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Antibodies to macrophage stimulating protein (MSP): specificity, epitope interactions, and immunoassay of MSP in
M H Wang1, A Skeel, T Yoshimura
1Immunopathology Section, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.
Abstract:
Macrophage stimulating protein (MSP) is a member of a family of proteins characterized by a triple disulfide loop structure (kringle). We developed antibodies to human MSP for detection in Western blots, quantification in biological fluids, and neutralization of activity. Immunogens included native MSP, reduced and alkylated alpha and beta chains, and peptides of MSP regions with minimal sequence similarity to other kringle proteins. We found three antibody categories based on interaction with the following types of epitope: primary sequence, discontinuous (dependent on disulfide bonds), and cryptic (not exposed in native MSP). None of the antibodies reacted with related kringle proteins. A specific sandwich ELISA was developed for measuring human MSP. The mean serum concentration was 4 nM. Serum MSP did not increase over a 24-h period in response to intravenous lipopolysaccharide, indicating that MSP is not an acute phase protein. These findings are consistent with the hypothesis that regulation of MSP activity is by conversion of pro-MSP to MSP rather than by rapid changes in rates of synthesis.
Insights
Researchers developed specific antibodies and an ELISA to measure macrophage stimulating protein (MSP). Findings suggest MSP is not an acute phase protein, with regulation likely occurring via pro-MSP conversion.
Area of Science:
- Biochemistry
- Immunology
Background:
- Macrophage stimulating protein (MSP) belongs to a protein family with a characteristic triple disulfide loop (kringle) structure.
- Understanding MSP regulation and developing detection methods are crucial for its study.
Purpose of the Study:
- To develop antibodies for detecting and quantifying human MSP.
- To neutralize MSP activity and investigate its role as an acute phase protein.
Main Methods:
- Generation of antibodies using various MSP immunogens (native, chains, peptides).
- Characterization of antibody epitope specificities (primary sequence, disulfide-dependent, cryptic).
- Development of a sandwich ELISA for serum MSP quantification.
Main Results:
- Developed antibodies that specifically recognize human MSP without cross-reacting with related kringle proteins.
- Established a sandwich ELISA with a mean detectable serum MSP concentration of 4 nM.
- Demonstrated that serum MSP levels do not increase after lipopolysaccharide administration, indicating it's not an acute phase protein.
Conclusions:
- The developed antibodies and ELISA are effective tools for MSP research.
- MSP is likely regulated by conversion from its precursor (pro-MSP) rather than rapid synthesis changes.
- MSP does not appear to be an acute phase reactant protein.