| The Hepatitis B (HB) infected by Hepatitis B virus (HBV) is a worldwide infectious disease. Hepatitis B surface antigen (HBsAg) located on the HBV surface is the major diagnostic marker in HBV detection. The most popular serological method for detection of HBsAg is enzyme-linked immunosorbent assay (ELISA) based on sandwich method.Magnetic affinity immunoassay (MAIA) using magnetic particles as a carrier was developed in 1980's, the method has the advantages of both enzyme immunoassay and the magnetic separation using an external magnetic field. MAIA also exhibits high sensitivity, reliability, toxic- and radioactive-free in the process of antigen/antibody detection. Herein, a method based on the MAIA for detection of HBsAg using GoldMag (Fe3O4/Au) particles as a carrier was set up and the chemiluminescence immunoassay (CLIA) was also used in this detection system because of its extremely high sensitivity and rapid performance. The research work included the following two parts.Using GoldMag particle as a carrier, an immunoassay based on sandwich method for detection of HBsAg was developed and validated by testing quantitative HBsAg panels. The serum (or plasma), GoldMag particles coupled with anti-HBsAg monoclonal antibody and HRP-labeled anti-HBsAg polyclonal antibody were incubated simultaneously at 37℃for 25 min, then the sandwich complex on GoldMag particle surface was washed and incubated with 3, 3', 5, 5'-tetramethylbenzidene (TMB) peroxidase solution, the absorbance of solution was measured at 450 nm. The results indicated that the relative standard deviation (RSD) for within- and between-batch assay were about 1.20 to 7% (n=5). The sensitivity for HBsAg subtypes, adr, adw and ay, were found to be 0.5 ng/mL, 0.5 ng/mL and 1.0 ng/mL, respectively. 203 serum samples from clinic blood donor were tested using this method and the results were consistent with the commercial kit.Chemiluminescence substrate (Luminol-H2O2) was introduced in the immunoassay system in which paraiodophenol (PIP) was used as an enhancer for chemiluminescence. The 1:1000 dilution of HRP-labeled antibody, 25 min for reaction time and 0.1 mol/L CBS buffer (pH 9.5) were selected as the optimal reaction conditions for chemiluminescence, and the concentration of Luminol, H2O2 and PIP were selected to be 5×10-4 mol/L, 1×10-3 mol/L and 1×10-3 mol/L, respectively. A calibration curve was determined and the linear range was 4～1100 pg/mL (R2 = 0.996). The detection limit for HBsAg was found to be 8.6×10-4 ng/mL. It was 1000 times lower than the detection limit of the ELISA using microtiter plate as carrier, which was reported to be 0.5～1 ng/mL. The precision of the method was determined by measuring 11 standard solutions successively and the relative standard deviation (RSD) was 6.8%.All the study above indicates that the immunoassay for HBsAg using GoldMag particles is more sensitive, reliable, easier to perform and without radioactive pollution. Therefore, it provides potential application for HBsAg testing in the near future.
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