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Objective To investigate the distribution characteristics of serum sIgE antibodies against four allergenic protein components of egg white in children with egg allergy,and then clarify the clinical application value of single component-resolved diagnostics of egg allergy.Methods Serum samples from 197 children with egg allergy were collected.The levels of serum sIgE antibodies against four major allergenic protein components of egg white,including ovomucin,ovalbumin,ovotransferrin,and lysozyme,were detected by the light-excited chemiluminescence assay(LiCA),and the distribution characteristics of sIgE antibodies were analyzed.Results The positive rates of serum sIgE antibodies against ovalbumin,ovomucin,ovotransferrin,and lysozyme in 197 chlidren with egg allergy were 77.16%(152/197),70.56%(139/197),35.02%(69/197),and 18.27%(36/197),respectively.The positive rate of serum sIgE antibody against both ovomucin and ovalbumin was 30.45%.There was a weak correlation between the levels of sIgE antibodies against egg and the cumulative levels of sIgE antibodies against four allergenic protein components(r=0.266 8,P＜0.05).There were signifi-cant individual differences in the levels of serum sIgE antibodies against four allergenic protein components of egg white in the children with egg allergy.Conclusion There is individual heterogeneity in the levels of serum sIgE antibodies against four components of egg white in the children with egg allergy.The detection of sIgE antibodies against egg white components can distinguish different forms of egg allergies,which is of great value for the accurate diagnosis and precise desensitization of children's egg allergy.
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Objective To establish a candidate reference measurement procedure for the detection of serum 25-hydroxy vitamin D3[25(OH)D3]based on isotope dilution liquid chromatography tandem mass spectrometry(LC-MS/MS).Methods Isotope standard solution was used as internal standard,liquid-liquid extraction was used for pre-treatment,and positive ion electrospray ionization mode was used for monitoring.The accuracy,precision,linear range,limit of quantitation,detection limit and relative matrix effect of method were verified based on documents of the America Clinical and Laboratory Standards Institute(CLSI)such as C62-A and EP15-A3.Candidate reference measurement procedure and mass spectrometry routine procedure were used to detect 40 clinical serum samples,and to evaluate the consistency of the two methods.Results The analysis time of the candidate method was 15 min.Isometric elution of chromatography was used to effectively separate the isomer 3-epi-25(OH)D3,with good specificity.RELA comparison sample was measured,with a bias of less than 1.5%.The intra-batch precision and inter-batch precision ranged from 0.75%to 2.31%and 1.28%to 2.01%,respectively.The limits of quantification and detection were 0.85 ng/ml and 1.84 ng/ml.It had good linearity in the concentration range of 2.5～220 ng/ml,and there was no relative matrix effect and carrier contamination.The correlation between the mass spectrometry routine procedure and candidate reference procedure was good(r=0.982),while the deviation at low concentration samples exceeded the allowable total error±25%in the external quality assessment of the National Center for Clinical Laboratories.Conclusion A candidate reference measurement procedure for serum 25(OH)D3 technology based on LC-MS/MS was successfully established,and the analytical performance met the requirements,which could be used for quantitative traceability by clinical conventional methods.
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Objective:To prepare 68Ga-2-(4, 7-bis(carboxymethyl)-1, 4, 7-triazonan-1-yl)pentanedioic acid (NODAGA)-YHWYGYTPQNVI (GE11) and evaluate its feasibility of PET imaging for pancreatic cancer. Methods:GE11 peptide was conjugated with NODAGA and then labeled with 68Ga. The labeling yield, radiochemical purity, hydrophilicity, stability and specificity in vitro were determined. Human pancreatic cancer BxPC3 nude mice models ( n=9) were established. MicroPET imaging was then obtained after 30 and 90 min, and mice were sacrificed at 90 min to acquire the radioactivity distribution of main organs and tumors. Pair t test was used to analyze the data. Results:The labeling yield was (73.5±5.4)% and radiochemical purity was more than 98%. After incubation 120 min in mouse serum at 37 ℃, radiochemical purity was more than 92%. The uptake was specific in BxPC3 cell lines. MicroPET images showed that 68Ga-NODAGA-GE11 could accumulate quickly in tumor. Value of tumor uptake was significantly higher than that of normal pancreas at 90 min ((1.38±0.25) vs (0.49±0.07) %ID/g; t=12.67, P<0.05), and the radio-uptake of blood, muscle and bone was lower than that of tumor. Conclusions:68Ga-NODAGA-GE11 is easy to be prepared with high radiochemical purity and good stability, and can specifically target BxPC3 xenograft tumor. However, due to the high uptake in the kidneys and liver, the value of 68Ga-NODAGA-GE11 in PET imaging for pancreatic tumor needs further study.
