An indirect ELISA method for quantitative detection of anti-IgE autoantibodies
10.3760/cma.j.issn.1009-9158.2019.09.012
- VernacularTitle: 间接ELISA定量检测抗IgE自身抗体方法的建立
- Author:
Yue YIN
1
;
Hong DANG
2
;
Chuang GAO
2
;
Xia PENG
1
;
Yuting LIANG
1
;
Huanjin LIAO
1
;
Li LI
1
Author Information
1. Department of Laboratory Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
2. Department of Dermatology, Shanghai General Hospital, Shanghai 200080, China
- Publication Type:Journal Article
- Keywords:
Immunoglobulin E;
Autoantibodies;
Enzyme-linked immunosorbent assay;
Urticaria
- From:
Chinese Journal of Laboratory Medicine
2019;42(9):782-786
- CountryChina
- Language:Chinese
-
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 IgE was 5.0×10-4 mg/ml; serum dilution was 1∶300; enzyme-labeled antibody dilution was 1∶100 000. 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≤171 μmol/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.
