AffinityImmuno Products

Introduction

IDO1 is a heme-containing enzyme that carries out the first and rate-limiting step of tryptophan breakdown along the kynurenine pathway, converting the amino acid, along with related substrates such as serotonin and tryptamine, into N-formylkynurenine. By locally depleting tryptophan and generating kynurenine pathway metabolites, IDO1-expressing dendritic cells, monocytes, and macrophages can slow the proliferation of nearby T cells, push them toward apoptosis, and favor differentiation of regulatory T cells, a mechanism central to peripheral immune tolerance. This same pathway helps prevent autoimmunity and protects the fetus from maternal immune attack during pregnancy, but tumors can co-opt it: IDO1 is expressed by tumor, stromal, or endothelial cells in a large fraction of human cancers, where its immunoregulatory tyrosine motifs sustain its own expression in dendritic cells and help tumors evade immune surveillance, a pattern linked to worse prognosis. IDO1 activity also has antimicrobial and antioxidant roles, restricting the growth of intracellular pathogens by starving them of tryptophan.

Although the IDO1 inhibitor epacadostat did not succeed in combination with pembrolizumab in its pivotal trial, IDO1 remains an active research target, with newer work exploring biomarker-guided patient selection and non-enzymatic IDO1 functions — making quantitative IDO1 measurement useful for understanding why past checkpoint-combination trials underperformed and for informing next-generation approaches.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format to measure human IDO1. Antibody specific to human IDO1 is pre-coated onto the microtiter plate wells, where it captures the target from added standards or samples. A biotin-conjugated detection antibody, also specific to human IDO1, is then introduced and binds a second epitope on the captured analyte. Avidin-conjugated horseradish peroxidase (HRP) is subsequently added to each well, linking to the biotin and completing the detection complex. Upon addition of TMB substrate, a colorimetric signal develops exclusively in wells containing human IDO1, the biotin-conjugated antibody, and Avidin-HRP. The enzymatic reaction is stopped by the addition of sulfuric acid stop solution, and absorbance is read at 450 nm (± 10 nm). Sample concentrations are determined by comparing well absorbance values against a standard curve.