E antigen is a new antigen found in the positive serum of hepatitis B surface antigen (HBsAg). He may be closely related to the infectivity of hepatitis B. Li Yu et al. Reported that the sensitivity of ELISA to detect e antigen was 150-300 times higher than that of agar immunodiffusion. Test procedure:
1. Extraction of patient's immunoglobulin IgG (e antibody): The crude extract obtained by processing the above-mentioned antibody-positive human serum with a saturated ammonium sulfate precipitation method was collected through a column (DEAE-Cellulose) to collect the IgG-positive portion, and then flowed through a normal human serum column. IgG (e antibody) was obtained, concentrated to 5ng / ml and stored in the refrigerator.
2. Preparation of enzyme-labeled IgG (e antibody): Using glutaraldehyde two-step method, 10 mg HRP was cross-linked with 5 mg IgG (e antibody), which was divided into vials and stored at low temperature (-20 ° C).
3. ELISA detection of e antigen:
a. IgG (e antibody) coated polystyrene micro-reaction plate, overnight at 4 ° C (IgG concentration 25r / ml).
b. Wash 3-5 times.
c. Add patient serum to be tested (1:10 dilution, add two wells per specimen, 0.1ml per well, and incubate at 37 ° C for 2h).
e. Wash 3-5 times.
f. Add enzyme-labeled IgG (e antibody) and incubate at 37 ° C for 2h (dilution of enzyme-labeled IgG at 1: 640).
g. Wash 3-5 times.
h. Add substrates and hydrogen donors (H2O2 and OPD) and place in the dark for 30min.
i. Add 2mol / L sulfuric acid to stop the reaction.
j. Positive and negative controls are set for each plate, and judged according to different color reaction.
k. After the appeal method tests, the specimens initially determined to be positive, take two, one with sufficient amount of e-antibody serum and the other with normal human serum, incubate at 37 ° C for 1h, and then determine e antigen according to the above method . Those who added e antigen body serum can be judged to be positive for e antigen when the color reaction is controlled (compared with normal serum).
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