remaining abstracts from the sixth annual hybridoma congress
TRANSCRIPT
Remaining Abstracts fromthe Sixth Annual
Hybridoma Congress
A NOVEL GENEFUSION SYSTEM FOR RAISING ANTIBODIES AGAINST ADEFINED PEPTIDE SEQUENCE. B.Löwenadler3,B.JanssonD.S.Paléus3,B.Nilssgn" E.Holmgren ,T.Moks,G.Palma , S
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Josephsonf" andM.Uhlén
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Kabigen AB, S-112 87 Stockholm, Sweden. Dept. ofBiochemistry, Royal Inst, of Technology, S-100 44 Stockholm,Sweden.
Methods to obtain specific antibodies against peptides usinggenefusions have been described by several laboratories. Wehave earlier presented a set of expression vectors in whichcDNA or synthetic sequences were fused to the gene coding forStaphylococcus aureus protein A ( EMBO J. vol 5, 9,2393-2398,1986). After expression in S.aureus or E.coli the fusionprotein could be harvested from the growth mediumrespectively released from the cells by osmotic shocktreatment or sonication and used for immunization. In thepresent work we have synthesised two short oligonucleotidesencoding the C-terminal part (amino acid 57 to 70) of humaninsulin-like growth factor 1 (IGF-1) and cloned them into anew set of fusion vectors (pEZZS and pEZZ18) based on twosynthetic IgG binding domains (Z) of protein A. The smallsize fusionprotein (M 16 kd) thus obtained was harvesteddirectly from the culture medium after expression in E.coliand purified in a one step procedure by affinitychromatography on Sepharose bound IgG. Rabbits immunized withthe genefusion protein gave rise to peptide specificantibodies reactive both with a synthetic C-terminal peptideas well as with the native human IGF-1 protein. This wasdemonstrated by a solid phase RIA and western blottingexperiments. Our results indicate that the genfusion systemmay comprise a rapid and efficient way of producingantibodies against short peptides encoded by syntheticoligonucleotides, thereby representing a versatile geneticalternative to immunization with synthetic peptides.
DETECTION OF MIRCOMETASTASIS WITH -CtKCLONAL ANTIBODIESIN CANCER PATIENTS
I. FunJte, G. Schlimock, G. RiethmullerInstitute of Irrnujiology, Goethestr. 31, 8000 Munich 2, F.R.G.
The detection of early micrometastasis or disseminatedsingle tumor cells is almost impossible with conventionaldiagnostic procedures. Our approach is to screen bone
marrow aspirates obtained at primary surgery with themonoclonal antibody CK 2, which recognizes the human cyto-ceratin component no. 18. This antibody is highly specificfor cells of epithelial origin as demonstrated in a doublestaining procedure with an anti T200 antibody reacting witha leukocyte common antigen. Furthermore, no positive reac-
tion of MAb CK 2 was seen in bone marrow samples of patientswithout carcinoma (N = 75).In 9.5%
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20.5% of patients with either colorectal or breastcancer without distant metastasis, single tumor cells couldbe detected in bone marrow. Significant correlations bet-ween these disseminated tumor cells and some conventionalrisk factors were found. In a first step towards úimi-notherapy, we were able to demonstrate that infused MAb17-lA can label single tumor cells in bone marrow in vivo.Furthermore, CK 2 positive cells disappeared four to twelveweeks after the first infusion with MAb 17-lA. To analyzethe biological and prognostic relevance of these cells, longterm observation of the patients is required.