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 activity against C1. Here we describe the quantitation of FJ in human serum 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 in 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 and functionally. Furthermore, anti-FJ specifically retained serum components antigenically related with urine FJ. Taking into account this reactivity, we have developed an inhibition enzyme-linked immunosorbent assay (ELISA) useful for measuring FJ levels in normal human serum. This immunoassay involves preincubating polyclonal anti-FJ with different dilutions of normal human serum to quantitatively reduce the antibody available to bind to purified FJ-coated microtiter plates. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using peroxidase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of purified FJ. Conditions for optimization of this quantitative assay have been assessed, including trials with different blocking agents, of which nonfat milk gave the best results. Preliminary experiments showed the existence of paradoxical effects, that is, high nonspecific binding at high serum dilutions. We have eliminated these effects by including high ionic 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jiménez-Clavero
M A
MA
Unidad de Inmunología, Hospital la Paz, Madrid, Spain.
González-Rubio
C
C
Fontán
G
G
López-Trascasa
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D003174:Complement C1 Inactivator Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D003169:Complement Inactivator Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D011817:Rabbits; D012016:Reference Values; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003174:Complement C1 Inactivator Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; C116021:factor J protein, human