Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures.
Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.
Hartmann
H
H
ABION GmbH, Jülich, Germany.
Lübbers
B
B
Casaretto
M
M
Bautsch
W
W
Klos
A
A
Köhl
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods; D015926:Complement C3a / Q000032:analysis* / Q000235:genetics; D015936:Complement C5a / Q000032:analysis* / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D015926:Complement C3a; D015936:Complement C5a