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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 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 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 .