Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor.
We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method.
Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo, Japan. usami@nihs.go.jp
Makoto
M
Usami
Katsuyoshi
K
Mitsunaga
Yasuo
Y
Ohno
D015374:Biosensing Techniques; D047628:Estrogen Receptor alpha; D006801:Humans; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D011994:Recombinant Proteins