Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA™ 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 μm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 × 106 and 5.20 × l04 cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 × l06 to 1.64 × l010 cells/mL for P. aeruginosa, and 5.20 × l04 to 1.04 × l09 cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6–2.5) × l05 antibodies for one P. aeruginosa cell and (2.2–2.7) × l08 antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.