Variability in exposure of epitope G40-R43 of domain i in commercial anti-beta2-glycoprotein I IgG ELISAs.
BACKGROUND
A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β2glycoprotein I (β2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β2GPI opens up, thereby exposing G40-R43.


OBJECTIVE
To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays.


METHODS
Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β2GPI irrespective of its conformation. These antibodies were tested in commercial anti-β2GPI assays (A-E).


RESULTS
In assay A, both antibodies showed equal reactivity towards β2GPI, indicating that all the β2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43.


CONCLUSIONS
Exposure of epitope G40-R43 on β2GPI is highly variable between commercial anti-β2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.
Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands.
Leonie
L
Pelkmans
Hilde
H
Kelchtermans
Philip G
PG
de Groot
Stephane
S
Zuily
Veronique
V
Regnault
Denis
D
Wahl
Vittorio
V
Pengo
Bas
B
de Laat
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D016736:Antiphospholipid Syndrome / Q000175:diagnosis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult; D053482:beta 2-Glycoprotein I / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D011933:Reagent Kits, Diagnostic; D053482:beta 2-Glycoprotein I