Chemiluminescent Assay Of Allergen SIgE Nano Magnetic Particle

Objectives A nano magnetic particle chemiluminescent immunoassay was established for the clinical detection of allergens, in order to provide an accurate, fast, and variety allergens screen method for China patients. The sensitivity, accuracy, specificity, repeatability and stability of this assay were verified.Methods Allergens with high incidence and serious symptoms were chosen as the research objects according to the epidemiological survey results of Chinese allergy patients. The optimum concentration of chemiluminescent substrate solution, immune reaction conditions, the appropriate particle size of nano magnetic particles, the best working concentration of nano magnetic particles linking with streptavidin, the best working concentration for connection of biotin and allergens, and the best working concentration of HRP-labeled mouse anti-human IgE were determined based on the signal-to-noise ratio. The standard curve was determined according to the WHO classification criteria for serum IgE concentration grade. The performance of the established method has been evaluated in laboratory and clinical trials according to "Guidelines of in vitro diagnostic reagents analysis performance assessment" and "Guidelines of in vitro diagnostic reagent clinical trial".Results 1 Chemiluminescent reaction system:substrate A solution (luminol 0.4mg/ml) and B solution (urea peroxide 0.2mg/ml). This luminescent reaction system has highly sensitive of HRP (minimum detection 0.01 ng/ml). The luminescence value shall be measured after add A and B substrates solution and react 5 minutes (room temperature, avoid light), but detection result is effective in 5-30 minutes.2 Two-step reaction is applied, and the optimum conditions for each step is 37℃,15 minutes.3 Nano magnetic particles with the size of 100 nm has the best effect. The best working concentration of streptavidin is 2.5μg/ml.12 kinds of biotin labeled allergens have the best concentration respectively. The best work concentration of HRP-labeled mouse anti-human IgE is 0.25μg/ml.4 According to the clinical requirement select 0.35～100IU/ml as the standard curve range.5 The sensitivity, accuracy, specificity, repeatability and stability of the in vitro allergen test method meet the national requirements for in vitro diagnostic reagents.6 According to the results of 1202 cases clinical trials, the method present consistent rate and diagnostic agreement rate above 90% with the results of Uni-CAP system, also artemisia pollen and house dust mites allergens were found as the most important allergens in China.Conclusions 1 A nano magnetic particle chemiluminescent immunoassay has been established, and its sensitivity, accuracy, specificity, repeatability and stability were proved to meet the requirements of design, regulations and clinical requirements.2 The result of clinical trial shows that the method can replace the expensive imported products; because of the applying of local raw allergen materials, the method is more suitable to the diagnose of Chinese allergy patients, in the meanwhile, simple operation, low cost and fast test speed are the advantages of the method.3 By the clinical trial shows that Artemisia pollens and house dust mites are proved as the most important allergens in China.