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Selection and characterisation of FVIII-specific single chain variable fragments
A Naumann1, A K Scherger, J Neuwirth
1Christoph Königs, University Hospital Frankfurt, Goethe University, Department of Paediatrics, Molecular Haemostasis and Immunodeficiency, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany, Tel. +49/(0)69/630 18 30 30, Fax +49/(0)69/630 18 39 91,
Hamostaseologie
|October 31, 2013
Summary
Developing standards for FVIII-specific IgG subclasses is crucial for monitoring immune tolerance induction (ITI) in haemophilia A patients. This research isolated and characterized FVIII-specific antibodies to aid in ITI outcome prediction.
Area of Science:
- Immunology
- Hematology
- Biotechnology
Background:
- Inhibitory anti-FVIII antibodies are a major complication in haemophilia A treatment.
- Immune tolerance induction (ITI) is a strategy for eradicating these inhibitors.
- FVIII-specific IgG subclass distribution may correlate with ITI success.
Purpose of the Study:
- To develop FVIII-specific IgG standards for quantifying IgG subclasses in patient plasma.
- To investigate the role of IgG subclass distribution in ITI outcomes for haemophilia A.
- To create tools for assessing prognostic factors in ITI therapy.
Main Methods:
- Isolation of FVIII-specific single chain variable fragments (scFvs) using phage display.
- Characterization of scFv binding specificity to FVIII domains.
- Cloning and expression of a selected scFv (1G10) into four human IgG subclasses (hIgG1-4).
- Purification of recombinant 1G10-hIgG standards.
Main Results:
- Successfully isolated FVIII-specific scFvs, including 1G10 binding the FVIII A2 domain.
- Engineered and expressed four distinct 1G10-hIgG subclass standards.
- Established purified standards for quantifying FVIII-specific IgG subclasses.
Conclusions:
- The developed 1G10-hIgG standards enable absolute quantification of FVIII-specific IgG subclasses.
- These standards will facilitate studies on the prognostic value of IgG subclass distribution in ITI for haemophilia A.
- This work contributes to a better understanding of immune responses in haemophilia A treatment.
