Specificity of three anti-complement factor 3 monoclonal antibodies.
Monoclonal antibodies which recognize specific C3 fragments may be used to distinguish C3 cleavage products bound to organisms. We defined the specificity of three commercially available monoclonal antibodies by Western immunoblot analysis, enzyme-linked immunosorbent assay, and a quantitative flow cytometric technique. Two monoclonal antibodies with specificity for (i) an erythrocyte-bound C3d epitope or (ii) an erythrocyte-bound C3c epitope retained their specificity in all assays. However, the third monoclonal antibody with selectivity for erythrocyte-bound C3bi failed to retain specificity for C3bi bound to non-erythrocyte surfaces in each of our assays; binding instead to all C3 fragments containing the C3g domain. We postulate that erythrocyte C3b-binding surface proteins may alter the availability of certain C3b epitopes and influence observed anti-C3 monoclonal antibody specificity. We conclude that the specificity of monoclonal antibodies for C3 fragments should be confirmed with assays which do not employ erythrocytes or other surfaces bearing C3 receptors. Also, our quantitative flow cytometric technique is a potentially valuable tool for the enumeration of particle-bound C3 fragments.
Henwick
S
S
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38101-0318.
Hetherington
S V
SV
Hostetter
M K
MK
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D003176:Complement C3 / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D003176:Complement C3