$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. ELISA for Alginate Quantitation
1. Using a micropipette, add 50 µL of the collected sample to an untreated 96-well plate. Add 50 µL of coating buffer (carbonate/bicarbonate buffer pH 9.6) to the wells. Incubate the plate at 37 °C for 2 h.
NOTE: For a positive control, a known concentration of D-Mannuronic acid and/or a known stable alginate-producing strain (e.g. PAO581- PAO1mucA25) can be used. For a negative control, use a 0.85% NaCl solution and/or PAO1ΔalgD (in-frame deletion of the algD gene renders the bacteria completely unable to produce any alginate). Multiple wells can be made from each sample to increase the number of technical repeats to aid in statistical analysis. Prepare 3−5 wells of each sample.
2. Using a squirt bottle, wash the plate wells 2x with 1x phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBS-T) by filling the wells, and then draining them by flipping the plate over.
3. Using a micropipette, add 200 µL of blocking buffer (10% skim milk in PBS-T) to the wells. Incubate at 4 °C overnight.
NOTE: Wrap the plate in paraffin film or shrink wrap to help avoid any evaporation in the refrigerator.
4. Using a squirt bottle, wash the plate wells 2x with PBS-T by filling the wells, and then draining them by flipping the plate over.
5. Using a micropipette, add 100 µL of diluted primary antibody (mouse anti-alginate monoclonal antibody) to the wells and incubate at 37 °C for 1−2 h.
NOTE: Primary antibody (mouse anti-alginate monoclonal antibody) was provided at a concentration of 0.5 µg/mL (dilution of 1:2,000 of 1 mg/ mL stock) in antibody diluent solution (1% skim milk in 1x PBS).
6. Using a squirt bottle, wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping the plate over.
7. Using a micropipette, add 100 µL of diluted secondary antibody to the wells and incubate at 37 °C for 1−2 h.
NOTE: Secondary antibody used was pierce goat anti-mouse poly-horseradish peroxidase (HRP) antibody to a concentration of 0.25 µg/mL (dilution of 1:2,000 of 0.5 mg/mL stock) in antibody diluent solution.
8. Using a squirt bottle, wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping the plate over.
9. Using a micropipette, add 100 µL of TMB (3,3',5,5'-Tetramethylbenzidine)-ELISA (Enzyme-Linked Immunosorbent Assay) solution (Table of Materials) and incubate at room temperature for 30 min in the dark.
NOTE: The color of a positive reaction would be a shade of blue.
10. Using a micropipette, add 100 µL of stop solution (2 N sulfuric acid).
NOTE: The color will turn from blue to yellow when the stop solution is added. Color is stable for up to 30 min after the reaction is stopped.
11. Using a plate reader, read the OD at 450 nm.
12. Produce a standard curve by measuring the OD450 of serial dilutions of known concentrations of D-Mannuronic acid (1,024 µg/mL, 512 µg/ mL, 256 µg/mL, 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, and 8 µg/mL). Repeat 2x. From these readings, extract a linear equation.
NOTE: The standard curve needs to be done only once. The linear equation extracted from it can be used for later testing.
1. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from the linear equation by OD600 to obtain the total amount of alginate per OD600.