Immunodetection of small molecule biomarkers

Metabolite biomarker case study

Anti-L-kynurenine antibody for IHC and ELISA detection

kynurenine pathway biomarker

Essential amino acid L-Tryptophan is converted to L-Kynurenine either through indoleamine-2,3-dioxygenase 1/2 (IDO1/2) or tryptophan-2,3-oxygenase (TDO2) enzymes. Abnormal tryptophan metabolism along the kynurenine pathway has been shown to promote tumor immune espace as well as neurodegenerative disorders and infectious diseases.
Direct detection of L-Kynurenine is therefore critical to stratify patients harboring exaggerated tryptophan catabolism and to assess the efficacy of drug candidates targeting IDO1, IDO2 and/or TDO2.

Objectives:

 


1 - Development of anti-L-Kynurenine antibody (mouse monoclonal)

1/ Immunogen synthesis

2/ Antibody production

3/ Antibody validation

Synthesis of L-Kynurenine conjugate

Synthesis of an immunogenic
L-Kynurenine/protein carrier adduct

Production of monoclonal anti-L-Kynurenine antibody

5-week mouse immunization &
monoclonal antibody (mAb) development

Validation of anti-L-Kynurenine antibody

Evaluation of antibody's specificity and binding affinity by ELISA

Benefits:


2 - IHC validation of L-Kynurenine antibody

1/ IHC : Kynurenine postivive tumor

2/ IHC : Kynurenine negative tumor

L-Kynurenine detection by IHC in colon cancer tissue L-kynurenine IHC staining validation


IHC detection of L-Kynurenine in human paraffin-embedded colon cancer tissues. Staining protocol: pH=6 antigen retrieval, overnight incubation with anti-L-Kynurenine antibody (1/500 dilution), polymer-conjugated secondary Ab, DAB revelation)

Benefits:


3 - Development of a novel immunoassay for L-Kynurenine quantification

1/ Standard ELISA protocol
- without derivatization

2/ ImmuSmol's ELISA protocol
- with derivatization

3/ ELISA cross-validation by LC/MS in human plasma samples

Standard ELISA method

ImmuSmol ELISA protocol

LC-MS cross-validation of L-Kynurenine detection in human plasma

Benefits:


References