FACTOR-J, AN INHIBITOR OF THE COMPLEMENT CLASSICAL PATHWAY - THE QUANTITATION BY AN ELISA INHIBITION ASSAY IN NORMAL HUMAN SERUM

Factor J (FJ) is a protein present in human serum, with inhibitory act ivity against C1. Here we describe the quantitation of FJ in human ser um by means of an ELISA inhibition assay. We have purified FJ from the urine of a normal donor following a previously published method with slight modifications. Polyclonal anti-FJ antibodies have been raised i n rabbits immunized with a single dose of purified antigen injected in multiple sites. IgG from polyclonal FJ antiserum, coupled to a solid matrix (Affi-Prep gel) was able to adsorb purified FJ antigenically an d functionally. Furthermore, anti-FJ specifically retained serum compo nents antigenically related with urine FJ. Taking into account this re activity, we have developed an inhibition enzyme-linked immunosorbent assay (ELISA) useful for measuring FJ levels in normal human serum. Th is immunoassay involves preincubating polyclonal anti-FJ with differen t dilutions of normal human serum to quantitatively reduce the antibod y available to bind to purified FJ-coated microtiter plates. Binding o f remaining antibody to the microtiter plate is measured spectrophotom etrically using peroxidase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of purified FJ. C onditions for optimization of this quantitative assay have been assess ed, including trials with different blocking agents, of which nonfat m ilk gave the best results. Preliminary experiments showed the existenc e of paradoxical effects, that is, high nonspecific binding at high se rum dilutions. We have eliminated these effects by including high ioni c strength (0.4 M NaCl) in the sample incubation solution. Sensitivity and reproducibility parameters have also been established. FJ levels have been measured for the first time in sera from 86 healthy donors. A wide range of FJ levels was observed, with a mean value of 5.4 +/- 2 .8 mg/L.