Premium
Studies on immunological assay of vitamin‐K dependent factors.
Author(s) -
Mikami S.,
O'Brien D. P.,
Mellars G.,
Goodall A. H.,
Tuddenham E. G. D.
Publication year - 1986
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1986.tb02963.x
Subject(s) - factor ix , immunoradiometric assay , polyclonal antibodies , chemistry , medicine , microbiology and biotechnology , haemophilia b , radioimmunoassay , vitamin d and neurology , antigen , haemophilia , endocrinology , haemophilia a , immunology , biology , pediatrics
S ummary . Two‐site immunoradiometric assays (IRMAs) for factor IX antigen (IX:Ag) were developed using a monoclonal antibody (RFF‐IX/1) on the solidphase and either another monoclonal antibody (RFF‐IX/4) or a human polyclonal inhibitor antiserum as tracer (M‐M and M‐I IRMA respectively). The lower sensitivity limits of these two assays for IX: Ag in normal reference plasma were 4 × 10 −4 (M‐M IRMA) and 2 × 10 −4 (M‐I IRMA) units/ml. In 20 samples of normal plasma, levels of factor IX coagulation activity (IX:C) and of factor IX antigen measured by both IRMAs were highly correlated. Mean values of approximately 1·0 units/ml were obtained in all three assays. In normal serum, IX: Ag levels were lower with means of 0·84 (M‐M IRMA) and 0.83 (M‐I IRMA) units/ml. 4/25 patients with haemophilia B were CRM neg., two were CRM+ and the remaining 19 patients were CRM R variants. In two of these, IX: Ag was detectable by M‐I IRMA whilst IX:C and IX:Ag measured by M‐M IRMA were undetectable. In plasma from a fetus subsequently terminated on eugenic grounds, IX:C and IX:Ag by both M‐M and M‐I IRMA were undetectable. In warfarin‐treated plasma ( n = 12), the level of IX:C was low (mean 0·39 units/ml). The levels of IX: Ag measured by M‐M IRMA (mean of 0·80 units/ml) and by M‐I IRMA (0.70 units/ml) showed a discrepancy. M‐M IRMA reflects thereal amount of IX:Ag in warfarinized plasma because both monoclonal antibodies bind to epitopes distant from the light chain carboxylated region. Western blotting of denatured factor IX demonstrated that RFF‐IX/1 binds an epitope that is lost after XIa activation. RFF‐IX/4 binds the heavy chain.