An Enzyme Immunoassay (ELISA) for the Quantitation of Human Factor VII

Abstract

SummaryA new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab′)2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21 to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VIIC was observed. In “compensated” DIC (n = 5), levels of F VII Ag varied from 60 to 186%, with significantly higher F VII C levels (from 143 to 189%). In contrast, in “decompensated” DIC (n = 7), low F VII Ag and F VII C levels were observed (from 7 to 27%). In patients with deep-vein thrombosis (n = 25), high levels of F VII Ag (from 102 to 136%) and F VII C (from 110 to 150%) were demonstrated. In surgical patients, no significant difference was observed before and one day after intervention.

Evaluation of factor VII antigen in factor VII congenital deficiencies with a new ELISA assay

“…The reproducibility and accuracy of the test proposed by Boyer et a1 (5) have been confirmed by us, as illustrated by the low coefficients of variation. In addition, the ELISA assay is more rapid than the Inhibitor Neutralization Assay.…”

“…Another advantage of the RIA assay is the direct evaluation of F VI1:Ag in plasma samples, whereas the INA can be considered an indirect method of assay since it is based upon two incubations of the antibody: the first with the sample and the second with the pooled normal plasma [6]. In addition, it is important to note that classic immunoprecipitation methods (electroimmunoassay or radial immunodiffusion) are not suitable for F VI1:Ag assay in plasma samples since the levels of factor VII protein are very low (<500 ng/ Recently Boyer et a1 [5] proposed an ELISA method for measuring F VII protein. In our study, this method was employed to estimate levels of F VII:Ag either in normals or in homozygotes and heterozygotes for F VII congenital deficiency.…”

“…With the introduction of the ELISA F VI1:Ag assay by Boyer et a1 [5], a method of similar intrinsic features was proposed with the additional advantage of being quick and easy to perform.…”

A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals.

“…21 Factor VII antigen concentration was determined using a commercially available enzymelinked immunosorbent assay kit (Diagnostica Stago, Franconville, France). 29 Values are expressed as a percentage of a standard. The coagulant activity of some other vitamin K-dependent coagulation factors, such as factors II and X, were measured by standard techniques 21 ; factor IX was measured as described in Reference 25 except for substitution of factor IX for factor VIll-deficient plasma.…”

Treatment of Factor VII Deficiency with Recombinant Factor VIla

“…The laboratory evaluation of factor VII deficiency relies on assays of plasma factor VII coagulant activity (VII:C) using animal thromboplastins as well as immunologic quantitation of factor VII antigen [2,13] Patients have been categorized as to the plas ma level of factor VII antigen (VII:Ag) or cross-reacting material (CRM-= low or ab sent antigen; CRMR = reduced antigen; CRM+ = normal antigen). CRM" defects pre sumably result from defective factor VII bio synthesis or accelerated clearance in vivo, CRM+ defects are structurally abnormal fac tor VII molecules, and CRMR defects may result from a combination of these mecha nisms.…”