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.

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