An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

Summary

Two monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG₁ was used to coat polystyrene tubes, the second one, an IgG₂a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

Key words

Monoclonal antibodies - von Willebrand’s disease - Hybridomas - Immunoradiometric assay

• References

• 1 Kohler G, Milstein C. Derivation of specific antibody producing tissue culture and tumor cell lines by cell fusion. Eur J Immunol 1976; 6: 511-519
• 2 Fazekas de St-Groth S, Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immunol Methods 1980; 35: 1-21
• 3 Avner P, Arnaud D, Sultan Y, Maisonneuve P, Jeanneau Ch. Monoclonal antibodies against the human factor VIII von Willebrand molecule: characterization and potential for screening of von Willebrand patients. In “Developments in Biological Standardisation” Karger; Basle: 1984. (in press)
• 4 Devare SG, Stephenson JR. Biochemical and immunological characterization of the major envelope glycoprotein of bovine leukemia virus. J Virol 1977; 23: 443-447
• 5 Ruggeri ZM, Mannucci PM, Jeffcoate SL, Ingram GI C. Immunoradiometric assay of factor VIII related antigen with observations in 32 patients with von Willebrand’s disease. Br J Haematol 1976; 33: 221-232
• 6 Weiss HJ, Hoyer LW, Rickless FR, Varma A, Rogers J. Quantitative assay of a plasma factor deficient in von Willebrand’s disease that is necessary for platelet aggregation; relationship to decreased factor VIII procoagulant activity and antigen content. J Clin Invest 1973; 52: 2708-2715
• 7 Laurell CB. Antigen antibody crossed electrophoresis. Anal Biochem 1965; 10: 358-361
• 8 Ruggeri ZM, Zimmerman TS. Variant von Willebrand’s disease. Characterization of two subtypes by analysis of multimeric composition of FVIII/von Willebrand factor in plasma and platelets J Clin Invest 1980; 65: 1318-1325
• 9 Sultan Y, Simeon J, Caen JP. Detection of heterozygotes in both parents of homozygous patients with von Willebrand’s disease. J Clin Pathol 1975; 28: 309-316
• 10 Ruggeri ZM, Pareti FI, Mannucci PM, Ciavarella N, Zimmerman TS. Heightened interaction between platelets and factor VIII/von Willebrand’s disease. N Engl J Med 1980; 302: 1047-1051