A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

This in vitro, 96-well-plate-based protocol can be used to test the inhibitory effect of antibodies on novel antifungal agents on the metabolic activity of fungal cells in Candida biofilms. Compared to other methods, this is a highly sensitive, accurate, high-throughput, reproducible, user-friendly, and cost-effective technique for evaluating the effect of antibodies or antifungals on the development of Candida biofilms. This assay can be used for assessing the efficacy of novel drug-based or antibody-based therapeutics, as well as vaccine candidates against Candida biofilms, which are associated with severity of invasive candidiasis.

This method can be applied for testing the effect of novel antifungal agents or antibodies during biofilm formation or maturation in different settings using different fungal strains or antibody sources. Demonstrating this procedure will be Mr.Pankaj Chandley, a PhD research scholar working in my laboratory. To begin, add 100 microliters of Candida tropicalis culture to a 96-well microtiter plate.

Using a multi-channel pipette, fill the last two columns with 100 microliters of RPMI 1640 MOPS medium alone. Cover the microtiter plate with a lid and aluminum foil and incubate the plate for 24 hours at 37 degrees Celsius under stationary conditions. The next day, aspirate the medium carefully using a multi-channel pipette.

Invert the plate gently on blotting sheets and tap to remove any residual medium. Wash the plate with 200 microliters of 1X PBS using a multi-channel pipette. Aspirate the PBS carefully using a multi-channel pipette, and wash twice with PBS.

To remove excess PBS, air dry the plate for 30 minutes at room temperature inside a biological safety cabinet. Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium. Add 100 microliters of the selected serum dilution to each well of the 96-well microtiter plate.

In column 10, add only RPMI 1640 MOPS medium for the fungus-only positive control. Add a one to 50 dilution of serum to all wells in column 11 to serve as the no-fungus plus serum negative control. After covering the plate with a lid and aluminum foil, incubate the plate for 24 hours at 37 degrees Celsius.

The next day, after aspirating the serum carefully using a multi-channel pipette, tap the inverted plate gently to remove any residual serum. Repeat the PBS wash three times, and air dry the plate for 30 minutes at room temperature inside a biological safety cabinet to dry any excess PBS. Then, dissolve 25 milligrams of XTT in 50 milliliters of filter-sterilized Ringer's lactate.

Aliquot 10 milliliters in separate tubes. Cover it with aluminum foil and store it at minus 80 degrees Celsius. Next, dissolve 8.6 milligrams of menadione in five milliliters of acetone.

And after distributing 50 microliters in 100 separate micro tubes, store the aliquots at minus 80 degrees Celsius. Take 10 milliliters of XTT and add one microliter of menadione to obtain a one micromolar working solution. Add 100 microliters of the XTT menadione solution per well of the 96-well microtiter plate.

Next, after covering the plate with a lid and aluminum foil, incubate the plate for two hours at 37 degrees Celsius in the dark. Finally, transfer 80 microliters of the colored supernatant from each well into a fresh 96-well plate, and read the plate at 490 nanometers. Serum from Sap2-parapsilosis-immunized mice could prevent the maturation of preformed Candida tropicalis biofilms by 65%as compared to 45%in serum from Sap2-albicans, and 55%in Sap2-tropicalis-immunized mice.

In general, biofilm inhibition by Sap2 immune serum was significantly higher than that by sham immune serum, which is 16%and 13%in pre-immune serum. Biofilm inhibition was close to negligible on using PBS and no serum control. Utmost care should be taken while performing the washing steps to prevent disruption of biofilms.

The XTT solution should be prepared just before use, and added to the plate immediately. Following this procedure, users can evaluate the effect of novel antifungal agents, vaccine candidates, or antibodies during Candida biofilm formation and maturation in different research settings using different fungal strains.