Sensitive and unambiguous measurement of the activated contact activation factor XII

Introduction The advent of chromogenic and fluorogenic low molecular weight peptide substrates has revolutionized protease measurements in terms of sensitivity but has not fully resolved issues related to selectivity. When measuring closely related proteases often the sum of their activities rather than the activity of a single protease is determined as the reaction between low molecular weight substrate and protease is focused on the protease’s active site only. Adding specific inhibitors to increase the assay’s sensitivity has been described as a valid approach but this increases the complexity of the assay. Here we describe the use of solid phase-bound corn trypsin inhibitor (CTI) to selectively measure activated factor XII (FXIIa) after formation of the immobilized CTI-FXIIa complex and its immunological detection by a FXII zymogen-specific antibodies.

Method CTI (Enzyme Research Laboratories) was diluted to 10 µg/mL in phosphate-buffered saline (PBS) and coated at 4°C overnight to a NUNC Maxisorp F96 plate (100 µL/well). After a washing step with PBS containing 0.05% Tween 20 (PBST), the plate was blocked by incubation with PBS containing 10 mg/mL human serum albumin and 2 mM benzamidine (200 µL/well, room temperature, 60 min). The washed plate was then incubated with serial dilution series of standard and samples. Purified FXIIa (Enzyme Research Laboratories) was used to establish the assay calibration curve. After a final washing step, the CTI-FXIIa complex formed was detected by an anti-human FXII IgG peroxidase conjugate (The Binding Site). Finally, bound peroxidase activity was measured with SureBlue (KPL).

Results The five-point log-log calibration curve ranged from 0.6 to 10 ng FXIIa/mL. Purified activated factor XI and kallikrein, present at 2-fold and 3-fold excess respectively, demonstrated essentially no influence on the concentration-response curve obtained for FXIIa. Similarly, the presence of a human immunoglobulin G concentration of as high as 2.5 mg/mL did not alter the characteristics of the FXIIa concentration-response curve.

Conclusion The CTI-FXIIa complex formation assay for the measurement of FXIIa showed adequate sensitivity and high human immunoglobulin G levels did not interfere. Mixing experiments demonstrated the high assay selectivity as excesses of the related proteases activated FXI and kallikrein demonstrated no effect on the FXIIa calibration curve. This high selectivity is obtained by the combined effects of the specific binding to CTI followed by the specific binding of the anti-FXII detection antibody, which is used to measure the amount of complex formed bound to the solid phase.

Publication History

Article published online:
20 February 2023