Simultaneous detection and epitope mapping of anti-factor VIII antibodies.
The development of antibodies (Abs) against infused factor VIII (FVIII) is currently one of the most serious complications in the treatment of patients suffering from haemophilia A. Improved prevention and eradication of these anti-FVIII Abs remain a challenge for both clinicians and scientists. Here we describe an immunoassay to simultaneously detect and map the epitope specificity of haemophilia A patients' inhibitors by screening plasma against both heavy and light chains (HC and LC) of human plasma-derived FVIII (pFVIII). The format used was a two-site sandwich assay, where one monoclonal antibody (mAb) specific for the HC or LC was first immobilized on beads, and then incubated with the different forms of pFVIII. After incubation with patients' plasma samples, binding was revealed by a phycoerythrin-labeled secondary Ab. Samples from haemophilia patients with autoantibodies (autoAb) or alloantibodies (alloAb) were screened in this format. The former preferentially recognized the LC, whereas the latter were directed against both LC and HC. This technology appears attractive as it is fast and requires only 100 microl of patient's plasma. Furthermore, not only are anti-FVIII Abs detected, but information on their epitopic specificity is also obtained.
Lavigne-Lissalde
Géraldine
G
CNRS FRE 3009, Cap Delta/Parc Euromédecine, Montpellier, France.
Tarrade
Catherine
C
Lapalud
Priscilla
P
Chtourou
Sami
S
Schved
Jean-François
JF
Granier
Claude
C
Villard-Saussine
Sylvie
S
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000097:blood*; D018604:Epitope Mapping; D005169:Factor VIII / Q000276:immunology*; D005240:Feasibility Studies; D005602:France; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007518:Isoantibodies / Q000097:blood*; D051379:Mice; D013557:Switzerland
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007518:Isoantibodies; D005169:Factor VIII