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Immunodetection of small molecule biomarkers

Metabolite biomarker case study

Anti-L-kynurenine antibody for IHC and ELISA detection

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

  • To develop a monoclonal antibody against L-Kynurenine
  • To validate the antibody for L-Kynurenine detection by IHC
  • To set up a novel immunoassay for accurate L-Kynurenine quantitation in biological fluids

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

Benefits

  • High specificity: no cross-reactivity with L-Kynurenine analogs
  • Cross-species reactivity: Antibody can detect L-Kynurenine both in experimental models and human samples

2IHC validation of L-Kynurenine antibody

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

  • Compatibility with routine diagnostic technologies
  • Fast diagnostic of Kynurenine-positive cancer patients

3Development of a novel immunoassay for L-Kynurenine quantification

Benefits

  • Validation of an ELISA protocol 50 times more sensitive than standard protocol without derivatization, capable of substituting to LC/MS
  • High-throughput and fast results: 96-well plates, 4 hour lead-time

References

  • Cavia-Saiz M et al. The role of plasma IDO activity as a diagnostic marker of patients with colorectal cancer. Mol Biol Rep. 2014 Apr;41(4):2275-9. doi: 10.1007/s11033-014-3080-2. Epub 2014 Jan 17.
  • Chuang SC et al. Circulating biomarkers of tryptophan and the kynurenine pathway and lung cancer risk. Cancer Epidemiol Biomarkers Prev. 2014 Mar;23(3):461-8. doi: 10.1158/1055-9965.EPI-13-0770. Epub 2013 Dec 19.
  • Munn DH, Mellor AL. Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013 Mar;34(3):137-43. doi: 10.1016/j.it.2012.10.001. Epub 2012 Oct 25.
  • Schwarcz R ety al. Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci. 2012 Jul;13(7):465-77. doi: 10.1038/nrn3257.
  • Pilotte L et al. Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase. Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2497-502. doi: 10.1073/pnas.1113873109. Epub 2012 Jan 30.
  • Favre D et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med. 2010 May 19;2(32):32ra36. doi: 10.1126/scitranslmed.3000632.

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