Comparative analysis of macroscopic and microscopic optical absorbance in hemagglutination assay.

We report a comparative study of macroscopic and microscopic optical absorbance in hemagglutination (HA) assay. Red blood cells (RBC) have unique optical absorbance properties with characteristic peaks including Soret, Qv, and Qo. In addition, RBC absorbs the light by showing dark contrast in bright field image in optical microscopy, implying that RBC tends to increase the local optical density (OD). By systematic analysis of macroscopic and microscopic measurements of ODs and UV-Visible (UV-Vis) spectroscopy, we developed a phenomenological model of RBC agglutination and non-agglutination. The chemical reaction in antigen-antibody reaction of RBCs is a catastrophic event such that the networking of RBC clumps is initiated at a critical RBC concentration. We analyzed the dependence of the RBC concentration on OD. At the critical concentration of RBCs, the ODs are dropped or saturated for RBC agglutination, on the other hand, the ODs keep increasing as the increase of RBC concentration for RBC non-agglutination. By the analysis of UV-Vis spectroscopy for HA assay, we provide an optimum wavelength as 480 nm to 520 nm, away from RBC characteristic peaks. For further validation, we demonstrated the OD-based HA assay for the detection of H1N1 influenza A virus. Our investigation described here provides an insight into how to utilize physical properties of RBCs for novel HA assay platform.