Original Articles
Copyright ©The Author(s) 2001. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 15, 2001; 7(2): 216-221
Published online Apr 15, 2001. doi: 10.3748/wjg.v7.i2.216
Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab’)2 fragment and staphylococcal enterotoxin A Lian
Jun Yang, Yan Fang Sui, Zhi Nan Chen
Jun Yang, Yan Fang Sui, Zhi Nan Chen, Department of Pathology, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
Jun Yang, graduated from the Fourth Military Medical University as a Bachelor of Medicine in 1993, as a Master of Medicine in 1998, and as a M.D. in 2001. Now he is a lecturer in the Fourth Military Medical University, majoring the molecular pathology of tumors.
Author contributions: All authors contributed equally to the work.
Supported by the National 863 Project of China, No.102-09-01-02 and National Natural Science Foundation of China, No.39770827
Correspondence to: Dr. Lian Jun Yang, Department of Pathology, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China. yangsi@sina.com
Telephone: +86-29-3374541 Ext.115
Received: November 15, 2000
Revised: November 28, 2000
Accepted: November 30, 2000
Published online: April 15, 2001

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 (2pyridyldithio) 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 IgG 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: SPDP is a good protein conjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18 F(ab’)2 fragment and SEA protein was prepared successfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti HCC mAbs or their fragments.

Keywords: carcinoma, hepatocellular/immunology, liver neoplasms/immunology, superantigens, enterotoxins, antibodies, monoclonal