Immunoassays of factor IX antigen using monoclonal antibodies

Abstract

Monoclonal antibodies to factor IX were produced by immunization of balb/c mice ith purified factor IX and fusion of spleen cells with SP-1 murine myeloma cells. Antibody producing hybrids were detected by an enzyme linked immunoassay (ELISA) and by coagulation inhibitor (Bethesda type) methods. Monoclonal antibodies with titers of > 1 .times. 105 tested by the ELISA and 5000-8000 inhibitor units were obtained. A new ELISA method was developed using 1 of these monoclonal antibodies to quantitate factor IX antigen (IXAg) in plasma samples from patients with hereditary factor IX deficiency. In addition a 2-site solid phase immunoradiometric assay (IRMA) for factor IX was established. The results of these new methods were compared with those obtained on the same plasma samples using conventional factor IX coagulation assays and the Laurell rocket method. The lower limit for the detection of IXAg by the ELISA was .apprx. 0.01 U/ml while that for the IRMA was about 0.001 U/ml (normal plasma = 1U/ml). The lower detection limit for IXAg using the Laurell rocket method was about 0.06 U/ml. The improved sensitivity of the new immunoassay enabled quantitation of low levels of IXAg in patients with moderately severe factor IX deficiency and confirmed the presence of excess IXAg compared to IX activity (IXC) in a relatively high proportion of cases (28 of 51 tested). Results of testing plasma from obligate carriers confirm the suggestion that measurements of IXAg and IXC may improve the classification of carrier status in this kindred.