Published online Oct 15, 1998. doi: 10.3748/wjg.v4.iSuppl2.143
Revised: August 21, 1998
Accepted: September 14, 1998
Published online: October 15, 1998
AIM: To prepare gastric cancer MDR associated monoclonal antibody MGr1, and to screen tumor drug resistance cell epitope library with MGr1, the refer to obtain novel cDNA fragment associated with tumor MDR.
METHODS: By hybridoma technique, gastric cancer MDR associated monoclonal antibody MGr1 was prepared using gastric cancer MDR cell strain SGC79 01/VCR as immunogen. Tumor drug resistance cell epitope library was screened and rescreened by using MGr1 as a probe. The cDNA fragment brought by the positive recombinant was sequenced forward and backward, then, homology analysis was undertaken within GenBank. In situ hybridization and Northern blot determined the expressing level of positive cDNA fragment in several types of tissues and cells.
RESULTS: 26 clones of hybridoma whose supernatant had the ability to bind SGC7901/VCR were obtained from 3268 clones after 21 times cell fusing. And one monoclonal antibody of interest, which was named MGr1, was selected out by immunohistochemistry and MDR reversal test of monoclonal antibody. The staining results showed that MGr1 stained much more on SGC7901/VCR than on SGC79 01. MGr1 could enhance the cytotoxicity of 5-Fu and ADR on SGC7901/VCR. And wes tern blot showed that the Mr of MGr1 was about 80000-110000, which could not be recognized by Pgp or MRP antibody. Therefor MGr1 could be regarded as gastric cancer MDR associated monoclonal antibody. About library screening, five bacterial clones showed having affinity to MGr1, and one clone ws confirmed to bring the positive recombinant after being screened repeatedly. The cDNA fragment brought by the positive recombinant, which was named MGr1-Ag, had 310 bp and showed no homology with other genes in GeneBank. Results of in situ hybridization suggested that, the normal tissues of liver, esophagus, stomach, colon did not express the MGr1-Ag. Results of Northern blot suggested that the Mgr1-Ag was expressed in SGC7901, SGC7901/VCR, EC109 and HCC7402. The expressing level of MGr1-Ag was very high in SGC7901/VCR, but low in EC109 and eve n low in SGC7901 and HCC7402.
CONCLUSION: Gastric cancer MDR associated monoclonal antibody MGr1 was successfully prepared; A novel cDNA fragment associated with gastric cancer MDR, MGr1-Ag, was obtained by screening tumor drug resistance cell epitope library with MGr1.