Neutrophil attractant protein-1-immunoglobulin G immune complexes and free anti-NAP-1 antibody in normal human serum.
After obtaining data indicating the presence of a neutrophil attractant protein-1 (NAP-1)-IgG complex in normal human serum, we developed sandwich ELISAs that could quantify NAP-1 and NAP-1-IgG in mixtures of the two moieties. The ELISA for free NAP-1 used a monoclonal capture antibody that did not bind NAP-1-IgG. The ELISA for NAP-1-IgG was based on omission of the anti-NAP-1 detection antibody (required for the free NAP-1 ELISA) and on interaction of phosphatase-conjugated anti-human IgG with the human NAP-1-IgG complex. Gel filtration of immunoaffinity-purified NAP-1-IgG showed that the bulk of the complex comprised a single IgG. Binding between NAP-1 and antibody is strong, since 8 M urea at neutral or alkaline pH did not release NAP-1. However, at pH 2.0 in 9 M urea approximately 15% of the total NAP-1 could be dissociated from the complex. NAP-1-IgG was detected in 18 of 26 sera from normal humans. The mean serum concentration was 58 ng of IgG-bound NAP-1/ml, with an SEM of 16 and a range from undetectable to 247 ng/ml. NAP-1-IgG concentrations in paired sera drawn at a 1-mo interval were remarkably constant. Using an ELISA for free NAP-1 with a detection limit of 200 pg/ml, we found no free NAP-1 in the 26 sera. Free anti-NAP-1-IgG autoantibody was found in 9 of 26 sera by direct ELISA. IgG anti-NAP-1 of all nine sera was polyclonal, comprising both kappa and lambda isotypes; predominant subclasses were IgG2 and IgG3. NAP-1-IgG did not compete with 125I-NAP-1 for binding to neutrophils, which suggests that IgG anti-NAP-1 is a molecular trap that prevents binding of NAP-1 to neutrophils after it diffuses from production sites into the circulation.
Sylvester
I
I
Immunopathology Section, National Cancer Institute, Frederick, Maryland 21702.
Yoshimura
T
T
Sticherling
M
M
Schröder
J M
JM
Ceska
M
M
Peichl
P
P
Leonard
E J
EJ
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000378:metabolism*; D001323:Autoantibodies / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D007074:Immunoglobulin G; D016209:Interleukin-8