Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F( ab′ )_2 fragment and staphylococcal enterotoxin A  

作　　者：Lian Jun Yang Yan Fang Sui Zhi Nan Chen Department of Pathology, Fourth Military Medical University. Xi’an 710032, Shaanxi Province, China 

出　　处：《World Journal of Gastroenterology》2001年第2期216-221,共6页世界胃肠病学杂志（英文版）

基　　金：Supported by the National 863 Project of China,No.102-09-01-02;National Natural Science Foundation of China,No.39770827

摘　　要：AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can AIM To prepare the conjugate of staphy- lococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')_2 fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS MAb HAb18 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP -40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')_2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')_2 fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2- pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry s method. The antibody activity of HAb18 F(ab')_2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS The lgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab')_2-SEA conjugate. SDS -PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in Hab18 F(ab')_2-SEA conjugate and HAb18 F (ab')_2,i.e., the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab')_2-SEA conjugate was 0.182±0.012, that of negative control was 0.033 ±0.009, and there was significant difference between them (P<0.05). CONCLUSION SP

关 键 词：carcinoma  hepatocellular/immunology liver neoplasms/immunology SUPERANTIGENS ENTEROTOXINS antibodies  MONOCLONAL 

分 类 号：R735.7[医药卫生—肿瘤]