title: Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.
abstract: BACKGROUND
  Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA).
  
  
  METHODS
  Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens.
  
  
  RESULTS
  Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA.
  
  
  CONCLUSIONS
  This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease.
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan Kyowa Hakko Kirin Co., Ltd, Tokyo Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Yasutake
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan Kyowa Hakko Kirin Co., Ltd, Tokyo Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Hiura
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yanagawa
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Makita
authors.affiliation: Kyowa Hakko Kirin Co., Ltd, Tokyo Japan.
authors.forename: Etsuji
authors.initials: E
authors.lastname: Kaneko
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tomino
mesh_terms: D000373:Agglutinins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D005690:Galactose / Q000172:deficiency*; D005922:Glomerulonephritis, IGA / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood*; D007678:Kidney Glomerulus / Q000378:metabolism / Q000473:pathology*; D037102:Lectins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000373:Agglutinins; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D007070:Immunoglobulin A; D037102:Lectins; D005690:Galactose
keywords: ELISA; IgA nephropathy; galactose-deficient IgA1; helix aspersa agglutinin (HAA); immunofluorescence; monoclonal antibody