Related Experiment Videos
Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping
B Nilsson1, D Grossberger, K Nilsson Ekdahl
1Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden.
The Biochemical Journal
|March 15, 1992
Summary
This study maps complement component C3 (C3) epitopes using cDNA expression, revealing distinct locations for denatured versus bound C3 fragments. Findings differentiate epitopes on C3 fragments found on erythrocytes from those in fluid phase or denatured C3.
Area of Science:
- Immunology
- Molecular Biology
- Complement System Research
Background:
- Previous studies identified complement component C3 (C3) epitopes, termed C3(D), present in denatured and surface-bound C3 and its fragments.
- These C3(D) epitopes were previously detected using antibodies raised against denatured C3.
Purpose of the Study:
- To precisely localize C3(D) epitopes recognized by specific monoclonal and polyclonal anti-C3(D) antibodies.
- To differentiate between epitopes exposed on erythrocyte-bound C3 fragments versus fluid-phase or denatured C3.
Main Methods:
- Generated C3 cDNA fragments (200-300 bp) via DNAse I digestion.
- Expressed C3 cDNA fragments as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector.
- Utilized Western-blot analysis with monoclonal and polyclonal anti-C3 antibodies, followed by cDNA sequencing to map epitope locations.
Main Results:
- Polyclonal anti-C3(D) antibodies reacted with fusion proteins corresponding to C3c (residues 1477-1510) and C3d fragments (residues 1117-1155, 1234-1294).
- Monoclonal antibodies specific for EAC3b/EAC3bi (residues 1312-1404) and EAC3d (residues 1082-1118) bound to specific fusion protein segments.
- Antibodies recognizing only denatured C3 bound to fragments (residues 973-1026, 1477-1510), distinct from those on erythrocyte-bound C3.
Conclusions:
- Identified specific C3 segments (residues 1082-1118, 1117-1155, 1234-1294, 1312-1404) harboring C3(D) epitopes expressed on erythrocyte-bound C3 fragments.
- Determined that C3 segments (residues 973-1026, 1477-1510) contain C3(D) epitopes exclusively exposed in denatured C3, hidden in physiological fragments.