Biochemical Assay Kit for Human IDO2 (hIDO2)
NFK Green™ Assay Technology
Indoleamine 2,3-dioxygenase 2 (IDO2) is an enzyme that converts L-tryptophan into NFK, which is the first step in the kynurenine pathway of tryptophan degradation. IDO2 is a drug target for long COVID, rheumatoid arthritis and cancer immunotherapy. Due to the low tryptophan-metabolizing activity of IDO2, the biochemical assay kit for human IDO2 uses an alternative probe, known as NFK Green+™, to detect its activity. This probe has been optimized for detection of 5-methoxy-L-tryptophan degradation by IDO2, which is a tryptophan metabolite made by fibroblasts and epithelial cells and is more efficiently converted by IDO2 compared to L-tryptophan. The human IDO2 (hIDO2) biochemical assay kit can be used to determine the potency of IDO2 inhibitors.
Schematic representation of the NFK Green+™ assay principle. After enzymatic conversion of 5-methoxy-L-tryptophan (5-MTP) into 5-methoxy-N-formylkynurenine (5-MNFK), the reaction is stopped by addition of NFK Green+™, which yields a fluorescent complex that can be detected at the indicated wavelengths.
Dose-response curve for the reference IDO2 inhibitor in the NFK Green+™ hIDO2 assay.
Kits contents
| Component | Description | Stock conc. in kit | Conc. in assay |
|---|---|---|---|
| Enzyme | hIDO2 | 9.7 μM in storage buffer | 200 nM |
| Substrate | 5-MTP | 4 mM in MilliQ | 1 mM |
| Probe | NFK Green+™ | Ready to use | 1x |
| Reference compound | IDO5L | 10 mM in 100% DMSO | 10 μM |
| IDO2 assay buffer | Reaction buffer | Ready to use | – |
Advantages of NFK Green™
Detection
NFK Green+™ assays can be read out using a fluorescence reader with monochromators or filters that can handle excitation at 400 nm and emission detection at 535 nm. PerkinElmer Envision® multilabel plate readers with 400/25 (excitation; M400w) and 535/25 (emission; M535) filters and the D425/D490 dual mirror have already been successfully used to measure the signal of NFK Green+™ assays in 384-well format.
Assay Flowchart
