An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Take a microplate containing the fungus Candida tropicalis.

The cells secrete Sap2, a proteolytic enzyme that cleaves a transmembrane glycoprotein called Msb2, removing its inhibitory domain.

This induces the fungus to adhere to the bottom and grow into a microcolony of yeast and filamentous forms. The cells secrete soluble components and biopolymers, initiating biofilm formation.

Add a serum sample containing Sap2-specific antibodies to selected wells, and incubate in the dark.

The antibodies bind to Sap2 and neutralize its activity, impeding cell viability and biofilm maturation.

In untreated wells, the biofilm matures into a dense network of cells and extracellular matrix.

Aspirate the liquid, wash the biofilm, and air-dry the plate. Add a chromogenic substrate, and incubate in the dark.

Viable cells, utilizing membrane-bound oxidoreductases, reduce the substrate to a colored product.

Transfer the colored supernatant and read the absorbance.

A lower absorbance in the serum-treated wells indicates antibody-mediated biofilm inhibition.