Preparation of factor VIII-deficient plasma by immunoadsorption

Area of Science:

• Biochemistry
• Hematology
• Immunology

Background:

• Factor VIII is crucial for blood coagulation.
• Hemophilic plasma is often used as a reference in factor VIII assays.
• Developing reliable and accessible reagents for coagulation factor assays is important.

Purpose of the Study:

• To develop an immunoadsorbent for purifying human factor VIII.
• To evaluate the suitability of the purified plasma as a substitute for hemophilic plasma in factor VIII assays.
• To assess the reusability of the immunoadsorbent.

Main Methods:

• Coupling rabbit and human antibodies against factor VIII to Sepharose CL-2B to create an immunoadsorbent.
• Passing normal human citrate plasma through the immunoadsorbent to remove factor VIII and fibrinogen.
• Using the treated plasma, supplemented with fibrinogen, to calibrate a one-stage factor VIII assay.
• Comparing the performance of the artificial reagent with hemophilic plasma.

Main Results:

• The immunoadsorbent effectively removed factor VIII and fibrinogen from plasma.
• Other coagulation factors were well-recovered in the eluted plasma.
• The factor VIII-deficient plasma, when supplemented, yielded parallel lines in log-log plots against factor VIII procoagulant activity (VIII:C), similar to hemophilic plasma.
• The immunoadsorbent demonstrated stability and reusability for affinity chromatography.

Conclusions:

• An immunoadsorbent can be successfully prepared for factor VIII purification.
• The resulting artificial reagent serves as a viable substitute for hemophilic plasma in factor VIII assays.
• This method offers a reproducible and potentially more accessible approach for determining factor VIII procoagulant activity.