Ilama VHH as a Substitute for Rabbit Polyclonal Antibodies in ELISpot Application

Area of Science:

• Immunology
• Biotechnology
• Antibody Engineering

Background:

• Enzyme-Linked-Immunosorbent-Spot (ELISpot) assays are crucial for detecting low-level immune responses, particularly in monitoring therapy-induced immune activation.
• The established mouse interferon-gamma (mIFN-γ) ELISpot assay utilized a monoclonal capture antibody and a rabbit polyclonal detection antibody.

Purpose of the Study:

• To replace the existing rabbit polyclonal detection antibody in the mIFN-γ ELISpot assay with a monoclonal alternative.
• To leverage llama immune libraries and phage display technology for novel antibody discovery.

Main Methods:

• Immunization of a llama with recombinant mIFN-γ and construction of an immune VHH library.
• Phage display panning against the recombinant antigen, followed by ELISA screening of 190 clones to identify specific mIFN-γ binders.
• Reformatting of selected VHH clones into VHH-hFc fusions and evaluation in the mIFN-γ ELISpot assay.

Main Results:

• Screening identified 27 specific mIFN-γ binders from 190 clones, with 24 unique sequences grouped into four families.
• Two VHH-hFc fusion candidates, H3 and G4, demonstrated promising performance in ELISpot assays.
• The G4 candidate proved to be a viable and effective substitute for the original polyclonal detection antibody, improving assay performance.

Conclusions:

• VHH antibodies show significant potential for enhancing ELISpot assay applications.
• The developed monoclonal VHH antibody (G4) offers a robust and reproducible alternative to polyclonal antibodies for mIFN-γ detection.
• This advancement contributes to more reliable monitoring of immune responses in research and clinical settings.