Preparation And Identification Of Monoclonal Antibody Against Human Gastric Cancer Carcinoma

Bal/c mice were immunized with human gastric cancer cell line AGS. The research prepared the monoclonal antibody (McAb) against human gastric cancer carcinoma with the lymphocytes hybridoma technique and identified the elementary properties including: hybridoma chromosomes, titers of the McAb, subclass of the McAb and specific reaction.1. Culture of human gastric cancer cell line AGSAGS cells were cultured in vitro with the general methods. Proliferation-promoting rule of AGS cells was studied by protracting the growth curve. AGS cells were cryo-preserved with DMSO as cryoprotectants in manual way. After freeze-thaw cell viability was 96.31% in AGS cells. The results indicates that the cryo-preservation with DMSO as cryoprotectants in manual way is an effective way for cryo-preserving AGS cells.2. Hybridoma preparation, selection and clone(1) Preparation: 5 female Balb/c mice were immunized once every three weeks with human gastric cancer cell line AGS. Concentration optimization of coated antigen and antibodies in indirect ELISA were 2×10~5/ml and 1:10~4, respectively. SP2/0 cells and spleen cells fused with the help of PEG4000 after 3d when the titers of antibodies was 1:10~4. Hybridoma clones cultured in HAT were observed one week later. Fusion rate was 51.04% .(2) Selection: Hybridoma were selected by indirect ELISA. The result suggests that P/N of the supernatant of 1B4 were more than 2.1 in three indirect ELISA, that is to say, hybridoma 1B4 secretes antibodies.(3) Clone: Every cell was picked out of hybridoma 1B4 with micromanipulation and cultured in 96 culture plate. Antibodies only were found in hybridoma 1B4. The antibodies were called McAb 1B4.3. Identification of the elementary properties of hybridoma cell 1B4 and McAb 1B4(1) The average of the hybridoma cell 1B4 is 92 which accords with the characteristic of fusion cell.(2) The titers of antibodies in supernatant of hybridoma cell 1B4(McAb 1B4) is 1:10~4.(3) It was revealed that McAb 1B4 belonged to IgGl subclass.(4) The results of immunocytochemical method and indirect ELISA all suggested that McAb 1B4 was negative to Hela cell line, HepG cell line and lung cells from human embryo, but positive to AGS cell line. It shows that McAb 1B4 may be specific to human gastric cancer.