Immune receptor based reporter gene assay for detection of allergens and respiratory pathophysiology and pathogenesis

Principle of the cell-based test system. After specific ligand binding to the receptor, an intracellular signaling cascade activates NF-kB and induces the expression of the reporter gene (secreted alkaline phosphatase SEAP).

The reporter gene product (SEAP) catalyzes the hydrolysis of the substrate to a colored end product measured photometrically.

At Fraunhofer IGB we have developed a cell-based reporter gene assay that allows compounds to be screened for their interaction with innate immune receptors such as toll-like receptors or NOD-like receptors. For this assay, the appropriate receptor complex was stably transfected and expressed in NIH3T3. The activation of the receptor complex leads to NF-kB mediated induction of the reporter gene, e.g. a secreted alkaline phosphatase or Gfp. Immuno-modulating and allergenic properties of compounds thus can be detected via simple HTS-compatible photometric assays based on enzymatic conversion of a chromogenic substrate or Gfp. The assay has been patented and is certified according to GLP.


The mouse fibroblast cell line (NIH3T3) is stably transfected with human pattern recognition receptor(s) (PRR) e.g. toll like-receptor(s) (TLR) and an inducible reporter gene plasmid (reporter gene secreted embryonic alkaline phosphatase (SEAP)).

Field of application:

Toll-like-receptors (TLRs) play a pivotal role in innate an adaptive immunity and they are broadly distributed in various cell types like mucosal epithelial and endothelial cells, B cells, T cells, monocytes/macrophages, dendritic cells as well as natural killer cells. TLRs recognize pathogen-associated molecular patterns (PAMPs) such as components of viruses, bacteria or fungi as well as allergens. This, in turn, leads to immune responses via chemokines and chemokine receptors, up-regulation of co-stimulatory molecules like type I interferons and pro-inflammatory cytokines. In addition TLRs are also able to recognized and react to endogenous danger-associated molecular patterns (DAMPS) in the non-infectious inflammation response like intracellular and nuclear proteins (heat-shock proteins, HMGB1), purin metabolites (ATP, adenosine, uric acid) as well as nucleic acids (DNA, RNA).

Thus they are potent targets for manipulation of the immune system. TLR agonists enhance the activities of the immune system. They can be used as immune-therapeutics in the treatment and prevention of cancer, allergies and infections. In addition they are good adjuvants for vaccines by boosting the adaptive immune response. TLR antagonists on the other hand play a therapeutic role in suppressing overactive immune responses: They are able to reduce or suppress excessive inflammatory processes, chronic infections and allergies as well as autoimmune disorders and immune abnormalities in HIV.

In acute infectious lung-pathological diseases such as tuberculosis and infection with respiratory syncytial virus TLRs have been shown to play a protective role. In particular TLR2 is associated with infections with Gram-positive bacteria like mycobacteria, TLR 4 is associated with infections with Gram-negative bacteria like Pseudomonas aeruginosa and TLR3 and TLR9 are associated with viral respiratory tract infections. In these cases TLR agonists can be potential immune-therapeutics and adjuvants. TLR antagonists can be applied in chronic inflammatory airway diseases such as allergic asthma and chronic obstructive pulmonary diseases. In the case of allergic rhinitis and asthma TLR2, TLR4 (e.g. hay fever against Ambrosia), TLR7, TLR8 as well as TLR9 (e.g. house dust mite allergy) are interesting targets for immune-therapeutics. Using e.g. TLR8 activators Th2-responses to allergens could be reversed to restore homeostasis of the immune system. TLR2, TLR3, TLR4 and TLR9 are linked to cigarette smoke-induced inflammation and thus contribute to chronic obstructive lung disorders. In addition to using the described assay for screening of new immune-therapeutics, the allergic activity of different TLR ligands can also be determined. Beside acute and chronic inflammations and infections TLRs are also contributing to cancer. Regarding cancer like lung tumor and non-small cell lung carcinoma respectively TLR3, TLR4 as well as TLR9 contribute to these pathologies.

Endpoints/Outcome parameter

The cell-based test system allows the identification of TLR agonists and antagonists as well as the quantification of agonistic properties (effective concentration, EC50) and antagonistic properties (inhibitory concentration, IC50) of the analyzed compounds and allergens.

Readout parameter

The cell-based test system allows a simple HTS-compatible photometric readout via enzymatic conversion of a chromogenic substrate or Gfp. This allows a fast and easy quantitative identification of TLR agonistic and antagonistic ligands.

Quality management and validation

These assays can be performed according to GLP. They are currently certified within category 9. Receptor-specific natural ligands serve as compound controls. Unspecific binding can be detected using corresponding control cells lacking the specific receptor complex.


  • A. Burger-Kentischer, I.S. Abele, D. Finkelmeier, K.-H. Wiesmüller and S. Rupp (2010) Journal of Immunology Methods A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signaling pathways and the detection of pyrogens. 358:93-103

  • K. Nothelfer, E.T. Arena, L. Pinaud, M. Neunlist, B. Mozeleski, I. Belotserkovksy, C. Parsot, P. Dinadayala, A. Burger-Kentischer, R. Raqib, P.J. Sansonetti and A. Phalipon (2014) Journal of Experimental Medicine B Lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection. 211(6):1215-29

  • Patent: Burger-Kentischer et al. Cell-based Pyrogen Assay; DE 10 2006 031 483; EP 2 041 172