In Vivo Capture Assays

BD Biosciences offers in vivo capture assay sets for measuring cellular production of mouse cytokines: IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF. Specific cytokine-binding monoclonal antibodies form complexes with target cytokines that accumulate in serum and are assayed by enzyme-linked immunosorbent assay (ELISA).


Features

  • Direct measurement of in vivo production of cytokines using in vivo capture assays with biotin-labeled, cytokine-binding monoclonal antibodies that produce a long-lived, soluble cytokine–anti-cytokine complex
  • Increased sensitivity of detection of serum cytokines, as much as 50- to 100-fold compared to conventional ELISA detection
  • Direct correlation of detected cytokine to the amount of cytokine secreted

Measuring cytokine production in vitro versus in vivo

Analysis of the types and levels of cytokines that are produced in vivo is often difficult. Many cytokines have short in vivo half-lives and do not accumulate to detectable levels as measured in serum samples. Cytokine mRNA accumulation by cells in vivo and cytokine protein secretion by cells re-stimulated in vitro are relatively easy to measure. However, mRNA levels and in vitro cytokine protein secretion may not fully reflect cytokine protein secretion in vivo.

How the assay works

The BD Pharmigen™ in vivo capture assay directly measures the in vivo production of cytokine proteins. In this assay, mice are injected with 10 μg of a no azide/low endotoxin (NA/LE), biotin-labeled antibody that binds to the targeted cytokine protein as it is secreted, forming a long-lived soluble complex that accumulates in the blood. Mice are bled, and the complex is captured from serum in ELISA plate microwells coated with antibody that binds to a different epitope of the same cytokine. The complex is detected by streptavidin-horseradish peroxidase followed by the addition of a chromogenic solution.

The amount of cytokine detected is directly proportional to the amount secreted, regardless of the site of production. The BD Pharmigen in vivo capture assay does not inhibit cytokine-dependent processes because only a fraction of the secreted cytokine is captured by the injected NA/LE biotinylated antibody.

This figure demonstrates the measurement of in vivo IL-4 and IFN-γ production by IVC. BALB/c mice (5 per group) were injected intravenously with 10 μg each of biotin-anti–IL-4 mAb (Cat. No. 557781) and biotin-anti–IFN-γ mAb (Cat. No. 558491) 10 μg of anti-CD3 mAb (Cat. No. 553057). Mice were bled 4 hours later and serum levels of cytokine-biotin–anti-cytokine mAb complexes were determined by ELISA.

Set components:

  • In vivo capture mAb (biotinylated no azide/low endotoxin format)
  • Purified ELISA capture mAb
  • Recombinant standard
  • Streptavidin-horseradish peroxidase enzyme and substrates

References

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