Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G.
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.
Hocchi
Kazuo
K
Division of Biochemistry, Niigata University Graduate School of Medicine and Dental Sciences, Niigata, Japan.
Ohashi
Tatsuya
T
Miura
Toshihide
T
Sasagawa
Kumiko
K
Sato
Yasuhito
Y
Nomura
Fumio
F
Tomonaga
Takeshi
T
Sunaga
Masahiko
M
Kojima
Ryo
R
Katayama
Katsuhiro
K
Kato
Toshiyuki
T
Sato
Toyoji
T
Komoda
Tsugikazu
T
Oda
Kimimitsu
K
D000469:Alkaline Phosphatase / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000097:blood*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D013501:Surface-Active Agents; D000469:Alkaline Phosphatase