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A simple and rapid radioimmunoassay for human interleukin-6
J V Peppard1, J J Rediske, J A Koehler
1Research Department, Ciba-Geigy Pharmaceutical Division, Summit, NJ 07901.
Insights
A sensitive radioimmunoassay was developed for human interleukin-6 (IL-6) using readily available materials. This assay accurately measures IL-6 levels in biological samples, correlating well with functional assays.
Area of Science:
- Immunology
- Biochemistry
Background:
- Interleukin-6 (IL-6) is a key cytokine involved in immune responses and inflammation.
- Accurate quantification of IL-6 is crucial for understanding its role in various physiological and pathological conditions.
Purpose of the Study:
- To develop and validate a sensitive radioimmunoassay (RIA) for human IL-6.
- To enable the measurement of IL-6 produced by human monocytes.
Main Methods:
- A polyclonal rabbit anti-human IL-6 antibody was used with iodinated IL-6.
- Immune complexes were precipitated using magnetic particles coated with anti-rabbit IgG.
- Two assay formats were optimized: an overnight assay (0.1-10 ng/ml working range) and a 2-hour assay (1.5-25 ng/ml working range).
Main Results:
- The developed RIA demonstrated high sensitivity with a working range of 0.1-10 ng/ml (IC50 ~1 ng/ml) for the overnight format.
- The assay showed excellent correlation with a standard B9.9 hybridoma proliferation assay for both recombinant and natural IL-6.
- The assay exhibited low variance (<12%) and no cross-reactivity with interleukins 1-5.
- IL-6 production by human monocytes in response to stimuli like LPS and IL-1 was successfully measured.
Conclusions:
- A robust and sensitive radioimmunoassay for human IL-6 has been successfully developed.
- This RIA is suitable for quantifying IL-6 in biological samples and can be used to study IL-6 production by human monocytes.
Abstract:
Studies were undertaken to develop a sensitive radioimmunoassay for human IL-6 using commercially available reagents. The assay utilized a polyclonal rabbit anti-IL-6 binding to iodinated IL-6; the reaction was carried out in solution and the immune complexes were precipitated using anti-rabbit IgG coupled to magnetic particles. Using a format where sample IL-6 was added in advance of the radiolabelled IL-6, the working range of the assay was found to fall between 0.1 and 10 ng/ml (IC50 around 1 ng/ml) for a 50 microliters sample volume, and was run overnight. However, the assay could be completed in 2 h, using a direct competitive format when less sensitivity is required (working range 1.5-25 ng/ml, IC50 12 ng/ml). The RIA correlated directly with a standard functional assay for IL-6 (proliferation of the mouse hybridoma B9.9) for both recombinant IL-6 and natural IL-6 from human monocytes. The total assay variance was less than 12% and no reactivity with interleukins-1-5 was found. Using the RIA, IL-6 produced in culture by human monocytes in response to various stimuli (LPS, IL-1, dibutyryl cAMP) was measured.