Cytokine Neutralization Assay

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In Vitro Pharmacology

Cytokines are secreted proteins and function as immune-modulating agents. Their secretions are strictly regulated in human body. Excessive production of cytokines may cause autoimmune diseases or cancer, where neutralizing antibodies targeting these cytokines may be potentially therapeutic. ProBio offers multiply assays to evaluate the biological activity of the cytokine protein and functionalities of cytokine neutralizing antibodies at drug discovery stage.

Types of cytokine neutralization assay

Target cell	Stimulator	Endpoint and readout	Application Scenario
Cell line or Primary cell	Cytokine	Biological response (e.g. proliferation, apoptosis or cytokine release) , or Intracellular signaling pathway (kinase phosphorylation)	Functional screening or characterization
Reporter gene cell line	Cytokine	Reporter gene activity	Functional characterization, batch release during CMC stage

Case 1: anti-TNFα antibody neutralization assay

Case 2: IL-6 neutralization assay

Interleukin-6 (IL-6) can be both pro- and anti-inflammatory. IL-6 would induce TF-1 cell proliferation via triggering JAK signaling pathway and STAT3 phosphorylation, which offers a good in vitro model for functionality assessment.

Method 1. TF-1 cell proliferation
- IL-6 stimulates proliferation of TF-1 cells
- Siltuximab neutralizes IL-6 induced proliferation of TF-1 cells
Method 2. Intracellular STAT3 Phosphorylation
- IL-6 stimulates STAT3 phosphorylation of TF-1 cells
- Siltuximab neutralizes IL-6 triggered STAT3 phosphorylation of TF-1 cells

Case 3: VEGF reporter assay

Vascular endothelial growth factor (VEGF) is a growth factor that stimulates the formation of blood vessels. In cancer, VEGF may cause angiogenesis and promote tumor progression. The VEGF neutralization assay utilizes a VEGFR reporter gene cell line where an interaction between VEGF and VEGFR would activate luciferase reporter gene expression. In the presence of VEGF antibody (Bevacizumab), VEGF-dependent luciferase reporter gene expression will be inhibited.