GPⅠb結合ドメインならびにC末端部分を認識する抗von Willebrand因子(vWF)モノクロナール抗体を用いたvWF抗原量の測定

Abstract

Sandwich enzyme-linked immunosorbent assays (ELISA) for von Willebrand factor antigen (vWF : Ag) were developed using three anti-vWF monoclonal antibodies (MoAbs), NMC-4, 2-2-9, and 40-1. In these ELISA, polyclonal rabbit anti-vWF antibody (IgG) was used as a captured antibody and the peroxidase-labeled MoAbs as the second antibody. MoAb NMC-4 recognizes platelet glycoprotein (GP) Ⅰb binding domain (residue 449-728 of vWF subunit), whereas the epitope of MoAb 2-2-9 locates on the residue 1926-(2050), and that of MoAb 40-1 on the disulfied-linked two polypeptides of the residue 1781- and 1926-(2050). The results obtained by NMC-4, 2-2-9, or 40-1 : ELISA showed a good correlation with those by the double polyclonal ELISA in 30 normal plasmas. In 7 patients with type Ⅰ von Willebrand disease (vWD), vWF : Ags measured by NMC-4, 2-2-9, and 40-1 : ELISA were 22.0±16.0 u/dl, 21.9±17.2 u/dl, and 24.3±19.2 u/dl respectively. In 7 patients with type ⅡA vWD, the respective values were 40.9±16.4 u/dl, 24.0±8.4 u/dl, and 40.1±16.8 u/dl. Thus, the significant lower value in type ⅡA patients was noticed when it was assayed only by 2-2-9 : ELISA. In 3 patients with type ⅡB vWD, those values were 53.3±8.6 u/dl, 51.0±8.0 u/dl, and 53.0±8.3 u/dl respectively, and no discrepant value was obtained. In 30 normal plasmas, the values of vWF : Ag measured by NMC-4 : ELISA were well correlated with both the activities of ristocetin cofactor (Rcof) and botrocetin cofactor (Bcof). In 7 patients with type Ⅰ vWD, the correlation coefficient between the vWF : Ag values by NMC-4 and Rcof or Bcof was r=0.7635 or r=0.5929 respectively. In 7 patients with type ⅡA vWD, Rcof in all these patients was too low to determine the correlation coefficient, but that value between vWF : Ag levels by NMC-4 ELISA and Bcof was r=0.5831. In 3 patients with type ⅡB vWD in the same family, the coefficient was r=0.9899 or r=0.9997 respectively. Thus, the values of vWF : Ag by NMC-4 ELISA appear to reflect Bcof activity rather than Rcof activity.