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

Abstract

Source: Al Ahmar, R., et al. Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate. J. Vis. Exp. (2020)

This video demonstrates a colorimetric immunoassay to quantify alginate, a protective exopolysaccharide produced by Pseudomonas aeruginosa. The procedure involves coating a microplate with the sample, applying specific antibodies, and using an enzyme-substrate reaction to generate a measurable color change. A standard curve is then used to determine the alginate concentration based on the sample’s color intensity.

Protocol

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)....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Step Ultra TMB-ELISA	Thermo Scientific	34028	via Fisher Scientific
Assay Plates 96-well	CoStar	2021-12-20
Carbonate-Bicarbonate Buffer	Sigma	C3041
Centrifuge Tubes (50 mL)	Fisher Scientific	05-539-13	via Fisher Scientific
Culture Test Tubes	Fisher Scientific	14-956-6D	via Fisher Scientific
Cuvette Polystyrene (1.5 mL)	Fisher Scientific	14955127	via Fisher Scientific
D-Mannuronic Acid Sodium	Sigma Aldrich	SMB00280
FMC Alginate	FMC	2133
Mouse Anti-Alginate Monoclonal Antibody	QED Biosciences	N/A	Lot # :15725/15726
Phosphate Buffered Saline Powder (PBS)	Sigma	P3813
Pierce Goat Anti-Mouse Poly-HRP Antibody	Thermo Scientific	32230	via Fisher Scientific
Round Toothpicks	Diamond		Any brand
Seaweed alginate (Protanal CR 8133)	FMC Corporation
Skim Milk	Difco	232100	via Fisher Scientific
SmartSpec Plus Spectrophotometer	BioRad	170-2525	or preferred vendor
Sodium Chloride (NaCl)	Sigma	S-5886
SpectraMax i3x Multi-mode MicroPlate Reader	Molecular Devices	i3x	or preferred vendor
Sulfuric Acid (2 Normal -Stop Solution)	R&D Systems	DY994
Tween 20	Sigma	P2287