Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy
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Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy. / Dähnrich, Cornelia; Komorowski, Lars; Probst, Christian; Seitz-Polski, Barbara; Esnault, Vincent; Wetzels, Jack F; Hofstra, Julia M; Hoxha, Elion; Stahl, Rolf A; Lambeau, Gérard; Stöcker, Winfried; Schlumberger, Wolfgang.
in: CLIN CHIM ACTA, Jahrgang 421, 05.06.2013, S. 213-8.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy
AU - Dähnrich, Cornelia
AU - Komorowski, Lars
AU - Probst, Christian
AU - Seitz-Polski, Barbara
AU - Esnault, Vincent
AU - Wetzels, Jack F
AU - Hofstra, Julia M
AU - Hoxha, Elion
AU - Stahl, Rolf A
AU - Lambeau, Gérard
AU - Stöcker, Winfried
AU - Schlumberger, Wolfgang
N1 - Copyright © 2013 Elsevier B.V. All rights reserved.
PY - 2013/6/5
Y1 - 2013/6/5
N2 - BACKGROUND: Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using recombinant PLA2R1 as a substrate has been widely available but lacks a finely graduated assessment of antibody concentrations.METHODS: In order to setup a standardized ELISA, the extracellular domain of human PLA2R1 was expressed in HEK293. The purified protein was used to form the solid-phase in an ELISA which was then employed to analyze sera from 200 patients with primary MN, 27 patients with secondary MN, 230 patients with other glomerular diseases, 316 patients with systemic autoimmune diseases, and from 291 healthy blood donors.RESULTS: At a set specificity of 99.9% the sensitivity of the anti-PLA2R1 IgG ELISA was found to be 96.5%. A similar sensitivity (98.5%) was obtained when binding of only subclass IgG4 was analyzed. The calibrated assay showed a good class correlation with the results of the RC-IFA, was robust and could be stored for several months without any loss of quality.CONCLUSION: The results demonstrate that the new test system is qualified for routine use and that it has an almost perfect agreement with both, the clinical characterization of the patients and the results generated with RC-IFA.
AB - BACKGROUND: Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using recombinant PLA2R1 as a substrate has been widely available but lacks a finely graduated assessment of antibody concentrations.METHODS: In order to setup a standardized ELISA, the extracellular domain of human PLA2R1 was expressed in HEK293. The purified protein was used to form the solid-phase in an ELISA which was then employed to analyze sera from 200 patients with primary MN, 27 patients with secondary MN, 230 patients with other glomerular diseases, 316 patients with systemic autoimmune diseases, and from 291 healthy blood donors.RESULTS: At a set specificity of 99.9% the sensitivity of the anti-PLA2R1 IgG ELISA was found to be 96.5%. A similar sensitivity (98.5%) was obtained when binding of only subclass IgG4 was analyzed. The calibrated assay showed a good class correlation with the results of the RC-IFA, was robust and could be stored for several months without any loss of quality.CONCLUSION: The results demonstrate that the new test system is qualified for routine use and that it has an almost perfect agreement with both, the clinical characterization of the patients and the results generated with RC-IFA.
KW - Adolescent
KW - Adult
KW - Aged
KW - Aged, 80 and over
KW - Autoantibodies
KW - Biological Markers
KW - Case-Control Studies
KW - Enzyme-Linked Immunosorbent Assay
KW - Female
KW - Gene Expression
KW - Glomerulonephritis, Membranous
KW - HEK293 Cells
KW - Humans
KW - Immunoglobulin G
KW - Male
KW - Middle Aged
KW - Protein Structure, Tertiary
KW - Receptors, Phospholipase A2
KW - Sensitivity and Specificity
U2 - 10.1016/j.cca.2013.03.015
DO - 10.1016/j.cca.2013.03.015
M3 - SCORING: Journal article
C2 - 23541686
VL - 421
SP - 213
EP - 218
JO - CLIN CHIM ACTA
JF - CLIN CHIM ACTA
SN - 0009-8981
ER -