D013154:Spleen / Q000166:cytology; D014468:Umbelliferones; D015003:Yeasts
C106879:7-hydroxycoumarin-3-carboxylic acid; D005456:Fluorescent Dyes; D037102:Lectins; D014468:Umbelliferones; D003208:Concanavalin A; D001360:Avidin; D001710:Biotin