A microtitre plate ELISA to measure thrombin-antithrombin complex using pan-specific antibodies

Area of Science:

• Biochemistry
• Immunology
• Hematology

Background:

• Thrombin-antithrombin complex (TAT) is a marker of coagulation activation.
• Accurate measurement of TAT is crucial for diagnosing and monitoring thrombotic disorders.
• Existing methods for TAT measurement may have limitations.

Purpose of the Study:

• To develop and validate a novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying plasma TAT levels.
• To compare the performance of the developed microtitre plate ELISA with a commercially available tube-based ELISA.
• To assess the assay's ability to differentiate between normal individuals and patients with disseminated intravascular coagulation (DIC).

Main Methods:

• Development of a sandwich ELISA using polyclonal antibodies against prothrombin and antithrombin.
• Immobilization of anti-prothrombin antibody on a solid phase for optimal TAT capture.
• Comparative analysis with a commercial tube-based ELISA (Enzygnost-TAT).
• Investigation of prothrombin interference and anticoagulant effects (citrate vs. EDTA).

Main Results:

• The microtitre plate ELISA demonstrated high correlation (r=0.88) with the commercial assay, irrespective of anticoagulant used.
• Both assays showed high internal consistency for plasma prepared with EDTA versus citrate (r > 0.90).
• Prothrombin levels can compete for capture antibody but do not critically affect the detection of significant TAT elevations.
• Both assays successfully discriminated between normal controls and patients with DIC.

Conclusions:

• The developed sandwich ELISA is a reliable and sensitive method for measuring plasma TAT levels.
• The assay is comparable to existing commercial methods and effective in identifying DIC.
• This assay provides a valuable tool for clinical diagnostics and research in coagulation disorders.