Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G

Kazuo Hocchi, Tatsuya Ohashi, Toshihide Miura, Kumiko Sasagawa, Yasuhito Sato, Fumio Nomura, Takeshi Tomonaga, Masahiko Sunaga, Ryo Kojima, Katsuhiro Katayama, Toshiyuki Kato, Toyoji Sato, Tsugikazu Komoda, Kimimitsu Oda

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3 Citations (Scopus)

Abstract

A convenient method for measuring immune complexes between tissue-non-specific 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 horse-radish peroxidase (HRP)-conjugated anti-human IgGI 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 IgGI constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.

Original languageEnglish
Pages (from-to)322-329
Number of pages8
JournalJournal of Clinical Laboratory Analysis
Volume21
Issue number5
DOIs
Publication statusPublished - 2007

Keywords

  • Arbitrary unit (AU)
  • ELISA
  • Immune complexes
  • Immunoglobulin G
  • Semiquantitative assay
  • Surface-active agent
  • TNSALP-IgG complexes
  • Tissue-nonspecific alkaline phosphatase

ASJC Scopus subject areas

  • Immunology and Allergy
  • Hematology
  • Public Health, Environmental and Occupational Health
  • Clinical Biochemistry
  • Medical Laboratory Technology
  • Biochemistry, medical
  • Microbiology (medical)

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  • Cite this

    Hocchi, K., Ohashi, T., Miura, T., Sasagawa, K., Sato, Y., Nomura, F., Tomonaga, T., Sunaga, M., Kojima, R., Katayama, K., Kato, T., Sato, T., Komoda, T., & Oda, K. (2007). Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G. Journal of Clinical Laboratory Analysis, 21(5), 322-329. https://doi.org/10.1002/jcla.20192