A new method for the estimation of C3d. Affinity clearance of C-determinant-bearing C3 molecules and fragments followed by estimation of C3d by ELISA.
A method is described to quantitate human complement fragment C3d. Test samples were treated with a predetermined excess of anti-C3c-Sepharose beads in the presence of EDTA to remove all the C-determinant-bearing C3 molecules or fragments. C3d left in the supernatant was then estimated by ELISA. Using this method, C3d could be estimated accurately in normal plasma samples. A good correlation (r = 0.93) was observed between C3d values obtained by this method and values obtained by the widely used method of Perrin and coworkers. The average C3d plasma concentration was 2.8 mg/l (SD = 0.7 mg/l, n = 21). The interassay coefficient of variation using a normal plasma pool (C3d 2.7 mg/l) was 8.3% and using normal plasma pools in which the C3d concentrations were raised to 10.3 and 17.4 mg/l by the addition of aged normal serum the levels were 8.0 and 7.5% respectively. Intra-assay coefficients of variation with these samples were 4.6, 3.0 and 2.8%, respectively. 16 patients with renal dysfunction had C3d levels in the range of 4.3-10.0 mg/l and 15 patients undergoing continued ambulant peritoneal dialysis had levels of 3.3-12.2 mg/l. The C3d content in peritoneal dialysate of patients undergoing dialysis varied from 9.3 to 383 micrograms/l.
Asghar
S S
SS
Department of Dermatology, University of Amsterdam, The Netherlands.
Schraag
B
B
Backhaus
A H
AH
Zorn
I
I
Venneker
G T
GT
Hannema
A J
AJ
D000918:Antibody Specificity; D002846:Chromatography, Affinity; D003176:Complement C3 / Q000032:analysis* / Q000302:isolation & purification; D003179:Complement C3b / Q000302:isolation & purification; D015933:Complement C3d; D004492:Edetic Acid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D006801:Humans; D007674:Kidney Diseases / Q000097:blood; D010531:Peritoneal Dialysis, Continuous Ambulatory; D013997:Time Factors
D003176:Complement C3; D000939:Epitopes; D003179:Complement C3b; D015933:Complement C3d; D004492:Edetic Acid