Quantifying Bacterial Alginate Using an Antibody-Based Enzyme-Linked Immunosorbent Assay

Take a microplate with a sample containing alginate, a protective exopolysaccharide produced by the bacterium Pseudomonas aeruginosa.

Prepare a serial dilution of an alginate-derived standard to quantify sample alginate concentration.

Add a coating buffer and incubate to facilitate adherence of the molecules to the well surface.

Wash to remove unbound molecules.

Incubate with a blocking agent to mask non-specific binding sites.

Wash, then incubate with primary antibodies specific to alginate.

Wash again, then incubate with enzyme-conjugated secondary antibodies that target the primary antibodies.

Wash and then incubate with a chromogenic substrate, which the enzyme oxidizes to produce a colored product.

Add an acidic solution that stops the reaction and enhances the color to enable colorimetric detection.

Using a microplate reader, measure the color intensity.

Create a standard curve from the intensities of the standard dilutions and plot the sample intensity to determine alginate concentration.

Using a micropipette, add 50 microliters of the collected sample to an untreated 96-well plate.

Then add 50 microliters of ELISA coating buffer to the wells. Incubate the plate at 37 degrees Celsius fo