In Vitro Antibody Quantification with Hyperspectral Imaging in a Large Field of View for Clinical Applications

Hyperspectral imaging (HSI) is a non-invasive, contrast-free optical-based tool that has recently been applied in medical and basic research fields. The opportunity to use HSI to identify exogenous tumor markers in a large field of view (LFOV) could increase precision in oncological diagnosis and surgical treatment. In this study, the anti-high mobility group B1 (HMGB1) labeled with Alexa fluorophore (647 nm) was used as the target molecule. This is the proof-of-concept of HSI's ability to quantify antibodies via an in vitro setting. A first test was performed to understand whether the relative absorbance provided by the HSI camera was dependent on volume at a 1:1 concentration. A serial dilution of 1:1, 10, 100, 1000, and 10,000 with phosphatase-buffered saline (PBS) was then used to test the sensitivity of the camera at the minimum and maximum volumes. For the analysis, images at 640 nm were extracted from the hypercubes according to peak signals matching the specificities of the antibody manufacturer. The results showed a positive correlation between relative absorbance and volume (r = 0.9709, p = 0.0013). The correlation between concentration and relative absorbance at min (1 µL) and max (20 µL) volume showed r = 0.9925, p < 0.0001, and r = 0.9992, p < 0.0001, respectively. These results demonstrate the HSI potential in quantifying HMGB1, hence deserving further studies in ex vivo and in vivo settings.