[Two mouse hybridomas secreting monoclonal antibodies against human liver carcinoma and their antibody specificity].

There has been much evidencesuggesting that human liver carcinomacells might have some tumor-associatedantigen(s). However,neither wellcharacterized human hepatoma associ-ated antigens were isolated nor mono-clonal antibodies againt this tumorwere reported in the literature.Thispaper describes the production of mono-clonal antibodies which seemed to bindmore or less specifically to human livercarcinoma cells.The Production and Screening of hy-bridomas The BALB/c myeloma cell linep3-NS1/1-Ag4-1 used for fusion wasmaintained in DMEM Containing 20%newborn calf serum,BEL-7402 humanliver carcinoma cell line used forimmunization and other cell lines usedfor screening or detection were all maintained in RPMI 1640 containing20% newborn calf serum.BEL-7402cells were first transfered to serum-free medium for 24hr,centrifuged andwashed 3 time with PBS before immuni-zation.Two BALB/c mice were eachimmunized intraperitoneally with about1×10~7 cells every week for 3 weeks.After an interval of three weeks,abooster dose of 1×10~7 cells was givenand spleen cells were collected threedays later. Immunized spleen cells (2×10~8)were fused with NSI cells (2×10~7)as described by Oi and Herzenbergin 0.8ml of 50% polyethylene glycol(Mw.4000) for I min at 37℃. Thecells were centrifuged,resuspended inHAT medium in DMEM and distri-buted to 1152 wells of microtiter plates.The production of antibodies was testedby indirect immunofluorescence assayaccording to Hosoi et al.Tissueculture cells or cryostat sections oncoverslips were used without fixing.They were washed 3 times with PBS,covered with supernatant of hybridomacultures and incubated for half an hour.They were washed again 3 times withPBS and treated with properly dilutedFITC-conjugated rabbit anti-mouseIgG.After half an hour,they werewashed 3 times with PBS and examinedunder a fluorescent microscope. In the first screening,we usedBEL-7402 cells,HeLa cells and mixedblood lymphocytes as target cells andobtained 53 out of 1152 hybridomaswhich secreted antibodies that werepositive to BEL-7402 cells but negativeto HeLa cells and lymphocytes.Thesecond screening of these 53 hybridomawas aimed at the sorting out of the hy-bridomas which produced antibodiesreactive to BEL-7402 and BEL-7405human liver carcinoma cells,but notreactive to BEL-7702 normal humanliver cells,SGC-7701 human stomachadenocarcinoma cells and TCA-8113human tongue squamous carcinomacells.Two hybridomas thus obtainedwere subsequently cultured in 24 wellmultiwell plates with I ml of mediumand cloned 5—8 times by limitingdilution method at 0.5 cell/well.Specificity of the monoclonal antibo-dies The two hybridomas which secretedmonoclonal antibodies against humanliver cancer cells were designated asHepama-1 and Hepama-2.Indirectmembrane immunfluoresence assaysdemonstrated that these two monoclonalantibodies reacted with five out of sixhuman liver carcinoma cell lines andwith five out of six human liver cancersurgical specimens,but not with 15other human tumor tissues and humantumor cell lines (Table.1). Fifteen kindsof normal human adult tissues (includingnormal adult liver) and nineteen kindsof fetal tissues (including fetal liver)were also found to be negative to these antibodies,except for a weak reactionto one of the three fetal testes byHepama-2(Table 2). The relativespecificity thusfar obtained with mono-clonal antibodies derived from thesetwo hybridomas seems to indicate thatthey are possibly directed against somecommon determinants of human he-patoma associated antigen(s).