A sensitive immunoassay for determination of hepatitis B surface antigen and antibody in human serum using capillary electrophoresis with chemiluminescence detection.
A sensitive and homogeneous immunoassay (IA) based on capillary electrophoresis (CE) with enhanced chemiluminescence (CL) detection has been developed for the determination of hepatitis B surface antigen (HBsAg) and antibody (HBsAb) in human serum. The conditions for the CL reaction and electrophoresis were investigated in detail using horseradish peroxidase (HRP) labeled HBsAg (HBsAg*) as a marker because of its catalytic effects on the luminol-hydrogen peroxide reaction. The CL reaction was enhanced by para-iodophenol and the CL detector was designed uniquely without any dead volume or diluents effect. The present method has been used for assaying HBsAg and HBsAb in human serum using a competitive format and a non-competitive format, respectively. Under the optimal conditions, the linear ranges were from 1 to 400 pmol/L (R=0.9988) for HBsAg and 2 to 200 mIU/mL (R=0.9981) for HBsAb. The detection limits were 0.4 pmol/L and 1 mIU/mL for HBsAg and HBsAb, respectively. The relative standard deviations of peak area were 4.2% and the errors of it were from -0.03% to +0.05% for 80 pmol/L HBsAg* (n=7). In this study, the free HBsAg* and the bound HBsAg* (HBsAg*-HBsAb) were separated in the separation capillary within 6 min using a borate run buffer. To verify the experimental reliability, the result was comparable with that of enzyme linked immunosorbent assay (ELISA) and demonstrated the feasibility of the CE-CL immunoassay method for clinical diagnosis.
Zhang
Yingxue
Y
Institute Analytical Science, Southwest University, Chongqing, China.
Zhang
Zhujun
Z
Yang
Feng
F
D000936:Antigen-Antibody Complex / Q000737:chemistry; D001667:Binding, Competitive; D019075:Electrophoresis, Capillary / Q000379:methods*; D005240:Feasibility Studies; D006509:Hepatitis B / Q000175:diagnosis; D006510:Hepatitis B Antibodies / Q000097:blood*; D006514:Hepatitis B Surface Antigens / Q000097:blood*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007124:Immunoenzyme Techniques; D007462:Iodobenzenes / Q000737:chemistry; D049449:Luminescence; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000936:Antigen-Antibody Complex; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007462:Iodobenzenes; C050181:4-iodophenol; D008165:Luminol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase