title: Application of a monoclonal antibody against a neoepitope on activated C4 in an ELISA for the quantification of complement activation via the classical pathway.
abstract: In order to study the activation of the complement system via the classical pathway we have attempted to raise antibodies specific for C4 activation products. Of 20 mouse monoclonal antibodies (mAbs) obtained, one appeared to react with an activation dependent epitope exposed on the activation products C4b, C4bi, C4c (C4b/c) as well as on iC4, but not on native C4. Using this antibody as a capture antibody and polyclonal biotinylated antibodies against C4 as detecting antibodies we developed an ELISA for the quantification of C4b/c in biological fluids. The lower limit of detection was approximately 0.025 nmol C4b/c per litre. Mean C4b/c levels in plasma samples collected from healthy volunteers in tubes containing 10 mM EDTA and 0.05% (w/v) polybrene, final concentrations, appeared to be 30 nmol/l. The potential of the ELISA procedure for evaluating complement activation in clinical samples was demonstrated.
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
authors.forename: G J
authors.initials: GJ
authors.lastname: Wolbink
authors.forename: J
authors.initials: J
authors.lastname: Bollen
authors.forename: J W
authors.initials: JW
authors.lastname: Baars
authors.forename: R J
authors.initials: RJ
authors.lastname: ten Berge
authors.forename: A J
authors.initials: AJ
authors.lastname: Swaak
authors.forename: J
authors.initials: J
authors.lastname: Paardekooper
authors.forename: C E
authors.initials: CE
authors.lastname: Hack
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000276:immunology / Q000628:therapy; D003167:Complement Activation / Q000276:immunology*; D003181:Complement C4 / Q000276:immunology*; D015935:Complement C4b / Q000276:immunology; D003171:Complement Pathway, Classical / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D007167:Immunotherapy; D007680:Kidney Neoplasms / Q000276:immunology / Q000628:therapy; D051379:Mice; D010446:Peptide Fragments; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D003181:Complement C4; D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; C033860:complement C4c; D015935:Complement C4b