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

Enzyme-Linked-Immunosorbent-Spot (ELISpot) is a highly sensitive technique capable of detecting low-level immune responses, offering critical insights into therapy-induced immune activation. Our mouse interferon-gamma (mIFN-γ) ELISpot assay was originally based on a monoclonal capture antibody and a rabbit polyclonal detection antibody. The objective of our study was to replace the polyclonal detection antibody with a monoclonal alternative, using a llama immune library and phage display technology. A llama was immunized with recombinant mIFN-γ, and an immune VHH library was constructed. The library underwent two rounds of panning using the recombinant antigen. Subsequently, 190 clones were screened by Enzyme-Linked-Immunosorbent Assay (ELISA), yielding 27 specific binders to mIFN-γ. Sequence analysis revealed 24 unique clones grouped into four families based on their CDR3-VH sequences. One representative clone from each family was reformatted as VHH-Human Fragment Crystallizable (VHH-hFc) fusion and produced recombinantly for testing in the ELISpot assay. The purified candidates were evaluated in pairs on native mIFN-γ from mouse splenocytes. Two candidates, H3 and G4, were selected for further trial. Comparative analysis of ELISpot performance showed that G4 is a promising substitute for the original rabbit polyclonal antibody, enhancing the overall performance of the mIFN-γ ELISpot assay. This study highlights the potential of VHH antibodies in ELISpot applications and supports their use as a robust, reproducible alternative to polyclonal antibodies.