Semi-continuous, label-free immunosensing approach for Ca2+-based conformation change of a calcium-binding protein.

A label-free immunosensing method based on the conformational change of calcium-binding protein (CBP) depending on analyte concentration was explored for semi-continuous analysis of free Ca(2+). Glucose-galactose-binding protein as a CBP and produced as a recombinant protein by Escherichia coli was used as the immunogen to produce monoclonal antibodies by hybridoma technology. We finally screened the 3-6F cell clone, which produced the desired antibody specific to a particular structural conformation of the protein that occurred only upon CBP-calcium complex formation. To construct an immunosensor, the antibody was immobilized via a secondary antibody on an Octet Red optical fiber-based label-free sensor. Calcium analysis was conducted on the sensor in combination with CBP previously added to the aqueous sample, which distinguished the sensor signal according to the analyte concentration. The immunosensor produced a signal in real time with a response time of approximately 15 min and could be reused for analyses of different samples in a semi-continuous manner. The minimum detection limit of the analyte under optimal conditions was 0.09 mM and the upper limit was about 5 mM (log-logit transformed standard curve linearity: R(2) > 98%). In sample tests with milk, the analytical performance of the sensor was highly correlated (R(2) > 99%) with that of the reference system based on the KMnO4 titration method (ISO 12081). Although the sensor showed cross-reactivity at high concentrations (>1 mM) of cations including zinc, iron, manganese, and copper, these ionic components were not traceable (<0.01 mM) in milk.

Toll-like receptor-based immuno-analysis of pathogenic microorganisms.

In this study, a novel mammalian cell receptor-based immuno-analytical method was developed for the detection of food-poisoning microorganisms by employing toll-like receptors (TLRs) as sensing elements. Upon infection with bacterium, the host cells respond by expressing TLRs, particularly TLR1, TLR2, and TLR4, on the outer membrane surfaces. To demonstrate the potential of using this method for detection of foodborne bacteria, we initially selected two model sensing systems, expression of TLR1 on a cell line, A549, for Escherichia coli and TLR2 on a cell line, RAW264.7, for Shigella sonnei (S. sonnei). Each TLR was detected using antibodies specific to the respective marker. We also found that the addition of immunoassay for the pathogen captured by the TLRs on the mammalian cells significantly enhanced the detection capability. A dual-analytical system for S. sonnei was constructed and successfully detected an extremely low number (about 3.2 CFU per well) of the pathogenic bacterium 5.1 h after infection. This detection time was 2.5 h earlier than the time required for detection using the conventional immunoassay. To endow the specificity of detection, the target bacterium was immuno-magnetically concentrated by a factor of 50 prior to infection. This further shortened the response to approximately 3.4 h, which was less than half of the time needed when the conventional method was used. Such enhanced performance could basically result from synergistic effects of bacterial dose increase and subsequent autocrine signaling on TLRs' up-regulation upon infection with live bacterium. This TLR-based immuno-sensing approach may also be expanded to monitor infection of the body, provided scanning of the signal is feasible.

Chemiluminometric immuno-analysis of innate immune response against repetitive bacterial stimulations for the same mammalian cells.

For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.