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Objective@#To develop and evaluate a beads-based light-initiated chemiluminescent assay (LICA) for quantitation of cow milk component (Bos d 5) specific IgG 4 antibody in human serum. @*Methods@#The sIgG 4 -LICA was performed by incubated serum samples with biotinylated allergens, emission beads coated with mouse anti-human IgG 4 antibody and streptavidin-coated sensitizer beads. The reaction conditions of sIgG 4 -LICA were optimized and the analytical performance was evaluated. @*Results@#The precision of intra-assay, within-day and inter-assay (coefficient of variation) were 1.78% to 3.13%, 6.65% to 8.41% and 7.94% to 12.30%, respectively. The functional sensitivity of this assay was 4.71 ng/mL. For the linear range, the sIgG 4 -LICA had a good linear relationship within the range between 28.13 and 1 800 ng/mL, and the linear regression equation was Y=0.98X-1.31(r 2 =0.997). Maximum dilution limit was 1∶64. The disturbing rates measured by adding hemoglobin, triacylglycerol, total bilirubin, acid resistance and biotin to human sera with different concentrations of Bos d 5 sIgG 4 were from -6.38% to 8.60%. @*Conclusions@#The sIgG 4 -LICA introduced in this study was demonstrated to have effective performance for quantitation of allergen-specific IgG 4 and can meet the need of clinical requirement.
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Objective@#To establish an analytical method for serum prolactin (PRL) based on the photoinduced chemiluminescence technology, and evaluate its performance. @*Methods@#A pair of PRL monoclonal antibodies were labeled with luminescent nanospheres and biotin respectively, and the double antibody sandwich detection system was formed with the serum prolactin and streptavidin-labeled photosensitive microspheres (universal photosensitive solution) under homogeneous conditions. The performance index and correlation of the detection system were evaluated. @*Results@#The precision of intra-assay and within-day (coefficient of variation) of the developed assay were 4.60% and 5.25%, respectively. The functional sensitivity was 2.48 μIU/mL, and its reportable results were ranged from 2.48 to 4 240 μIU/mL. The recovery rates of different PRL calibrators (42.2, 424, 4 240 μIU/mL) added to human sera were ranged from 96.25% to 102.93%. There was no interference from bilirubin＜20 mg/dL, hemoglobin＜200 mg/dL, triglyceride＜3 000 mg/dL and biotin＜20 ng/mL. Also, the light-initiated chemiluminescent assay for PRL (PRL-LICA) correlated well with Beckman Unicel Dxi 800 Access 2. @*Conclusions@#LICA showed effective performance for detecting PRL in human serum, and it could meet the basic requirements of clinical diagnosis.
