Purification of human IgG4 subclass with allergen-specific blocking activity

Area of Science:

• Immunology
• Allergen Immunotherapy
• Protein Purification

Background:

• Blocking antibodies (bAb) in allergen immunotherapy are primarily IgG1 and IgG4 subclasses.
• Study of IgG4 bAb has been hindered by a lack of effective purification methods.
• IgG4 antibodies play a significant role in the later stages of immunotherapy.

Purpose of the Study:

• To develop a rapid immunoaffinity chromatographic method for purifying IgG4 subclass antibodies from serum.
• To enable further research into the function and therapeutic potential of IgG4 blocking antibodies.

Main Methods:

• Utilized a two-step immunoaffinity chromatography approach with anti-IgG4 monoclonal antibodies (mAbs).
• Employed a strong-binding mAb for initial immunoadsorption and a weak-binding mAb for purification.
• Assayed blocking activity using IgE-sensitized human basophils and measured specificity via indirect ELISA.

Main Results:

• Achieved a 65-fold purification of serum IgG4, with eluates containing 82% IgG4 and a yield of approximately 30%.
• The purified IgG4 fraction retained nearly all allergen-specific antibody and demonstrated comparable blocking activity to the original serum.
• Immunoblot analysis confirmed the specificity of the purified antibody for Dactylis glomerata pollen antigens.

Conclusions:

• A rapid, effective immunoaffinity chromatographic method for purifying active IgG4 blocking antibodies has been established.
• This method overcomes previous limitations in studying IgG4 antibodies, facilitating further research in allergy and immunotherapy.
• The choice of monoclonal antibody affinity is critical for successful protein purification via immunoaffinity chromatography.