A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo.
A novel sandwich immunoassay for measurement of soluble fibrin in plasma has been developed. For immunization we used the synthetic heptapeptide Gly-Pro-Arg-Val-Val-Glu-Arg representing the amino terminus of the alpha-chain of human fibrin. A monoclonal IgG1 antibody was obtained by conventional hybridoma technology. To increase convenience, the sandwich immunoassay was developed for the Enzymun-Test systems which are based on streptavidin pre-coated tubes. The new assay was designed with the same fibrin specific antibody both in biotinylated and in peroxidase-labelled form. Fibrin in native plasma samples could only be detected after pre-incubation of the plasma with chaotropic ions. Test results were calculated using a standard curve comprising six fibrin standards (0-50 micrograms/ml). Precision of the method was satisfactory; intra-assay CVs using plasma samples ranged between 5.0% (23.7 micrograms/ml) and 12.4% (0.2 microgram/ml). CVs of interassay precision measurements using standards as samples range between 7.3% (25.0 micrograms/ml) and 11.4% (1.0 micrograms/ml). The lower detection limit was 0.12 microgram/ml. Investigations of normal range in 70 age-matched healthy individuals resulted in a mean of 1.12 micrograms/ml. Linearity was excellent; recovery of high fibrin plasma after dilution with normal plasma was always between 100 and 107%. Fibrin specificity was due to the monoclonal antibody 2B5 used and no cross reactivity with fibrinogen, fibrinogen split products or fibrin D-dimer was observed. Fibrin fragment E1 (studied by Dempfle CE, et al. Blood Coag Fibrinol 1993; 4: 79-86) and fragments X and Y showed moderate cross reactivity in the assay and caused some overestimation of fibrin at high fibrin split product concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Lill
H
H
Boehringer Mannheim GmbH, Biochemical Research Center Tutzing, Germany.
Spannagl
M
M
Trauner
A
A
Schramm
W
W
Schuller
D
D
Ofenloch-Haehnle
B
B
Draeger
B
B
Naser
W
W
Dessauer
A
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001777:Blood Coagulation; D005337:Fibrin / Q000032:analysis*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D005337:Fibrin