A rapid enzyme-linked immunosorbent assay with two modes of detection for measuring cytokine concentration.

Interleukin (IL)-6 and IL-8 were measured in 101 serum samples collected from eight intensive-care unit patients using a polystyrene-based stick enzyme-linked immunosorbent assay (STICKELISA) system. This system consisted of an immobilized-antibody ELISA stick and a noncontact spectrophotometer. Cytokine concentration was detected by two ways: first, rapidly and semi-quantitatively by naked-eye observation of the color change and second, quantitatively using the spectrophotometer for accurate concentration determination. The spectrophotometric assay enabled the quantitation of as little as 100 pg/mL cytokine and took only 45 min to complete. There was a good agreement between the STICKELISA observations and data obtained using a plate ELISA system. The agreement between STICKELISA naked-eye observation and plate ELISA determination was 94 and 85% for IL-6 and IL-8, respectively. The correlation coefficients between the STICKELISA spectrophotometric determination and plate ELISA determination were 0.88 and 0.91 for IL-6 and IL-8, respectively, in a 0.1-5 ng/mL cytokine concentration range. These results demonstrate that the STICKELISA system is a simple, rapid, and quantitative method for bedside cytokine measurement in critical-care settings.

The role of immune response to Staphylococcus aureus superantigens and disease severity in relation to the sensitivity to tacrolimus in atopic dermatitis.

BACKGROUND: Staphylococcus aureus-producing superantigens (SAgs), such as staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin-1 (TSST-1), are frequently observed in atopic dermatitis (AD). However, little has been done to establish the association of immune responses to SAgs and the therapeutic response to immunosuppressive drugs in AD. Therefore, we investigated the prevalence and role of SAgs in the pathophysiology and immunosuppressive drug sensitivity in AD patients. METHODS: We classified 29 patients into two groups on the basis of their clinical AD scores: a low-score group (n = 14) corresponding to mild to moderate patients and a high-score group (n = 15) corresponding to severe patients. We estimated the plasma anti-SEB or TSST-1 IgE of these patients and healthy subjects by ELISA. We also estimated individual drug sensitivity by determining drug concentrations that would give 50% inhibition (IC(50)) of peripheral-blood mononuclear cell (PBMC) proliferation in vitro. RESULTS: The levels of plasma anti-SEB or TSST-1 IgE in the severe patients were significantly higher than those in the mild to moderate patients (p < 0.05 and p < 0.01, respectively). When stimulated with concanavalin A in vitro, PBMCs in the severe patients exhibited low sensitivity to the suppressive efficacy of tacrolimus (FK506) as compared to the mild to moderate patients (p < 0.01). Furthermore, there was a significant correlation between the IC(50)s of FK506 and plasma anti-TSST-1 IgE levels (p < 0.01). CONCLUSIONS: We showed that PBMCs in severe AD patients exhibited lower sensitivity to FK506, and had higher plasma levels of anti-TSST-1 IgE as compared to the mild AD patients. SAgs appear to be one of the causes of decreased PBMC sensitivity to FK506, and therefore an alternative treatment would be useful based on the individual drug sensitivity data and anti-TSST-1 IgE levels.

Mixed bacterial infection model of sepsis in rabbits and its application to evaluate superantigen-adsorbing device.

BACKGROUND: Superantigens are suspected to be the potent and lethal pathogens of gram-positive sepsis, and a new therapy that targeted to superantigens are required. METHODS: A mixed infection model was developed in rabbits by the cecal ligation and puncture associated with the intraperitoneal injection of Staphylococcus aureus, which produces toxic shock syndrome toxin 1 (TSST-1). Animals were also hemoperfused with a superantigen-adsorbing device (SAAD), or a control column. RESULTS: The model animals revealed multiple organ failure and died 6-12 h after the injection of S. aureus. The plasma levels of TSST-1, but not of lipopolysaccharide (LPS), significantly (p < 0.01) and inversely correlated with mean arterial pressure (r = -0.63). Plasma TSST-1 level was significantly reduced and shock-onset time was significantly retarded in the SAAD treated group, although the survival time was not significantly affected. CONCLUSIONS: The animal model developed could serve as a model for sepsis. It is suggested that there is the potential application of SAAD in treating superantigen-related sepsis.