Surface Biotinylation-Tight Junction Permeability Assay

Surface Biotinylation-Tight Junction Permeability Assay 

PURPOSE AND RATIONALE This test uses the ex vivo model of comeal organ culture previously discussed combined with surface biotinylation technique. The comeal epithelium functions as a barrier that separates the internal ocular tissues from the external environment and is therefore vulnerable to chemical insult (Gipson and Sugrue 1994). The joint assessment of comeal organ culture and surface biotinylation is a measure of ocular toxicity that is intended to reveal disrupted tight junction barriers following chemical exposure. 

The method allows visualization of the epithelial barrier and penetration of small molecules into cell layers indicating disruption of tight junctions (Saitou et al. 1998). Slides are examined under the fluorescent microscope for bound biotin and rhodamine staining in deeper epithelial and stromal layers. Staining indicates penetration of biotin molecules into the tissue through disrupted tight junction barriers (Xu et al. 2004). Recovery from disruption is tracked on day 0,1,2,3, and 4. 

Comeal organ culture combined with objectively quantifiable assays for comeal epithelial barrier disruption reduces the high variability associated to the subjectively scored Draize Test. Although the surface biotinylation allows for an objective outcome measure, the scoring system is not yet quantitatively comparable for assessment of ocular irritancy to multiple test products. As it is utilized more extensively in varied laboratories with numerous test chemicals a standardized scoring system can be elicited similar to the familiar Draize Test. 

The concentration of test chemicals applied directly and dissolved in culture media may be modified to generate extended biotinylation of the comeal surface caused by test chemical exposure in a concentration-dependent manner (Xu et al. 2000). Xu et al. also suggests modifying exposure time of test chemicals from 2 minutes to longer test durations to examine further disturbed biotin surface labeling and stromal labeling which would indicate breakdown of tight junctions and penetration of biotin into the epithelial layer. 

---

Surface Biotinylation-Tight Junction Permeability Assay... 

---