Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab')(2) fragment and staphylococcal enterotoxin A.
OBJECTIVE
To prepare the conjugate of staphylococcal 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 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).


CONCLUSIONS
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.
Yang
L J
LJ
Department of Pathology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province,China. yangsi@sina.com
Sui
Y F
YF
Chen
Z N
ZN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D004768:Enterotoxins / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008113:Liver Neoplasms / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018089:Superantigens / Q000737:chemistry
D000911:Antibodies, Monoclonal; D004768:Enterotoxins; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D018089:Superantigens; C026066:enterotoxin A, Staphylococcal