An in-house ELISA method for quantification of circulating tissue factor.

Area of Science:

• Biochemistry
• Immunology
• Hematology

Background:

• Tissue Factor (TF) is a critical transmembrane glycoprotein initiating the coagulation cascade.
• TF regulates hemostasis, thrombosis, inflammation, and cellular immune responses.
• Accurate quantification of plasma TF is essential for understanding thrombotic disorders.

Purpose of the Study:

• To develop and validate a sensitive two-step direct sandwich ELISA for quantifying human plasma TF.
• To establish the assay's performance characteristics, including linearity, detection limit, and variability.
• To provide a reliable tool for measuring plasma TF in patients with thrombotic conditions.

Main Methods:

• Development of a two-step direct sandwich ELISA using specific monoclonal antibodies for TF capture and detection.
• Utilization of peroxidase-conjugated anti-TF IgG and tetramethylbenzidine/H2O2 substrate system.
• Validation of the assay with recombinant TF, assessing linearity, detection limit, intra-assay and inter-assay variability, recovery, and parallelism.

Main Results:

• The developed ELISA demonstrated linearity for TF concentrations between 10 to 4000 pg/ml.
• The assay achieved a low detection limit of 36.31 pg/ml.
• Excellent assay performance was confirmed with low intra- (2.50-9.23% CV) and inter-assay (5.65-13.57% CV) variability, satisfactory recovery (91.55-103.95%), and good parallelism.

Conclusions:

• A robust and sensitive ELISA for plasma TF quantification has been successfully developed.
• The assay exhibits high precision, accuracy, and sensitivity, suitable for clinical research.
• This validated assay is poised for application in measuring plasma TF levels in patients with thrombotic disorders.