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Soluble forms of membrane cofactor protein (CD46, MCP) are present in plasma, tears, and seminal fluid in normal
T Hara1, S Kuriyama, H Kiyohara
1Department of Immunology, Centre for Adult Diseases Osaka, Japan.
Abstract:
We have established an ELISA for determination of membrane cofactor protein (MCP, CD46) both solubilized from cell membranes and released in body fluids. In this assay, mouse MoAbs against MCP, M177 and M160 whose epitopes were different, were used as capture and detection antibodies, respectively. The NP-40 concentration in samples for MCP to be measured must be less than 0.05%. The detection limit of this MCP assay was 0.5 ng. The assay was used to quantify solubilized membrane MCP, and soluble MCP in normal human plasma, serum, urine, saliva, tears, and seminal fluid, and culture media of tumour cell lines. Soluble MCP was barely detected in the conditioned media of the cell lines. The levels of sMCP in plasma and serum were 10-60 ng/ml and that in tears, 0-50 ng/ml. Seminal fluid contained about 10-fold more soluble MCP than serum. Soluble MCP was not detectable by this assay in the other body fluids, suggesting that their MCP levels were less than the detection limit, if any.
Insights
We developed an ELISA to measure membrane cofactor protein (MCP, CD46) in body fluids and cell membranes. This assay accurately quantifies MCP levels, revealing higher concentrations in seminal fluid compared to serum.
Area of Science:
- Immunology
- Biochemistry
Background:
- Membrane cofactor protein (MCP, CD46) plays a crucial role in immune regulation.
- Quantification of soluble MCP (sMCP) in various biological samples is essential for understanding its physiological and pathological functions.
Purpose of the Study:
- To establish and validate an Enzyme-Linked Immunosorbent Assay (ELISA) for the accurate determination of MCP.
- To quantify MCP levels in different human body fluids and cell membrane preparations.
Main Methods:
- Development of a sandwich ELISA utilizing distinct mouse monoclonal antibodies (MoAbs) against MCP (M177 and M160).
- Optimization of assay conditions, including NP-40 concentration (<0.05%) and antibody epitope specificity.
- Validation of the assay with a detection limit of 0.5 ng.
Main Results:
- The assay successfully quantified solubilized membrane MCP and sMCP in human plasma, serum, urine, saliva, tears, and seminal fluid.
- Soluble MCP was detected at 10-60 ng/ml in plasma and serum, and 0-50 ng/ml in tears.
- Seminal fluid exhibited approximately 10-fold higher sMCP levels than serum, while sMCP was barely detected in tumor cell line culture media and undetectable in other tested body fluids.
Conclusions:
- The developed ELISA provides a reliable method for quantifying MCP in diverse biological matrices.
- The findings reveal differential distribution of sMCP across human body fluids, with significantly higher levels in seminal fluid.
- This assay facilitates further research into the biological roles and clinical implications of MCP.