High-affinity antibodies in a new immunoassay for plasma tissue factor: reduction in apparent intra-individual variation.
Tissue factor, the main initiator of blood coagulation, is shed into plasma by blood cells and endothelium. While studying such circulating plasma tissue factor with a commercially available immunoassay, we found unsatisfactory results and therefore developed a new and highly sensitive enzyme-linked immunosorbent assay (ELISA). High-affinity monoclonal antibodies raised against recombinant soluble tissue factor were used and the new assay had a detection limit of 40 fmol/L, approximately six-fold lower than existing assays. Normal ranges in 20 healthy donors were established in serum and in citrated EDTA and heparinized plasma. Tissue factor was also measured in three successive plasma samples from 43 patients with type 2 diabetes mellitus. In citrated plasma from healthy donors, tissue factor concentrations were 2.5 (1.0-9.3) pmol/L (median with range) and were not significantly different in diabetics. With a commercially available immunoassay, seven plasma samples were below the detection limit. Use of the new assay reduced intra-individual variation in diabetics from 49% to 14% and we conclude that high-affinity antibodies may markedly improve immunoassay performance.
van der Putten
Roy F M
RF
Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
te Velthuis
Henk
H
Aarden
Lucien A
LA
ten Cate
Hugo
H
Glatz
Jan F C
JF
Hermens
Wim Th
WT
D000293:Adolescent; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002648:Child; D002675:Child, Preschool; D003924:Diabetes Mellitus, Type 2 / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D010949:Plasma / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013925:Thromboplastin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D013925:Thromboplastin