Quantification of circulating D-dimer by peptide immunoaffinity enrichment and tandem mass spectrometry.
D-dimer is a product of the coagulation cascade and is associated with venous thromboembolism, disseminated intravascular coagulation, and additional clinical conditions. Despite its importance, D-dimer measurement has limited clinical utility due in part to the lack of reliable assays. The difficulty in developing an immunoassay that is specific for D-dimer arises from the inherent heterogeneity in its structure. In this report, we describe a highly specific method for the quantification of D-dimer level in human plasma. In our method, the reciprocally cross-linked peptide resulting from factor XIIIa-catalyzed dimerization of fibrin γ chains was selected to represent the D-dimer antigen. Using an antipeptide antibody, we enriched the cross-linked peptide from trypsin-digested plasma prior to quantitative analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The assay has a quantitative range of 500 pmol/L to 100 nmol/L in human plasma. In further characterization of the assay, we found that it exhibited good correlation with fibrinolytic activity in human donors and with thrombin generation and clot strength in an in vitro thromboelastography assay. These observations thus establish the biological relevance of the assay and suggest it may be a valuable biomarker in characterization and treatment of blood coagulation disorders.
Department of Molecular Biomarkers, Merck Research Laboratories, Rahway, New Jersey 07065, USA. weixun_wang@merck.com
Weixun
W
Wang
Nykia D
ND
Walker
Li-Ji
LJ
Zhu
Weizhen
W
Wu
Lan
L
Ge
David E
DE
Gutstein
Nathan A
NA
Yates
Ronald C
RC
Hendrickson
Martin L
ML
Ogletree
Michele
M
Cleary
Gregory J
GJ
Opiteck
Zhu
Z
Chen
D000906:Antibodies / Q000276:immunology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005176:Factor XIII / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry; D013917:Thrombin / Q000378:metabolism
D000906:Antibodies; D005338:Fibrin Fibrinogen Degradation Products; D010455:Peptides; C036309:fibrin fragment D; D005176:Factor XIII; D013917:Thrombin