ELISA for antiphosphatidylethanolamine antibody detection: high impact of assay buffer on results.
The aim of this study was to evaluate the influence on the results of the main variables of ELISA used for the detection of antiphosphatidylethanolamine antibodies (aPE). Forty sera from patients with either autoimmune disorders including antiphospholipid syndrome (APS) or the clinical features of APS only were assayed by ELISA performed under different conditions. Variables were sources of PE (egg yolk, soybean, bovine brain or Escherichiacoli), microtiter plates (plain or gamma irradiated) and buffer components-fetal calf serum (FCS), adult bovine plasma (ABP), adult bovine serum (ABS) or bovine serum albumin (BSA). aPE binding was decreased with PE from E. coli while the other tested PE gave comparable results. The influence of the type of plates was restricted to IgM isotype with slightly, but significantly higher optical densities with plain than with irradiated plates. Most importantly, the component buffer had the highest impact on the results as shown by a strong decrease of the signal by ABP or ABS. This inhibitory effect was confirmed by using mixtures of FCS or BSA with increasing concentrations of ABS. Partial delipidation of ABS resulted in a recovery of OD levels close to those obtained with FCS. This study is the first to demonstrate that aPE reactivity is dependent on the lipid concentration of the buffer component. These results highlight the need for standardization of aPE-ELISA for a better understanding of their clinical significance.
Sanmarco
Marielle
M
Laboratoire d'Immunologie, Hôpital de La Conception, Marseille, France. msanmarco@ap-hm.fr
D000328:Adult; D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000097:blood* / Q000276:immunology*; D016736:Antiphospholipid Syndrome / Q000276:immunology; D001327:Autoimmune Diseases / Q000276:immunology; D002021:Buffers; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology; D007075:Immunoglobulin M / Q000032:analysis / Q000276:immunology; D008055:Lipids / Q000032:analysis; D008297:Male; D008875:Middle Aged; D010714:Phosphatidylethanolamines / Q000276:immunology*; D044967:Serum / Q000737:chemistry
D017152:Antibodies, Antiphospholipid; D002021:Buffers; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008055:Lipids; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine