A colorimetric method for detection of specific ligand binding.
The binding of IL-2 and IL-3 to the factor-dependent cell lines CTB6 and 32D, respectively, was determined using biotinylated ligand detected by the addition of a streptavidin/alkaline phosphatase conjugate and amplified with a phosphatase amplification system. Binding of both ligands was detectable after incubation with as little as 20 fmol of ligand and could be inhibited with a 10-fold molar excess of nonbiotinylated ligand. No binding was observed when biotinylated ligand was incubated with a receptor negative cell line (PC-12) and IL-2 was unable to compete with biotinylated IL-3 binding to 32D cells, further demonstrating specificity. These studies indicate that biotinylated ligands can be used as a nonradioactive method to detect specific, high-affinity cell surface receptors.
Wang
H
H
Cellular Biochemistry Research and Development, Life Technologies, Inc., Gaithersburg, Maryland.
Beckner
S K
SK
D000469:Alkaline Phosphatase; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism*; D007377:Interleukin-3 / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010744:Phosphoric Monoester Hydrolases; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D007376:Interleukin-2; D007377:Interleukin-3; D008024:Ligands; D015375:Receptors, Interleukin-2; D016185:Receptors, Interleukin-3; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases