Detection of autoantibodies against alpha-2-macroglobulin-like 1 in paraneoplastic pemphigus sera utilizing novel green fluorescent protein-based immunoassays.
Autoantibodies
/ blood
Diagnosis, Differential
Enzyme-Linked Immunosorbent Assay
/ methods
Green Fluorescent Proteins
/ genetics
HEK293 Cells
Humans
Paraneoplastic Syndromes
/ blood
Pemphigoid, Bullous
/ blood
Pemphigus
/ blood
Recombinant Fusion Proteins
/ genetics
Sensitivity and Specificity
alpha-Macroglobulins
/ genetics
Alpha-2-macroglobulin-like 1
Autoimmunity
Diagnostic immunoassays
Paraneoplastic pemphigus
Journal
Journal of dermatological science
ISSN: 1873-569X
Titre abrégé: J Dermatol Sci
Pays: Netherlands
ID NLM: 9011485
Informations de publication
Date de publication:
Jun 2020
Jun 2020
Historique:
received:
13
02
2020
revised:
31
03
2020
accepted:
16
04
2020
pubmed:
23
5
2020
medline:
15
4
2021
entrez:
23
5
2020
Statut:
ppublish
Résumé
Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies. To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera. We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates. A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values. Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.
Sections du résumé
BACKGROUND
BACKGROUND
Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies.
OBJECTIVES
OBJECTIVE
To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera.
METHODS
METHODS
We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates.
RESULTS
RESULTS
A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values.
CONCLUSIONS
CONCLUSIONS
Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.
Identifiants
pubmed: 32439251
pii: S0923-1811(20)30141-9
doi: 10.1016/j.jdermsci.2020.04.005
pii:
doi:
Substances chimiques
A2ML1 protein, human
0
Autoantibodies
0
Recombinant Fusion Proteins
0
alpha-Macroglobulins
0
enhanced green fluorescent protein
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Evaluation Study
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
173-178Informations de copyright
Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest None declared.