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An indirect ELISA method for quantitative detection of anti-IgE autoantibodies / 中华检验医学杂志
Chinese Journal of Laboratory Medicine ; (12): 782-786, 2019.
Article in Chinese | WPRIM | ID: wpr-756505
ABSTRACT
Objective The accurate measurement of anti-IgE levels in patients' serum helps for make diagnosis of chronic urticaria (CU). An indirect ELISA method for quantitative detection of IgG anti-IgE, were established. Methods The serum samples and the clinical data of 75 first-diagnosed CU patients and 120 healthy controls were collected at Shanghai General Hospital during the year of 2018. In the indirect ELISA system to measure the IgG (anti-human IgE), the antigen, Human IgE Myeloma, was coated on the plate;Omalizumab (a humanized anti-human IgE antibody) was the standard, and horse radish peroxidase (HRP)-labeled anti-human IgG was the tracer. The optimum concentrations of serum and HRP-labeled anti-human IgG were determined by chessboard titration, and method evaluation was conducted. The comparison of serum anti-IgE levels in CU patients and healthy people were analyzed by Mann-Whitney U test and chi-square test. Receiver operating characteristic (ROC) curves were applied to establish the diagnostic performance of serum anti-IgE for CU. Results The coating concentration of IgEwas 5.0 × 10-4 mg/ml; serum dilution was 1300; enzyme-labeled antibody dilution was 1100000. Standard curve was in good linearity with R2=0.996. The intra-and inter-assay coefficient of variation were 3.9%-7.5% and 6.0%-8.2%, and the recovery rate of low and high concentration samples were 95.9% and 108.4%, respectively. When hemoglobin≤1.3 g/L, triglyceride≤4.6 mmol/L, bilirubin≤171μmol/L, no interference were observed. The limit of blank, limit of detection, and limit of quantitation were 5.8 × 10-4, 1.8 × 10-3, and 2.0 × 10-3 mg/ml. The linearity range was from 2.0 × 10-3 to 354.4 mg/ml. No Hook effect was found until anti-IgE reached 354.4 mg/ml. The anti-IgE remained stable after serum storage at 4℃overnight or treated with 6 repeated freeze-thaw cycles. The anti-IgE levels in CU patients [19.0(1.9, 58.6)mg/ml] were significantly higher than those in healthy controls [0.7(0.002, 11.1)mg/ml] with P<0.001. When cut-off value was set as 15.3 mg/ml, in this method, the positive rate of CU patients (54.7%) was significantly higher than these of healthy controls (18.3%) (P<0.001, AUC=0.736). Conclusions The indirect ELISA method for serum anti-IgE measurement was successfully established. Anti-IgE autoantibodies in serum would be used in the diagnosis of CU.

Full text: Available Index: WPRIM (Western Pacific) Type of study: Diagnostic study Language: Chinese Journal: Chinese Journal of Laboratory Medicine Year: 2019 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Type of study: Diagnostic study Language: Chinese Journal: Chinese Journal of Laboratory Medicine Year: 2019 Type: Article