Development And Application Of A Light-initiated Chemiluminescent Immunoassay For The Determination Of SIgE Against Egg White Allergens With Component-resolved Diagnosis

Objective:The accurate quantitation of serum sIgE level is of great instructive significance in determining the diagnosis and therapy of food allergy.Also,component-resolved diagnosis(CRD)has become a trend,and it reflects important value in precision detection and specific immunotherapy.In this study,we take the four allergens in egg white as the research object,and intend to establish a homogeneous immunoassay to determine the serum sIgE level.Also,from the perspective of CRD,we evaluate the clinical value of single allergen in predicting food allergy.Methods:1.The initial establishment of a light-initiated chemiluminescent assay(LICA)in determining sIgE against egg white allergen:Based on LICA,the four allergens of egg white were biotinylated by Biotin,and the chemiluminescer beads were coated by anti-humanIgEantibody,afterincubatingwithserumsample,the streptavidin-coated sensitizer beads were added,thus constituting the reaction system.We then optimized the concentration and incubation time,and initially established LICA to quantitate sIgE in serum.2.Performance characteristics of LICA in determining sIgE against egg white allergen:We evaluted the assay performance characteristics,i.e.,sensitivity,precision and accuracy.Moreover,we selected 8 patients allergic to single allergen and made correlation analysis with ImmunoCAP,thus further verifying the accuracy of the method.3.The clinical value of four allergens in predicting food allergy,respectively:In order to evaluate the clinical value of single allergen in predicting food allergy,70 egg white allergic patients and 30 healthy volunteers were chosen,and ImmunoCAP were used as standard,we operated the receiver operating characteristic(ROC)curves,and calculated sensitivity,specificity,and area under the ROC curve(AUC).Also,we made combination of the components to select the combination method with the best diagnostic value.Results:1.The initial establishment of LICA in determining sIgE against egg white allergen:We optimized the working conditions,and determined serum dilution as1:10,concentration of antigen as 0.665μg/ml,concentration of anti-IgE antibody as1μg/ml,concentration of chemibeads as 20μg/ml,and the first-stage incubation time as 30 min.2.Performance characteristics of LICA in determining sIgE against egg white allergen:The intra-assay precision and inter-assay precision(%coefficient of variation)ranged from 1.83 to 4.13%and 2.70%to 8.70%,respectively.Also,positive sera and negtive sera could be differentiated even at large dilution level,exhibiting excellent sensitivity.In the accuracy assay,the recovery rate was 90.29%to 117.93%.When comparing with ImmunoCAP,it showed good corelation(r~2=0.929).3.The clinical value of four allergens in predicting food allergy,respectively:In predicting food allergy,ovomucoid(Gal d 1)and ovalbumin(Gal d 2)had the same AUC of 0.81,the specificity were 90.0%and 93.3%,and the sensitivity were 61.4%and 58.6%,respectively.However,when applying Gal d 1 and Gal d 2 in combination analysis,they showed a higher sensitivity of 72.4%and exhibited better diagnostic value.Conclusion:Based on CRD,we established a light-initiated chemiluminescent assay to determine sIgE against egg white allergen.This method showed a good performance characteristics and had a certain clinical applied value.It was expected to play a role in the field of molecular diagnosis of food allergy.