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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Validation of a quantitative SPR assay for recombinant FVIII
A N McCormick1, M E Leach, G Savidge
1The Haemophilia Centre, St Thomas' Hospital, London, UK. aine.mccormick@gstt.sthames.nhs.uk
Clinical and Laboratory Haematology
|January 24, 2004
Summary
A new surface plasmon resonance assay accurately quantifies recombinant factor VIII (rFVIII) products. This method, using monoclonal antibodies, shows high precision and reusability for monitoring rFVIII production.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Accurate quantification of recombinant factor VIII (rFVIII) is crucial for therapeutic product development.
- Existing assays may have limitations in precision and applicability for specific FVIII products.
Purpose of the Study:
- To develop and validate a quantitative surface plasmon resonance (SPR) assay for recombinant FVIII (rFVIII).
- To assess the assay's precision, reusability, and suitability for monitoring rFVIII production.
Main Methods:
- Immobilization of anti-FVIII monoclonal antibody (ESH4) onto a carboxymethyldextran surface for SPR analysis.
- Quantification of rFVIII using a sandwich assay format with a secondary antibody (ESH8) for signal enhancement.
- Validation using the NIBSC sixth International rFVIII concentrate standard.
Main Results:
- The assay demonstrated high precision with inter- and intra-assay coefficients of variation (CVs) of 2.75% and 1.5% respectively when using signal enhancement.
- The immobilized antibody chip retained binding capacity after at least 75 regeneration cycles.
- The assay was not suitable for quantifying plasma-derived FVIII products.
Conclusions:
- The developed SPR assay provides a precise and reusable method for quantifying recombinant FVIII products.
- This assay may be valuable for the quality control and monitoring of rFVIII manufacturing processes.
- The assay's limitations exclude its use for plasma-derived FVIII quantitation.
