title: Standardized antigen preparation to achieve comparability of anti-beta2-glycoprotein I assays.
abstract: BACKGROUND
  The Sydney classification for diagnosis of the antiphospholipid syndrome (APS) first introduced the determination of anti-beta2-glycoprotein I (anti-beta2-GPI)-antibodies in serum as laboratory criteria. In this context, widely differing results of anti-beta2-GPI assays are a concerning issue. Considerable efforts have been made to optimize ELISAs, however little attention was hitherto spent to the antigen preparation. We evaluated the influence of different beta2-GPI preparations on the ability to separate ill and healthy patients and on the comparability of anti-beta2-GPI-assays.
  
  
  METHODS
  Microplates were coated with various beta2-GPI preparations and anti-beta2-GPI IgG- and IgM-ELISAs were performed for 21 APS patients and 21 controls using the monoclonal calibrators HCAL and EY2C9. Subsequently, by use of a surface plasmon resonance (SPR) biosensor, affinity constants for the HCAL- and EY2C9-interaction with each beta2-GPI preparation were determined and antigen binding of sera of APS patients and controls was studied.
  
  
  RESULTS
  All ss2-GPI preparations showed good discrimination ability ill vs. healthy but poor inter-assay comparability in the ELISAs. Affinity constants for HCAL and EY2C9 were independent of the beta2-GPI variant (K(A) 0.105 - 0.200 and 0.449 - 1.04 x 10(10)M(-1); K(D) 50.0 - 95.5 and 9.61 - 22.3 x 10(-11)M, respectively). In the biosensor, reactivity to the different beta2-GPIs was negligible for the controls and varied considerably for patients.
  
  
  CONCLUSIONS
  Inter-assay comparability of anti-beta2-GPI ELISAs is highly dependent upon the beta2-GPI preparation. Only agreement on one common beta2-GPI preparation will improve the requested inter-assay comparability.
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Str. 22, D-81675 München, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Müller
authors.forename: Alice
authors.initials: A
authors.lastname: Schlichtiger
authors.forename: Gudrun
authors.initials: G
authors.lastname: Balling
authors.forename: Udo
authors.initials: U
authors.lastname: Steigerwald
authors.forename: Peter B
authors.initials: PB
authors.lastname: Luppa
authors.forename: Markus
authors.initials: M
authors.lastname: Thaler
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D016736:Antiphospholipid Syndrome / Q000175:diagnosis*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D020349:Surface Plasmon Resonance; D053482:beta 2-Glycoprotein I / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D053482:beta 2-Glycoprotein I