Tissue sample acquisition and preparation

For the first time, we directly investigated the residual blood contamination in various tissues using a novel differential injection of the mAb and stable-isotope-labeled-mAb (SIL-mAb), enabling accurate evaluation of the positive bias and removal efficiency using whole body transcardial perfusion. Optimal conditions for perfusion were identified and adopted for removal of blood contamination in various tissues for protein quantification. Based on our extraction study with real tissue samples, the model biotherapeutics was found being present in various tissues with free form (30-60%) and bound form (40-70%), which was supported by evidences on acid dissociation, incubation, and detergent-extraction experiments. A strong, detergent-cocktail buffer was found to be optimal to extract biotherapeutics and cell membrane associated proteins from tissues for quantification. This strategy is novel because Optimal whole body transcardial perfusion was used to eliminate residual blood contamination for targeted quantification of biotherapeutics in various tissues. Aalso, an exhaustive and reproducible extraction method using detergent cocktail buffer has been developed to secure the accuracy and reliability for quantification of both biotherapeutics and its antigen in various tissues.

 

Update date: December, 2022