Novel monoclonal antibodies against mouse C3 interfering with complement activation: description of fine specificity and applications to various immunoassays.
The role of complement proteins in various pathophysiological settings has been studied primarily using mouse models of disease. However, the specific contribution of C3-derived fragments to these biologic processes has not been addressed in a rigorous manner because of a lack of antibodies that can selectively recognize mouse C3 or any of its degradation fragments. Here we report the generation and characterization of a panel of rat monoclonal antibodies reacting with mouse C3 and its degradation products. We describe their performance in various immunological assays such as ELISA, Western blotting, flow cytometry and immunohistochemistry. Of all the antibodies generated, one selectively recognized the C3a anaphylatoxin, and all other reacted with C3c. Furthermore, two monoclonal antibodies preferentially reacted with the cleaved C3 fragments C3b/iC3b/C3c but not native C3. Except for the one recognizing C3a, all antibodies were suitable for detecting C3 deposited on cells and tissues, two effectively inhibited the hemolytic activity of mouse complement and one enhanced C3-deposition to the cell membrane. These novel monoclonal antibodies may serve as useful reagents for elucidating functions mediated by C3-derived fragments in various pathophysiological conditions.
Mastellos
Dimitrios
D
Department of Pathology and Laboratory Medicine, University of Pennsylvania, 401 Stellar Chance, Philadelphia, PA 1910, USA.
Prechl
József
J
László
Glória
G
Papp
Krisztián
K
Oláh
Eszter
E
Argyropoulos
Emelia
E
Franchini
Silvia
S
Tudoran
Ruxandra
R
Markiewski
Maciej
M
Lambris
John D
JD
Erdei
Anna
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D003167:Complement Activation; D003176:Complement C3 / Q000032:analysis* / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006825:Hybridomas / Q000276:immunology; D007118:Immunoassay; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000166:cytology / Q000276:immunology
D000911:Antibodies, Monoclonal; D003176:Complement C3