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Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a
M D Wewers1, H A Pope, D K Miller
1Department of Medicine, Pulmonary and Critical Care Division, Ohio State University, Columbus 43210.
Journal of Immunological Methods
|October 15, 1993
Summary
A new pro-interleukin-1 beta (IL-1 beta) enzyme-linked immunosorbent assay (ELISA) accurately quantifies pro-IL-1 beta. This specific assay overcomes limitations of conventional ELISAs that underestimate pro-IL-1 beta, especially intracellular levels.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Conventional enzyme-linked immunosorbent assays (ELISAs) for interleukin-1 beta (IL-1 beta) may underestimate pro-IL-1 beta concentrations.
- Pro-IL-1 beta is the precursor to mature IL-1 beta and plays a critical role in inflammatory responses.
- Accurate quantification of pro-IL-1 beta is essential for understanding IL-1 beta biology and related diseases.
Purpose of the Study:
- To develop and validate a novel sandwich ELISA specifically designed to quantify pro-IL-1 beta.
- To compare the performance of the pro-IL-1 beta specific ELISA with a conventional IL-1 beta ELISA.
- To assess the accuracy of both assays in measuring intracellular and extracellular pro-IL-1 beta.
Main Methods:
- Development of a sandwich ELISA using antibodies targeting the amino-terminus and carboxy-terminus of pro-IL-1 beta.
- Comparison of the pro-IL-1 beta specific ELISA with a conventional IL-1 beta ELISA using purified pro-IL-1 beta and mature IL-1 beta.
- Enzymatic cleavage of pro-IL-1 beta to assess assay responses to processed IL-1 beta.
- Quantification of intracellular pro-IL-1 beta in monocytes and macrophages using both ELISA methods.
Main Results:
- The pro-IL-1 beta specific ELISA accurately detects intact pro-IL-1 beta without cross-reacting with mature IL-1 beta.
- Conventional IL-1 beta ELISAs showed increased signals upon enzymatic processing of pro-IL-1 beta, while the specific ELISA showed decreased signals.
- The pro-IL-1 beta specific ELISA detected significantly higher intracellular pro-IL-1 beta levels in monocytes and macrophages compared to conventional ELISAs (5-10 fold higher).
- No pro-IL-1 beta was detected in supernatants of monocytes or macrophages, despite detectable mature IL-1 beta, indicating intracellular localization.
Conclusions:
- A pro-IL-1 beta specific ELISA is crucial for accurate quantification of pro-IL-1 beta, overcoming the underestimation by conventional IL-1 beta ELISAs.
- Conventional IL-1 beta ELISAs are prone to overestimating mature IL-1 beta due to cross-reactivity with processed forms.
- Intracellular pro-IL-1 beta is the predominant form in monocytes and macrophages, highlighting the need for specific assays to study its cellular regulation and release.