A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3.
A monoclonal antibody raised against SDS-denatured C3 was shown to react with both solid-phase C3a and unfragmented C3. However, in the fluid phase the antibody was found to bind only to C3a and not to native C3. These findings indicated that the antibody could be used in an assay to detect C3a in human EDTA-plasma without prior separation of C3a from native C3. A simple and rapid competition ELISA was developed which monitored soluble C3a. 200 microliter of C3a (8 ng) was absorbed to plastic wells over night at 4 degrees C. Thereafter, 50 microliter of sample and 50 microliter of constant amounts of monoclonal antibody conjugated with beta-galactosidase, were incubated for 60 min at 37 degrees C. After washing, the colour reaction was started by adding nitrophenyl-galactopyridine to the wells. The microtitre plate was incubated at 37 degrees C for 30 min and the staining intensity was quantified at 405 nm. The assay detected both C3a and C3ades arg. A strong correlation was obtained between the new technique and an RIA which used an acid precipitation step for the separation of C3a prior to the determination of C3a (r = 0.9). Significantly higher levels of C3a were detected both in plasma from patients with immune complexes (93 +/- 9 ng/ml; P less than 0.1) and in plasma from patients treated in blood oxygenators (140 +/- 19 ng/ml; P less than 0.05) than in plasma from normal subjects (74 +/- 4 ng/ml). The results were not affected by repeated freezing and thawing of the plasma samples.
Nilsson
B
B
Blood Centre, University Hospital, Uppsala, Sweden.
Svensson
K E
KE
Inganäs
M
M
Nilsson
U R
UR
D000818:Animals; D000911:Antibodies, Monoclonal; D003176:Complement C3 / Q000032:analysis*; D015926:Complement C3a; D003165:Complement System Proteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011489:Protein Denaturation; D011863:Radioimmunoassay / Q000379:methods
D000911:Antibodies, Monoclonal; D003176:Complement C3; D007202:Indicators and Reagents; D015926:Complement C3a; D003165:Complement System Proteins