Open AccessDevelopment of an ELISA method for detecting immune complexes between tissue‐nonspecific alkaline phosphatase and immunoglobulin G
Author(s) -
Hocchi Kazuo,
Ohashi Tatsuya,
Miura Toshihide,
Sasagawa Kumiko,
Sato Yasuhito,
Nomura Fumio,
Tomonaga Takeshi,
Sunaga Masahiko,
Kojima Ryo,
Katayama Katsuhiro,
Kato Toshiyuki,
Sato Toyoji,
Komoda Tsugikazu,
Oda Kimimitsu
Publication year - 2007
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.20192
Subject(s) - alkaline phosphatase , horseradish peroxidase , chemistry , antibody , microbiology and biotechnology , absorbance , immunoglobulin g , monoclonal antibody , immunoassay , conjugated system , chromatography , enzyme , biochemistry , immunology , biology , organic chemistry , polymer
A convenient method for measuring immune complexes between tissue‐nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP‐IgG) would be highly useful for routine analyses. Here, we identified a surface‐active agent that would dissolve membrane but not dissociate TNSALP‐IgG complexes. Next, we developed an enzyme‐linked immunosorbent assay (ELISA) method for detecting TNSALP‐IgG complexes with two monoclonal antibodies (MoAbs): 3‐29‐3R was coated on assay plates and captured TNSALP‐IgG from a specimen; an horseradish peroxidase (HRP)‐conjugated anti‐human IgG1 then reacted with captured TNSALP‐IgG to form an “immunocomplex sandwich.” The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP‐IgG concentration. The ELISA values of patient sera known to contain TNSALP‐IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP‐IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3‐29‐3R MoAb and HRP‐conjugated anti‐human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP‐IgG complexes in human serum. J. Clin. Lab. Anal. 21:322–329, 2007. © 2007 Wiley‐Liss, Inc.