Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions.
We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.
Kelly
Kimberly A
KA
Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.
Reynolds
Fred
F
Weissleder
Ralph
R
Josephson
Lee
L
D001839:Bombesin; D002450:Cell Communication; D002460:Cell Line; D016650:Fluorescein-5-isothiocyanate; D006241:Haptens; D006735:Horseradish Peroxidase; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D018796:Immunoconjugates; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D018004:Receptors, Bombesin / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism
D006241:Haptens; D018796:Immunoconjugates; D010455:Peptides; D018004:Receptors, Bombesin; D011956:Receptors, Cell Surface; D006735:Horseradish Peroxidase; D016650:Fluorescein-5-isothiocyanate; D001839:Bombesin