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

Area of Science:

• Optical Imaging
• Biomedical Optics
• Medical Diagnostics

Background:

• Hyperspectral imaging (HSI) is a non-invasive optical technique increasingly used in research.
• HSI offers potential for identifying exogenous tumor markers, enhancing oncological diagnosis and surgical precision.
• Quantifying specific biomarkers like high mobility group B1 (HMGB1) is crucial for cancer research.

Purpose of the Study:

• To establish the proof-of-concept for HSI's ability to quantify antibodies in vitro.
• To assess the correlation between HSI-measured relative absorbance and the volume/concentration of a target antibody.
• To evaluate HSI's sensitivity in detecting varying amounts of anti-HMGB1.

Main Methods:

• Used anti-HMGB1 labeled with Alexa fluorophore (647 nm) as the target molecule.
• Performed serial dilutions of the antibody in phosphatase-buffered saline (PBS).
• Analyzed images at 640 nm to correlate relative absorbance with antibody volume and concentration.

Main Results:

• Demonstrated a positive correlation between relative absorbance and antibody volume (r=0.9709, p=0.0013).
• Showed strong correlations between concentration and relative absorbance at both minimum (1 µL, r=0.9925, p<0.0001) and maximum (20 µL, r=0.9992, p<0.0001) volumes.
• Confirmed HSI's sensitivity and accuracy in quantifying HMGB1 levels.

Conclusions:

• Hyperspectral imaging shows significant potential for quantifying specific antibodies like HMGB1.
• The findings support further investigation of HSI for ex vivo and in vivo applications in oncology.
• HSI could become a valuable tool for precise cancer diagnosis and surgical guidance.