The hemagglutination inhibition assay (HAI) is a classical method used worldwide in many analytical applications, including pathogen identification, vaccine production monitoring, and detection and characterization of pathogen-neutralizing antibodies (n-Ab). This is also a World Health Organization (WHO) reference method for the global surveillance of influenza viruses, which provides the information needed for the annual reformulation of the flu vaccine. HAI is a simple and inexpensive method that is performed without sophisticated equipment. However, it has to be carried out with fresh red blood cells (RBCs), a highly variable, unstable, and hard to mass-produce reagent, which impairs assay reproducibility. Here, we used the tests employed for influenza surveillance as a model to develop synthrocytes©, a synthetic reagent that could substitute animal erythrocytes in HAI. Contrary to previous examples exploiting sophisticated production paths to generate therapeutic synthetic RBCs, we founded production on the identification of microparticles able to generate different sedimentation patterns when agglutinated or not, which is the main requirement for HAI testing. Upon incorporation of influenza-binding receptors and optimization of production and assay conditions, synthrocytes succeeded in binding influenza A(H1N1) and B viruses as erythrocytes do, but were faster and more stable. Synthrocytes were finally employed in an HAI-like assay to detect the WHO reference reagents for influenza surveillance. Our results show that it is possible to substitute erythrocytes in classical HAI by a highly tuneable and potentially mass-produced synthetic reagent, which should facilitate worldwide HAI standardization with minimal equipment or training requirements.
Keywords: characterization of virus-neutralizing antibodies; fast detection tests; flu viruses; hemagglutination inhibition assay (HAI); influenza surveillance; method standardization; synthetic erythrocyte.