Table 2.
Assays used to detect estrogenic activity.
| Assay Category (Symbol)/Description | Example a |
|---|---|
| Animal test (A) | |
| Animal tests quantitate reproductive, developmental and behavioral effects in animals. | Medaka assay |
| Zebrafish assay | |
| Rainbow trout assay | |
| Rodent uterotrophic assay | |
| Xenopus assay | |
| Cell assay (C) | |
| Cell assays quantitate cell growth and proliferation. | Cell counter assay |
| Cell density/viability assay (SRB assay/AlamarBlue assay/MTS assay/MTT assay/WST-8 assay) | |
| Dye exclusion method (Trypan blue assay) | |
| E-screen assay | |
| Flow cytometry | |
| Ligand-binding assay (L) | |
| Ligand-binding assays quantitate the receptor–ligand interaction. | Assay with ERs in cells |
| Assay with ERs in tissues (uterus, etc.) | |
| Assay with recombinant ERs | |
| Assay with Venus fluorescent protein | |
| Competitive enzyme immunoassay | |
| Fluorescence polarization assay | |
| Molecular docking | |
| QSAR | |
| Protein assay (P) | |
| Protein assays quantitate protein amounts and functions. | ChIP assay |
| ELISA | |
| Immunoassay (ICC, IHC) | |
| Western blotting (ERα/ERβ) | |
| Western blotting (Akt/ERK) | |
| Reporter-gene assay (R) | |
| Reporter-gene assays quantitate the transcription upon ligand-dependent binding of the receptor to an estrogen response element on DNA. | CALUX assay |
| GFP-based assay | |
| Luciferase-based assay | |
| MVLN cell assay | |
| YES assay | |
| Transcription assay (T) | |
| Transcription assays quantitate the transcription of ER or marker genes. | DNA microarray assay |
| Northern blotting | |
| RT-PCR | |
| Yeast two-hybrid assay (Y) | |
| Yeast two-hybrid assays quantitate the ligand-dependent interaction between the receptor and the transcriptional activator. | GAL4-based assay |
| Whole hERα-based assay |
a The examples are adapted from Kiyama (2017) [37]. For details of estrogenic chemicals analyzed by each assay, see Kiyama and Wada-Kiyama (2015) [35]. Abbreviations: CALUX: chemically activated luciferase expression; ChIP: chromatin immunoprecipitation; ELISA: enzyme-linked immunosorbent assay; ER: estrogen receptor; ERK: extracellular signal-regulated kinase; GFP: green fluorescent protein; ICC: immunocytochemistry; IHC: immunohistochemistry; MTS: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; QSAR: quantitative structure-activity relationship; RT-PCR: reverse transcription polymerase chain reaction; SRB: sulforhodamine B; WST-8: (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium); YES: yeast estrogen screen.