Affinity-based selection of anti-Feline Leukaemia Virus p27 monoclonal antibodies for efficient lateral flow assay development

Classic diagnostic modalities—including RT-PCR and proviral DNA PCR—present certain drawbacks, highlighting the need of alternative diagnostic techniques, such as lateral flow immunoassays (LFIA), valued for their rapidity and sensitivity. Said desirable features depend on the efficiency of the ternary interaction wherein the antigen bridges immobilized capture and labelled detection antibodies. However, the cost and the throughput of current screening methods, for finding monoclonal antibody (mAbs) pairs efficient at establishing this type of ternary interactions, remains as a universal bottleneck for LFIA development. Traditional sandwich enzyme-linked immunosorbent assays (ELISA), although extensively employed in mAb screening procedures, is burdened by its high consumption of labelled mAbs and the potential alteration of antigen conformation upon plate immobilization, often yielding suboptimal surrogates for native interactions within the final LFIA setup. In this study, we developed an orthogonal approach integrating ELISA and Spectral Shift (SST) that jointly enable the minimisation of reagent consumption (mAbs, antigens, labelling reagents) to comprehensively profile a panel of mAbs against a bona fide antigen of Feline Leukaemia Virus FeLV p27. This facilitated the quick selection of two low nanomolar mAbs with distinct epitope recognition, for which we validated a ternary complex formation in a LFIA prototype. This strategy enables advanced, resource-efficient LFIA development for FeLV and other critical antigens, such as H5 Avian Influenza Virus or African Swine Fever Virus, in addition to major human pathogens including Helicobacter pylori or Mpox Virus, facilitating sensitive diagnostics and streamlined quality control.