title: Specificity of two monoclonal antibodies against a synthetic glycopeptide, an analogue to the hypo-galactosylated IgA1 hinge region.
abstract: Increased levels of hypo-galactosylated immunoglobulin (Ig)A1 (HG-IgA1) in IgA nephropathy (IgAN) have been detected using a Helix aspersa agglutinin lectin enzyme-linked immunosorbent assay (ELISA). In this study, we developed monoclonal antibodies to evaluate the HG-IgA1 in IgA nephropathy, aiming to gain a more consistent and reproducible assay. As an analogue to the HG-IgA1 hinge region, a 19 mer synthetic peptide with five GalNAc (sHGP) residues at positions 4, 7, 9, 11 and 15 [VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT (GalNAc)PSPS-NH2] was synthesized. Two monoclonal antibodies against sHGP (35A12 and 44H8) that reacted with human IgA were developed. Also, their reactivities to serum IgA from IgAN patients (n = 49), patients with other forms of kidney diseases (OKD, n = 48), and healthy controls (HC, n = 41) were evaluated using ELISA assays. The binding levels of the two monoclonal antibodies against serum IgA were significantly higher (all comparisons, p < 0.0001, Steel-Dwass non-parametric test) in IgAN patients compared to HC and OKD patients. In each individual, there was a close correlation of IgA binding levels between 35A12 and 44H8 (R(2) = 0.737). These results indicate that the monoclonal antibodies recognize similar epitopes in HG IgA1, which is found predominantly in IgAN patients. The developed antibodies are proposed as a clinically useful tool for IgAN screening.
authors.affiliation: Department of Clinical Engineering, School of Health Sciences, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi, 470-1192, Japan, yoshi-cake@mtj.biglobe.ne.jp.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Hiki
authors.forename: Hideo
authors.initials: H
authors.lastname: Hori
authors.forename: Kouichiro
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yuzawa
authors.forename: Nobuya
authors.initials: N
authors.lastname: Kitaguchi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D019540:Area Under Curve; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D005922:Glomerulonephritis, IGA / Q000097:blood / Q000175:diagnosis*; D006020:Glycopeptides / Q000276:immunology*; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood / Q000276:immunology*; D007674:Kidney Diseases / Q000097:blood / Q000175:diagnosis; D012372:ROC Curve
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D006020:Glycopeptides; D007070:Immunoglobulin A; C042441:glycosylated IgA