抗第Ⅷ因子活性を有する数種抗第Ⅷ因子モノクローナル抗体を用いた血友病A患者血漿中の第Ⅷ因子抗原量の測定

Abstract

Six monoclonal antibodies (MoAbs), NMC-Ⅷ/5-10, against factor Ⅷ (F. Ⅷ) were developed and two-site solid phase enzyme-linked immunosorbent assays (ELISAs) for factor Ⅷ antigen (F. Ⅷ : Ag) were performed using three anti-F. Ⅷ : Ag monoclonal antibodies, NMC-Ⅷ/5, Ⅷ/10 and C5 (mono-ELISAs). In these ELISAs, polyclonal anti-F. Ⅷ alloantibody (PolyAb) was used as a coating antibody and the peroxidase-conjugated MoAb as the second antibody. MoAbs NMC-Ⅷ/5-10 reacted with 80 kDa light chain and NMC-Ⅷ/5 and 6 reacted with 72 kDa light chain fragment by immunoblotting. The IgG subclass of these MoAbs was all IgG_1 and anti-F. Ⅷ inhibitory activity ranged from 2 to 160 Bethesda U/mg. C5 recognized the 54 kDa heavy chain fragment. The levels of F. Ⅷ : Ag in 20 normal individuals were 93±21 u/dl by Ⅷ-5-ELISA, 91±18 u/dl by Ⅷ-10-ELISA, 91±21 u/dl by C5-ELISA and 97±23 u/dl by the double polyclonal ELISA (poly-ELISA). Forty-nine cases with hemophilia A were divided into three groups with F. Ⅷ : Ag level by poly-ELISA ; Group 1 (<1 u/dl), Group 2 (1-49 u/dl), Group 3 (≧50 u/dl). F. Ⅷ : Ag was not detected by any mono-ELISA in 30 patients of Group 1. These patients were considered to lack F. Ⅷ protein. In 9 cases out of 13 in Group 2, the level of F. Ⅷ : C and F. Ⅷ : Ag correlated well (Group 2-A). The other 4 cases had higher level of F. Ⅷ : Ag than F. Ⅷ : C (Group 2-B). These patients were considered to have abnormal F. Ⅷ protein. Two cases out of 6 patients in Group 3 had extremely lower level of F. Ⅷ : Ag by C5-ELISA than of levels measured by other mono-ELISAs and poly-ELISA. These patients seem to have a molecular defect at or near the binding region of C5.