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Objective To develop a competitive immunoassay for quantitative determination of total immunoglobin E (tIgE) in human serum based on light-initiated chemiluminescent assay (LICA).Methods The LICA-tIgE assay was performed by incubating serum samples or calibrator with anti-human IgE antibody-coated chemiluminescet beads,biotinylated human IgE and streptavidin-coated sensitizer beads.The working conditions of this assay were optimized,analytical performance was detected and the correlation of tIgE results between LICA and Beckman Coulter IMMAGE 800 was evaluated.Results The precision of intra-assay and inter-assay (coefficient of variation) ranged from 5.50％ to 7.73％ and 6.45％ to 9.90％,respectively.The functional sensitivity of this assay was 12.65 IU/mL.The recovery rates measured by adding IgE calibrators to human sera with different IgE concentrations were ranged from 104.15％ to 109.37％.The disturbing rates measured by adding total bilirubin,hemoglobin and triacylglycerol to human sera with different IgE concentrations were ranged from-4.49％ to 8.46％.Also,the tIgE results of 111 patients measured by LICA correlated well with those by Beckman Coulter IMMAGE 800 (r2 =0.959).Conclusion LICA developed in this study for detecting tIgE of human serum showed effective perfomance and could meet the basic requirements of clinical diagnostic reagents.
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Objective:To prove hemocyanin is an important high-molecular-weight allergen from Eriocheir sinensis.Methods:The proteins were extracted from Eriocheir sinensis tissue with lysate extraction method .The protein components were analyzed by SDS-PAGE,two-dimensional electrophoresis and Western blot.Serum IgE from allergic donors identified a candidate protein,which was char-acterized by MALDI-TOF/TOF.The immune property of the candidate protein was tested using Dot-blot.Results: By SDS-PAGE and two-dimensional electrophoresis analysis,we prove that the native protein components were completely.According to the Western blot result,at least 18 components could react with the positive serum.Among them, two 70-80 kD proteins had the same isoelectric point.So,may be they were the same protein.MALDI-TOF/TOF analysis results showed that the suspected proteins were hemocyanin.A sensitization frequency of 61%was observed in Eriocheir sinensis patients by Dot-blot.Conclusion:Hemocyanin was identified as an important high-molecular-weight allergen from Eriocheir sinensis.
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Objective To improve the detecting sensitivity of serum specific IgE (sIgE) by improving the quality of coated antigen in shrimp. Methods The extracts from shrimp protein was prepared. Western blot assay was used to identify the major allergenic protein components. The protein components>55 ku were separated by Sephadex gel chromatography. SDS-PAGE technology was used to analyze proteins. Samples of shrimp protein and proteins>55 ku were used as the coat-ing antigen to coat 96 microplate respectively. Western blot assay and ELISA were used to evaluate preliminary sensitivity of the purified antigen for detecting sIgE. Results Immunoblot experiments showed that the protein>55 ku was the main aller-genic protein component of shrimp. Those >55 ku proteins were separated successfully by Sephadex gel chromatography, showing 10 identifiable bands in SDS-PAGE. Dot-pot immunoassay showed that proteins>55 ku used as coated antigens could improve the spots density of the weak serum. Meanwhile, the result of ELISA showed that sIgE detection value in-creased 92.9%in patients with shrimp allergy after coating effective antigens. Conclusion The detecting sensitivity of sIgE can be improved by using effective protein components of shrimp as coated antigens.
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Objective To establish homogeneous immunoassay for detecting serum cardiac troponin I (cTnI) by using light induced chemiluminescent immunoassay (LiCA). Methods Polyclonal antibodies of cTnI were coated on the receptor particles, monoclonal antibodies of cTnI were biotinylated, and the donor particles were coated with streptavidin, all of which were composed of LiCA reagents. The optimal test conditions and analytical performance of the detection method were studied. Results The method was rapid, sensitive, and detection time was 17.5 min.The analytical sensitivity was 0.045 ng/mL and the functional sensitivity was 0.053 ng/mL.The recovery rate was 104.96%-108.21%;The within-run and the between-run coefficients of variation were 3.88%-5.53%and 7.60%-8.75%, respectively. The interference rates for the endogenous substances were less than 10%. The reference value of cTnI was less than 1.05 ng/mL;Results of cTnI LiCA correlated well with direct chemiluminescence detection (r2 =0.979). Conclusions This approach can be used for the quantitative detection of serum cTnI, and it is homogeneous and is free of clean separation. It provides a convenient, highly sensitive detection platform for clinical practice.
