QUANTIFICATION OF C5A C5A(DESARG) IN BOVINE PLASMA, SERUM AND MILK/

Complement activation generates two potent inflammatory mediators from C5, C5a and its derivative C5a(desArg), which results from the remova l of the C-terminal arginine by ubiquitous carboxypeptidases. In this paper we describe the purification of milligram amounts of bovine C5a( desArg) by a simplified procedure, and the preparation of mouse monocl onal antibodies (MAbs) to C5a/C5a(desArg) which do not recognize nativ e C5.A MAb was used to develop a sandwich ELISA which made it possible to quantify levels of C5a/C5a(desArg) in bovine biological fluids. Sm all amounts (means +/- SEM) of C5a/C5a(desArg) were found in EDTA-plas ma (0.58 +/- 0.06 ng.mL(-1)). The anticoagulant EDTA was more efficien t than citrate or heparin in inhibiting in vitro activation of the com plement system. Complement activation occurred during coagulation sinc e the baseline concentration of C5a/C5a(desArg) (15.4 +/- 4.1 ng.mL(-1 )) was higher than in plasma. Zymosan, a potent activator of the compl ement cascade, was used to generate C5a/C5a(desArg). The time-course o f the reaction and the dose-effect of zymosan were investigated. Optim al conditions were incubation at 39 degrees C for 1 or 2 h with 2 mg o f zymosan per mL of serum. The maximal concentration of C5a/C5a(desArg ) attained in zymosan-activated serum was 4.28 +/- 0.14 mu g.mL(-1). N ormal milk (from healthy, uninflamed mammary glands) contained on aver age 0.12 ng of C5a/C5a(desArg).mL(-1) (range 0.02-0.19 ng.mL(-1)). The maximal amount of C5a/C5a(desArg) which was generated in milk with zy mosan was 1.1 ng.mL(-1) (range 0.68-2.17 ng.mL(-1)). In milk from quar ters with subclinical infections by coagulase-negative staphylococci, values were 0.18 ng.mL(-1) and 2.37 ng.mL(-1) for spontaneous and zymo san-generated C5a/C5a(desArg) concentrations, respectively. In milk fr om Escherichia coli endotoxin-induced mastitis, C5a/C5a(desArg) concen trations (means of four cows) before and after zymosan activation reac hed 6.5 ng.mL(-1) and 55 ng.mL(-1), respectively. These results indica te that a C5-convertase can operate in normal milk, that only minute a mounts of C5a/C5a(desArg) can be generated (less than 1/1 000 of plasm a potential), but that much higher concentrations are reached in milk during endotoxin-induced inflammation. The ELISA made it possible to d etermine normal ranges of C5a/C5a(desArg) in bovine blood plasma and i n milk, and is a valuable tool to define the variations of its concent rations in exudates during inflammatory reactions. (C) Inra/Elsevier, Paris.