Development Of Luminescent Oxygen Channeling Immunoassay For Total IgE And Specific IgE

ObjectiveAs the incidence of allergic diseases is constantly increasing, therefore, to establish an effective in vitro testing method for the prevention and treatment of allergic diseases has important significance. The objective of this study is to establish Luminescent Oxygen Channeling Immunoassay (LOCI) for the quantitative detection of total IgE, and make an methodology and clinical evaluation for this method. On this basis, the detection system was improved by using Chinese mitten crab as example, and the reaction system matrix, reaction conditions and modes of exploration were optimized.Methods1. To establish total IgE detection system, the receptor microspheres were coated with mouse anti-human IgE monoclonal antibody, polyclonal rabbit anti-human IgE antibody was labeled with biotin. The methodological evaluation was made for this detection system.2. Muscles of Chinese mitten crab were extracted, and SDS-PAGE was performed to analyze the components of crude protein. Then the crude proteins were labeled with biotin, the established detection system was used to measure the specific IgE.3. The reason of not obtaining a satisfactory result for measuring clinical specimens was analyzed. The biotinylated crab proteins and reagents such as receptor microspheres were analyzed by SDS-PAGE or dot-blot.Results1. The intra-assay and the inter-assay coefficients of variation were3.33%-5.83%and5.33%-9.40%, respectively; The sensitivity was2.75IU/ml; The recovery was98.11%-100.7%. By detecting123cases of normal serum, total IgE value shows the upper limit of normal reference values of the experimental method was225IU/ml; In the interference analysis, interference rate of hemolysis, jaundice and lipemia were all<10%. The30clinical specimens were detected by this established method and transmission immunonephelometry by using Beckman Coulter Image800, the correlation coefficient was0.962.2. Mitten proteins extracted from crab were analyzed by SDS-PAGE electrophoresis, crab protein crude extracts clearly visible in10major proteins, and the relative molecular weight were98kD,85kD,77kD,55kD,48kD,45kD,39kD,36kD and33kD. The extracted crab protein could react with serum of crab allergy suffers, which meaned the immune activity of crab protein extracted.3. Dot-blot results showed existence of specific IgE in the serum of clinical patients. After biotinylated, the proteins extracted still owed the immune activity. But the biotinylated crab proteins only showed85kD and77kD bands in SDS-PAGE assay.Conclusion1. This Luminescent Oxygen Channeling Immunoassay could be used for the quantitative measurement of serum total IgE. The method was simple, and the sensitivity and specificity could meet the clinical requirements.2. The extract protein was complete and it can use as the reagent.3. Protein components have lost in the processing of biotinylated may be the cause of this experiment could not effectively detect the serum specific IgE.