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Objective:To develop a luminescent oxygen channeling immunoassay for pregnancy associated plasm a protein A.Methods:The monoclonal antibody of PAPP-A was labeled with biotin ,the polyclonal antibody of PAPP-A was coated on receptor particles.The LOCI reagents also contained sensitizer particles coated with streptavidin.The optimal test conditions and analytical per-formance of the method were studied.Results:The within-run and the between-run coefficients of variation were 5.91%-7.94% and 6.14%-9.69%,respectively;the analytical sensitivity was 2.8 mU/L and the function sensitivity was 4.6 mU/L,good linear in 2.8 mU/L-8 000 mU/L range;the recovery rate was 96.7%-100.3%.The interference rate of hemolysis , icterus and triglycerides were less than 10%; there is no Hook effect of PAPP-A concentrations up to 8 000 mU/L;the correlation coefficient between clinical samples detection results and Time resolved fluoroimmunoassay analysis results was 0.974.Conclusion:This LOCI can be used for the quantitative of serum PAPP-A,and detection performance in line with the requirements of clinical diagnostic reagents .
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Objective:To analyze protein and allergic components in Eriocheir sinensis tissue with different extraction and processing methods , provide an optimal antigen extraction protocol for detecting specific IgE of Eriocheir sinensis.Methods: The proteins were extracted from tissue protein of Eriocheir sinensis with PBS extraction method ,acetone extraction method ,lysate extraction method,respectively;Extract heating and without heating tissue protein of Eriocheir sinensis with lysate extraction method .The total protein components were analyzed by SDS-PAGE and two-dimensional electrophoresis.The allergen components were identified by Western blot.Results:The binding strength of protein components extracted by different methods and sIgE in the immunoblot assay dif -fered.Protein components extracted by PBS extraction method were mainly 94,85,76,66,18 kD,by acetone extraction method were mainly 94,85,76,36 kD,while by lysate extraction method were mainly 200,125,51,43,38 kD.Protein components extracted by PBS extraction method reacted with patient serum were mainly 76,66,53,43,38 and 18 kD,by acetone extraction method were mainly 76, 66,53,43,38 and 18 kD,while by lysate extraction method are mainly 200,105,94,76,43,38 and 18 kD.Heating and without heating proteins and allergic components were similar.Conclusion:Lysate extraction method is more suitable for determination of specific IgE antigen preparation purpose.The binding activity of sIgE and heating or without heating allergen components is similar .
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Objective To investigate the allergization of milk high molecular weight proteins. Methods Thirty cas?es of patients with serum allergic to milk were selected. Their skin prick tests were positive. Results of serum specific IgE (sIgE) test were positive and≥1+. One case of healthy control with negative sIgE test and without history of allertgy, was in?cluded in this study. The serum samples were collected and frozen at-20℃. Sephadex G200 gel chromatography was used to obtain milk high molecular weight proteins. Western blot and ELISA methods were used to detect milk high molecular weight proteins and the activity of serum sIgE. Results Results of SDS-PAGE showed that high molecular weights proteins displayed by Sephadex G200 gel chromatography, mainly including three bands, the molecular weight of 67 ku, 80 ku and 160 ku. Western blot analysis showed that three kinds of high molecular weights proteins can react with milk allergy serum, and the most obvious appeared near the molecular weight 67 ku band. ELISA analysis showed that the positive response rate of high molecular weight proteins with milk allergic patient’s serum was slightly higher than that ofβ-lactoglobulin (46.7%and 43.3%, respectively). Conclusion The milk high molecular weight protein components can induce specific IgE antibod?ies, which have important sensitization in the process of milk allergy.
