Home » Therapeutic Antibody » Antibody Engineering » In vitro Pharmacology » Cytokine Neutralization Assay

Cytokine neutralization assay

Cytokines are secreted proteins with various immune functions. Their main role is to regulate the immune responses of immune cells (i.e. pro-inflammatory or anti-inflammatory). Their expressions are tightly regulated in human body. Persistent and excessive production of inflammatory cytokines may cause autoimmune diseases or cancer. Antibodies targeting inflammatory cytokines have superior therapeutic benefits in pre-clinical research and clinical trials.

GenScript ProBio cytokine neutralization assay platform offers customized cytokine neutralization assays assessing cell viability and downstream signaling transduction for evaluating the biological activity of the antibody drugs at early drug discovery stage.

Types of cytokine neutralization assay

Target cell Stimulator Assay type Advantage Disadvantage Application Scenario
1 Target cell line Human cytokine Target cell proliferation Relatively easy to conduct1, cost is relatively low The method is not sensitive enough Early drug screening or functional confirmation post-screening
2 Target cell line Human cytokine Intracellular signaling pathway activation (kinase phosphorylation) Relatively easy to conduct, assay sensitivity is relatively high Assay detection kit is relatively costly Early drug screening or functional confirmation post-screening
3 Target and reporter gene overexpression engineered cell line2 Ligand overexpression engineered cell line2 Reporter gene expression Cost effective, easy to conduct, high reproducibility Effort is required to construct cell lines and validate the assay method Early drug screening, batch-release during CMC

Key Features

  • Capability to design neutralization assay for most human cytokines
  • Experienced in establishing highly sensitive and reproducible cytokine neutralization assays
  • Free positive and negative control

Case 1: anti-TNFα antibody neutralization assay


  • TNFα shows cytotoxic effect against L929 cell line

  • Humira is able to neutralize the activity of TNFα, cytotoxic effect is significantly reduced

Case 2: Anti-IL-6 neutralization assay


  • The neutralization assay focusing on TF-1 cell proliferation is not sensitive enough due to a small assay window. But if cell proliferation is changed to STAT3 phosphorylation level, the neutralization assay becomes more sensitive with an assay window more than 10 fold.

  • IL-6 stimulates the growth of TF-1 cells

  • Anti-IL-6 antibody is able to neutralize the effect of IL-6 to stimulate the growth of TF-1 cells

  • IL-6 stimulates STAT3 phosphorylation of TF-1 cells

  • Anti-IL-6 antibody is able to neutralize the effect of IL-6 to stimulate the STAT3 phosphorylation of TF-1 cells

Case 3


The VEGF neutralization assay utilizes a VEGFR reporter gene assay cell line. The interaction between VEGF and VEGFR will activate the luciferase expression. When the interaction is disrupted, luminescence signal will decrease.