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A quantitative dot immunobinding assay for coagulation factor XII in plasma.
W A Wuillemin1, M Furlan, B Lämmle
1Central Hematology Laboratory, University of Bern, Inselspital, Switzerland.
Journal of Immunological Methods
|June 12, 1990
Summary
A new dot immunobinding assay accurately quantifies human coagulation factor XII (F XII) antigen (F XII:Ag) in plasma. This sensitive method allows for rapid analysis of large sample volumes, with results correlating well to clotting activity.
Area of Science:
- Biochemistry
- Hematology
- Immunology
Background:
- Human coagulation factor XII (F XII) plays a crucial role in the intrinsic pathway of blood coagulation.
- Accurate quantification of F XII antigen (F XII:Ag) is essential for diagnosing and managing hemostatic disorders.
- Existing methods for F XII quantification may lack sensitivity or throughput for large-scale studies.
Purpose of the Study:
- To develop and validate a sensitive dot immunobinding assay for quantifying human coagulation factor XII antigen (F XII:Ag).
- To assess the assay's performance characteristics, including sensitivity, precision, and correlation with functional F XII activity.
- To evaluate F XII:Ag levels in healthy adults and pregnant women.
Main Methods:
- A dot immunobinding assay was developed using nitrocellulose membranes.
- Plasma samples were dotted onto membranes, and F XII was detected using polyclonal antiserum and a radiolabeled antigen overlay (125I-F XII).
- Quantification was achieved by measuring bound radioactivity, with standards prepared from pooled normal human plasma (NHP) or purified F XII.
Main Results:
- The assay demonstrated high sensitivity, detecting F XII:Ag down to 0.12 ng, equivalent to 1% of NHP levels in 0.5 microliters of plasma.
- Intra-assay and inter-assay coefficients of variation (CV) were less than 5% and 16%, respectively.
- F XII:Ag levels in 50 healthy adults ranged from 12 to 47 micrograms/ml, showing a strong correlation (r = 0.93) with F XII clot promoting activity (F XII:C).
- Elevated F XII:Ag and F XII:C were observed in pregnant women, with F XII:Ag disproportionately higher than F XII:C.
Conclusions:
- The developed dot immunobinding assay provides a rapid, sensitive, and precise method for quantifying human F XII:Ag.
- The assay's high throughput and minimal reagent requirements make it suitable for analyzing large numbers of samples.
- Findings in pregnant women suggest potential alterations in F XII regulation during gestation.