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Updated: May 24, 2026
Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety
Published on: September 15, 2023
Reconstituting National Institute for Biological Standards and Control (NIBSC) chemokines
Margaret E Hoadley1, Sylvia Scarth, Stephen J Hopkins
1Brain Injury Research Group, Clinical Sciences Building, Salford Royal Foundation Trust, Salford M6 8HD, UK. margaret.hoadley@manchester.ac.uk
Insights
Discrepancies in immunoassay results for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were linked to preparation methods. Early protein addition is crucial to prevent loss of immunoreactivity during chemokine sample preparation.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Immunoassay results for cytokines like IL-8 and MCP-1 can vary significantly.
- Internal reference preparations from institutions like NIBSC may exhibit discrepancies.
- Chemokine stability is critical for accurate quantification in biological samples.
Purpose of the Study:
- To investigate the causes of discrepancies in immunoassay results for IL-8 and MCP-1.
- To determine the impact of preparation diluents and protein incorporation timing on chemokine stability.
- To establish best practices for preparing chemokine reference materials to ensure assay reliability.
Main Methods:
- Chemokine preparations (IL-8, MCP-1) were made using diluents with varying protein addition steps.
- Immunoreactivity of prepared chemokines was assessed using immunoassays.
- The effect of water exposure on chemokine preparations without added protein was evaluated.
Main Results:
- Significant discrepancies were observed between immunoassay results using different reference preparations.
- Even brief exposure to water, without additional protein, led to a loss of immunoreactivity.
- The timing of protein incorporation during sample preparation directly influenced chemokine stability and assay performance.
Conclusions:
- Early incorporation of protein into diluents is essential for maintaining chemokine integrity.
- Improper sample preparation, particularly inadequate protein stabilization, can lead to significant loss of immunoreactivity.
- Standardized protocols emphasizing early protein addition are vital for reliable IL-8 and MCP-1 immunoassay results.
Abstract:
We observed significant discrepancies between immunoassay results when using different internally prepared reference preparations for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from the National Institute for Biological Standards and Control (NIBSC). To evaluate the reasons for this we prepared the chemokines using diluents that incorporated protein at different steps. This showed that even brief addition of water to these preparations, in the absence of additional protein, resulted in loss of immunoreactivity in assays. The data obtained emphasise the importance of adding protein at an early stage of preparation to avoid loss of material and potential loss of activity.
