[Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection].
OBJECTIVE
To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection.


METHODS
The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA (HTS-ELISA). Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established.


RESULTS
The titers of mouse anti-sera reached 1:10(5);. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity (over 10(9); L/mol). Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg "d, y, r, w" epitopes, respectively. The sandwich ELISA was established through using #3-11 (Anti-HBsAg "a" epitope) as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions.


CONCLUSIONS
The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.
College of Life Sciences, Northeast Forestry University, Harbin 150040; Daqing MabCom, Inc., Daqing 163316, China.
Xiaolei
X
Zhang
College of Life Sciences, Northeast Forestry University, Harbin 150040; Daqing Institute of Biotechnology, Northeast Forestry University, Daqing 163316, China.
Yuzhe
Y
Nie
College of Life Sciences, Northeast Forestry University, Harbin 150040, China.
Xingguo
X
Lan
College of Life Sciences, Northeast Forestry University, Harbin 150040, China.
Yuhua
Y
Li
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000145:classification* / Q000276:immunology*; D006825:Hybridomas / Q000166:cytology; D007114:Immunization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012703:Serotyping / Q000379:methods*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006514:Hepatitis B Surface Antigens