PubMed · 15088782
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P Brandtzaeg. 2003. Mucosal immunity.. https://pubmed.ncbi.nlm.nih.gov/15088782/
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Soil-borne cereal mosaic virus (SBCMV) was first reported affecting wheat crops in Italy in 1960 and has spread subsequently to many other European countries, including the UK. SBCMV causes a serious disease of wheat, reducing yield by up to 70%; growing resistant varieties represents the only economical means of control. Real-time RT-PCR and PCR assays based on TaqMan chemistry were developed for the detection and quantitation of SBCMV and its vector, Polymyxa graminis. Each assay incorporated an RNA or DNA specific internal control to facilitate quantitation. Nucleic acid extracts from SBCMV-infected plants were diluted in a nucleic acid extract from a healthy plant and amplified by real-time PCR to produce a standard curve. The standard curve was used to quantify the amount of SBCMV and P. graminis in plant samples. The sensitivity of the real-time assays were compared to established serological quantitation and conventional PCR methods by testing a range of SBCMV-infected wheat varieties. The results indicate that real-time assays were a 1000 times more sensitive than ELISA for the quantitation of SBCMV, and a 100 times more sensitive than conventional PCR for the quantitation of P. graminis. Real-time assays enabled sensitive, reproducible and specific detection of both virus and vector in wheat tissues. The real-time assays are potentially useful tools for determining variations in virus and vector concentrations in plant tissue from wheat varieties differing in resistance to SBCMV.
The protein kinase glycogen synthase kinase-3 (GSK-3) is highly abundant in brain and involved in signal transduction cascades, particularly during neurodevelopment. We have previously found reduced GSK-3beta mRNA levels, protein levels and GSK-3 total (alpha+beta isoforms) activity in postmortem frontal cortex of schizophrenic patients in the Stanley Medical Research Institute's Brain Collection. To verify and extend these findings, GSK-3 parameters were now measured in the frontal cortex (BA9) and hippocampus obtained from the Rebecca L. Cooper Research Laboratories postmortem brain collection. Fifteen pairs of schizophrenic patients and matched control subjects have been studied. No significant differences in GSK-3alpha and GSK-3beta mRNA levels, GSK-3beta protein levels or total GSK-3 (alpha+beta) activity were found in the frontal cortex of the two diagnostic groups. Hippocampal GSK-3alpha and GSK-3beta mRNA levels were significantly lower (22% and 28%, respectively) in the tissue from the schizophrenic patients compared with the normal controls. Hippocampal GSK-3beta protein levels in the schizophrenic patients were 24% significantly lower than control values only after omission of three outlier subjects. Hippocampal total GSK-3 (alpha+beta) activity in the patients was 31% lower in the schizophrenic patients vs. control subjects. This difference was marginally significant. While our previous data on GSK-3beta in postmortem brain and the recent report that there is impaired AKT1-GSK-3beta signaling in schizophrenia suggest that changes in pathways involving protein kinases such as AKT1 and GSK-3beta in schizophrenia are complex, our present data do not provide strong evidence in support of the involvement of GSK-3beta in schizophrenia. Therefore, further investigation in a greater number of brain samples is warranted to better clarify the possible role of this enzyme in the pathophysiology of schizophrenia.
This paper puts forward a method to describe an equation of the within-plate uncertainty (relative standard deviation (R.S.D.) of measurements) as a function of analyte concentration in sandwich enzyme-linked immunosorbent assay (ELISA). A kit for thyroid stimulating hormone is taken as an example. The pipetting procedures of analyte solution and chromogen-substrate solution and absorbance inherent to the wells of a microplate are identified as major error sources and their variability is included as parameters in the uncertainty equation. These parameters can be determined by the experiments with distilled water. The theoretical R.S.D. is shown to be in good agreement with the results of the repeated experiments using the real samples. Since the theory gives a continuous plot of R.S.D. against concentration, the uncertainty structure of the ELISA kit can be recognized over a wide concentration range and the detection limit and quantitation range can easily be determined on the plot.