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

1. C. tropicalis biofilm formation

1. Prepare Candida biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1).
2. Add 100 µL of C. tropicalis culture (from 10⁶ cells/mL stock, prepared as above) to a 96-well microtiter plate using a multichannel pipette (Figure 2A). Keep the last two columns (11 and 12) as 'no fungus plus serum' and 'no fungus and no serum' negative controls by not adding fungal cells. Fill columns 11 and 12 with 100 µL of Roswell Park Memorial Institute (RPMI) 1640 morpholinepropanesulfonic acid (MOPS) medium alone.
3. Cover the microtiter plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 °C under stationary conditions.
4. The next day, aspirate the medium carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual medium.
5. Wash the plate with 200 µL of 1x phosphate-buffered saline (PBS) (per well) using a multichannel pipette. Add PBS very gently along the side walls of the well to avoid disrupting biofilms. Aspirate the PBS carefully using a multichannel pipette. Repeat the PBS wash 2x (a total of three washes).
6. To remove excess PBS, air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet.

2. Treatment of biofilm with antibodies

NOTE: Biofilms can now be processed for assessing the inhibition of biofilm maturation by antibodies. Murine serum was used as the source of polyclonal antibodies. Different groups of Sap2-immunized (Sap2-albicans, Sap2-tropicalis, and Sap2-parapsilosis) along with sham-immunized mice were bled retro-orbitally and serum was isolated. The presence of anti-Sap2 antibodies was confirmed using Sap2-specific enzyme-linked immunosorbent assay (ELISA).

1. Perform heat-inactivation of the serum (source of polyclonal antibodies) at 56 °C for 30 min before use to rule out the role of complement in the inhibitory activity. Heat-inactivate the serum before making serum dilution.
   NOTE: Use serum from sham-immunized mice, preimmune mice, and Sap2-specific antibody-depleted serum as additional controls. Antibody-depleted serum was prepared as per a previous study. Among the serial dilutions (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, and 1:12,800) for serum tested in this protocol, inhibition of biofilm maturation was observed at 1:25, 1:50, and 1:100; hence, 1:50 was selected to strike a balance between inhibition and serum consumption.
2. Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium (1:50). Use a common serum dilution (1:50) for all the serum samples to be tested for inhibition of biofilm maturation.
3. Add 100 µL of the selected serum dilution to each well of the 96-well microtiter plate. For each sample, add serum dilutions in duplicate, as per the layout attached (Figure 2B).
   1. In column 10, do not add serum dilution; add only RPMI 1640 MOPS medium for the fungus-only positive control.
      NOTE: Column 10, rows G1-G8 and H1-H8 initially had fungal cells in RPMI-MOPS. However, while RPMI-MOPS was added to column 10 even after 24 h, rows G1-G8 served as PBS control and rows H1-H8 as no-serum control after 24 h.
   2. In column 11, add a 1:50 dilution of serum to all wells to serve as the no fungus plus serum negative control.
   3. In column 12, do not add serum dilution to any well; keep this as the no fungus no serum negative control.
4. Cover the plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 °C.

3. Biofilm metabolic activity estimation

1. The next day, aspirate the serum carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual serum.
2. Wash the plate with 200 µL of 1x PBS (per well) using a multichannel pipette, adding the PBS along the side walls of the well to avoid disrupting the biofilms. Aspirate the PBS carefully using a multichannel pipette and repeat the PBS wash 2x (a total of three washes). Air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet to dry any excess PBS.
3. Preparation of XTT(2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)/menadione:
   1. Prepare XTT in sterile Ringers Lactate as a 0.5 g/L solution. Dissolve 25 mg of XTT in 50 mL of filter-sterilized Ringers Lactate. Aliquot 10 mL in separate tubes covered with aluminum foil and store at -80 °C.
   2. Prepare menadione as a 10 mM stock. Dissolve 8.6 mg of menadione in 5 mL of acetone and distribute 50 µL in 100 separate microtubes. Store the aliquots at -80 °C.
   3. Prepare XTT/menadione solution just before use by taking 10 mL of XTT and adding 1 µL of menadione to obtain a 1 µM working solution.
4. Add 100 µL of the XTT/menadione solution per well of the 96-well microtiter plate. Cover the plate with a lid and aluminum foil. Incubate the plate for 2 h at 37 °C in the dark.
5. Transfer 80 µL of the colored supernatant from each well into a fresh 96-well plate. Read the plate at 490 nm.
6. Calculate the mean of the absorbance values of the wells in column 10 (fungus-only positive control), which will serve as a reference value for calculating the percentage biofilm inhibition by each serum sample using equation (1).
   % Biofilm inhibition = 100 – × 100    (1)