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Objective To explore a novel quantitative detection method for the concentration of specific IgG (sIgG) in food intolerance, taking egg sIgG detection for the example. Methods A total of 173 patients underwent food allergen sIgG detection were included in this study, and 78 healthy subjects were used as negative controls. The microtiter plates were coated with biotinylated bovine serum albumin (BSA) and linked with streptavidin. Then, the biotinylated egg antigen and specimen were successively added into the wells of plate.After washing, enzyme labeled anti-human IgG was added to establish an antigen indirectly coated liquid-phase reaction patterns of ELISA method. The concentration of the biotinylated allergens and enzyme labeled antibody were optimized and the reaction conditions were determined. This method was used to detect the sIgG in serum samples. Results The biotinylated egg white was selected as antigen, the optimal dilution rate was 1∶2 000,and the most suitable enzyme labeled antibody dilution ratio was 1∶12 000 in this method. The within-run and the between-run coefficients of variation were 4.83%-8.55%and 4.88%-7.93%respectively. The specificity is preferable, the species-crossed reaction rates with crab, cow milk and goat milk were＜10%. There was a good correlation between the assay developed in this study and the food intolerance detection kit provided by United States BIOMERICA Inc ( =0.977X+8.45, r=0.961, P＜0.05). Conclusion The detection method can be used to detect serum sIgG for egg intolerance patients with easy operation, highly accuracy and specificity. Furthermore, it showed the capacity of excellent repeatability and flexibility potential, which provided a good foundation for developing a kind of“personalized”random combination of food varieties ELISA kit.
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Objective: To investigate the effect of human chorionic gonadotrophin (hCG) on the gene expression of migration inhibitory factor (MIF) in human peripheral blood mononuclear cell (PBMC). Methods: The healthy human PBMC was cultured with hCG at 37 ℃, 5%CO_2 for 2 hours. The mRNA of harvested cells was isolated. The MIF mRNA was detected by real-time RT-PCR. Results: In a certain range of doses, the mRNA expression of MIF significantly increased following the increase of hCG in a dose depandent manner, and it reached to a peak 1-2 hours after culture, then returned to the minimum level after 8 hours. Conclusion: In a certain range of doses, hCG can increase the mRNA expression of MIF. This effect is correlated with reacting time. It is suggested that hCG may involve in immune response by up-regulating the production of cytokines by PBMC.
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The paper has established an assessment system and a quantitative calculation method of the "implicit" environmental impact including environmental impact indicator, resources consumption indicator and energy consumption indicator. The quantitative calculation of the environmental impact indicator is based on the life cycle assessment system and the evaluation software BEES. The paper identifies normalization reference values and weights for 12 categories of the environmental impact. It also analyzes the environmental impact indicator in life cycle stages, raw materials, transportation, manufacturing, utilization, and end of life. A university refectory project is studied. The result has shown that human health, global warming and acidification are the first three environmental impacts in 12 categories. The environmental impact indicator per m2 of this project is 18.448×10-2 standard human equivalent weight. Moreover, 97.3 % of the total environmental impact occurs at the raw material stage, in which the most severe environmental impact is cancerous health effect; the global warming is the main impact at the transportation and manufacturing stages; the indoor air quality impact is at the usage stage.
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Object To study the effect of emodin, the active principle in rhubarb on the secretion of TNF ?, IL 1, IL 6, and [Ca 2+ ] i by rat peritoneal macrophage under different conditions Methods Hypernomic inflammation models of isolated rat peritoneal macrophage were prepared by lipopolysaccharide excitation and the levels of [Ca 2+ ] i and the proinflammatory cytokines TNF ?, IL 1 and IL 6 secreated determined by fluorescence spectrophotometry and bioassay Results Emodin showed a dual regulatory effect on the secretion of proinflammatory cytokines and [Ca 2+ ] i Conclusion It seemed that emodin has a biphasic immuno regulatory effect
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AIM To investigate the effect of puerarin (Pue) on tumor necrosis factor (TNF) ?, interleukin (IL) 6 secreted by neonatal rat cardiomyocytes during hypoxia/reoxygenation injury. METHOD The activities of TNF ? and IL 6 in the supernatants of cultured myocytes, which were sampled from different groups (control, model, and therapeutic groups with 1 g?L -1 Pue, 0 1 g?L -1 Pue, 0 01 g?L -1 Pue) at different time, were assayed by bioassay method. RESULTS TNF ? and IL 6 activity increased compared with that of control ( P