A Quantitative Immunochromatography Assay of Whole Blood Samples for Antigen-specific IgE—A New Method for Point of Care Testing for Allergens—

ABSTRACTBackgroundThe development of an inexpensive point-of-care testing system for antigen-specific IgE is greatly needed. We, therefore, tried to develop a quantitative enzyme immunochromatography assay system for antigen-specific IgE in fresh whole blood.MethodsWhole blood sample was mixed with a reagent containing detergent to lyse red blood cells, and the mixture was applied to an immunochromatography strip. The lysate was observed to migrate in the strip and was washed away by the substrate buffer. When the sample contained the specific IgE, the antigen-specific IgE line was clearly observed on the strip macroscopically.ResultsResults were obtained 20 minutes after the application of hemolysed blood sample to immunochromatography, and these results showed positive correlation with those obtained by the AlaSTAT system, which is one of the popular assay kits for specific IgE. The results were not affected significantly by the hematocrit value of the blood sample, by the kind of anticoagulant in the blood collection tube, or by the concentration of the total IgE, provided it was lower than 20000IU/ml.ConclusionsThese results indicate that our system is applicable for point-of-care testing for antigen-specific IgE

Role of the rasGAP-associated docking protein p62^(dok) in negative regulation of B cell receptor-mediated signaling

Antigenic stimulation of the B-cell receptor (BCR) is a central event in the immune response. In contrast, antigen bound to IgG negatively regulates signals from the BCR by cross-linking it to the inhibitory receptor FcγRIIB. Here we show that upon cross-linking of BCR or BCR with FcγRIIB, the rasGAP-associated protein p62^(dok) is prominently tyrosine phosphorylated in a Lyn-dependent manner. Inactivation of the dok gene by homologous recombination has shown that upon BCR cross-linking, p62^(dok) suppresses MAP kinase and is indispensable for FcγRIIB-mediated negative regulation of cell proliferation. We propose that p62^(dok), a downstream target of many PTKs, plays a negative role in various signaling situations

Tumor necrosis factor-alpha inhibition reduces CXCL-8 levels but fails to prevent fibrin generation and does not improve outcome in a rabbit model of endotoxic shock

The effects of a monoclonal antibody (mAb) to tumor necrosis factor-alpha (TNF-alpha) were examined in a rabbit model of endotoxic shock. Intravenous administration of lipopolysaccharide (100 microg/kg/hr) for 6 hours (n = 11) increased TNF-alpha levels. Fibrinogen was partially consumed, and fibrin deposits were seen in kidney and lungs at 24 hours. Mortality at 24 hours was 64%. Levels of interleukin-8 (aka CXCL-8) were notably increased. Mean arterial pressure (MAP) and leukocyte counts decreased, whereas creatinine levels were enhanced. The anti-TNF-alpha mAb (20 mg/kg i.v. bolus + 5 mg/kg/h i.v. for the first 90 minutes) (n = 10) efficiently inhibited the TNF-activity. Rabbits exhibited lower CXCL-8 levels; MAP improved, the decrease in leukocyte counts was partially prevented and creatinine levels were lower, but fibrinogen, fibrin deposits in kidneys and lungs and mortality, 55%, were similar to the LPS group. Rabbits that did not survive exhibited lower fibrinogen levels, more fibrin in kidneys and lungs and higher CXCL-8 and creatinine levels than survivors, while there were no differences in TNF-alpha, MAP and leukocytes. Thus, the inhibition of TNF-alpha, although beneficial through lowering CXCL-8 levels, is not enough to improve the outcome, which could be partly due to the inability to prevent the fibrin deposits formation in kidneys and lungs