Epitope mapping of SCLC-cluster-2 MAbs and generation of antibodies directed against new EGP-2 epitopes.
Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it difficult to generate and select antibodies against other potentially useful EGP-2 epitopes. Using PCR, we have generated mutant EGP-2 cDNA (delta EGP-2) from which the coding sequences for a putative immunodominant 6-kDa intra-chain loop structure has been removed. delta EGP-2 transfected COS-7 cells reacted with MM104, an antibody detecting a linear epitope on EGP-2, but were not recognized by any cluster-2 MAb. To generate new anti-EGP-2 antibodies we constructed another mutant EGP-2 protein (delta EGP-2) from which additional domains, irrelevant for antibody generation (signal peptide, trans-membrane and cytoplasmic domains), were removed. delta EGP-2 was introduced in a prokaryotic expression system that adds a polyhistidine affinity tag to the delta EGP-2 N-terminus, making possible one-step purification by immobilized metal-ion-affinity chromatography (IMAC). Western blot analysis showed that sera derived from mice immunized with purified delta EGP-2 had high-titer antibodies to reduced EGP-2 samples. We conclude that the availability of prokaryotic and eukaryotic EGP-2-expression constructs might facilitate the selection of new anti-EGP-2 MAbs otherwise difficult to obtain.
Helfrich
W
W
Department of Clinical Immunology, University Hospital Groningen, The Netherlands.
Koning
P W
PW
The
T H
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De Leij
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000032:analysis*; D015153:Blotting, Western; D018288:Carcinoma, Small Cell / Q000737:chemistry / Q000473:pathology*; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000071858:Epithelial Cell Adhesion Molecule; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000473:pathology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D018076:DNA, Complementary; D000071858:Epithelial Cell Adhesion Molecule; D000939:Epitopes