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Published on: March 2, 2011
Quantification of recombinant human interleukin-1 alpha by a specific two cell immunobioassay
R W Nadeau1, C M Ostrowski, G Ni-Wu
1Department of Drug Metabolism 86/842, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Insights
A novel immunobioassay detects picogram levels of recombinant human Interleukin-1 alpha (rhIL-1 alpha) in serum. This sensitive and specific assay utilizes a two-cell system for accurate quantification.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Interleukin-1 alpha (IL-1 alpha) is a key cytokine involved in inflammatory and immune responses.
- Accurate detection of IL-1 alpha in biological samples is crucial for research and diagnostics.
- Existing methods may lack the required sensitivity or specificity for picogram-level detection.
Purpose of the Study:
- To develop and validate a highly sensitive and specific immunobioassay for detecting picogram quantities of recombinant human IL-1 alpha (rhIL-1 alpha) in serum.
- To assess the performance characteristics of the assay, including sensitivity, precision, reproducibility, and specificity.
- To evaluate the stability and recovery of rhIL-1 alpha in serum and its application in animal studies.
Main Methods:
- A two-cell immunobioassay employing an IL-1 alpha specific mouse IgM monoclonal antibody for capture.
- Utilizing the EL-4 NOB-1 cell line, which produces IL-2 upon capture of IL-1 alpha.
- Measuring cell proliferation of the IL-2 dependent CTLL-2 line via thymidine incorporation to quantify IL-1 alpha.
Main Results:
- The assay demonstrated high sensitivity, detecting rhIL-1 alpha in the range of 1-50 pg/ml.
- The assay exhibited excellent specificity, precision, and reproducibility.
- Data confirmed the stability of rhIL-1 alpha in serum and efficient recovery, with successful application in rat serum samples.
Conclusions:
- The developed two-cell immunobioassay provides a sensitive and specific method for quantifying picogram levels of rhIL-1 alpha in serum.
- The assay's ease of use and robust performance make it a valuable tool for biological and immunological research.
- This method facilitates the study of IL-1 alpha dynamics in various physiological and pathological conditions.
Abstract:
The detection of picogram quantities of recombinant human IL-1 alpha in human and rat serum was accomplished by a sensitive and specific two cell immunobioassay. The specificity is provided by an IL-1 alpha specific mouse IgM monoclonal antibody which is non-neutralizing thus allowing for the addition of the EL-4 NOB-1 cell line directly to the IL-1 alpha monoclonal antibody complex. The above cell line is then converted to an IL-2 producer line in response to the captured IL-1 alpha. Supernatant from the EL-4 NOB-1 cells is then added to the IL-2 dependent CTLL-2 line and cell proliferation measured by thymidine incorporation. This assay has the advantage of specificity provided by the antibody capture step, sensitivity provided by the EL-4 NOB-1 line (1-50 pg/ml) and finally ease of maintenance of the responder cell line which requires no feeder cells or mitogens. Data are reported on the sensitivity, precision, reproducibility and specificity of the assay, the stability of rhIL-1 alpha in serum and the recovery of rhIL-1 alpha from serum. We also report on the use of this procedure to assay samples from rats given ascending doses of rhIL-1 alpha.
