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 microm 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 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)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) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) 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.

Airway mucosal thickening and bronchial hyperresponsiveness induced by inhaled beta 2-agonist in mice.

BACKGROUND: Patients with chronic persistent asthma require frequent use of inhaled beta(2)-agonist, which may result in aggravation of asthma symptoms. Our recent in vitro study has shown that beta(2)-agonist stimulates the growth of human airway epithelial cell lines. STUDY OBJECTIVE: To determine whether beta(2)-agonist likewise affects airway epithelial cell proliferation in vivo and, if so, what the mechanism of action is, we examined the effect of salbutamol on the morphology of murine airways. METHODS: Seventy-two BALB/c mice were administered aerosolized salbutamol using "flow-through" nose-only inhalation chambers at daily doses of 0.2 to 20 microg for up to 6 weeks. Morphology of tracheal mucosa, labeling of epithelial cells with 5-bromo-2'-deoxyuridine (BrdU), and bronchial responsiveness were assessed. RESULTS: Exposure to salbutamol increased the thickness of tracheal epithelial layer and the number of BrdU-positive epithelial cells in a dose- and time-dependent manner: the values in mice receiving 20 microg salbutamol for 6 weeks were 247% and 642%, respectively, of those in control animals receiving saline solution alone. These effects were inhibited by the mitogen-activated protein (MAP) kinase kinase inhibitors PD98059 and U0126. Salbutamol also caused a decrease in the provocative concentration of methacholine to achieve 400% of baseline enhanced pause. Combined treatment with inhaled budesonide attenuated salbutamol-induced airway morphologic changes and bronchial hyperresponsiveness. CONCLUSION: beta(2)-agonist stimulates proliferation of airway epithelial cells and produces airway wall thickening in vivo via MAP kinase-dependent pathway, and these effects are prevented by inhaled corticosteroid.