Synthesis of a group-selective antibody library against haptens.
A generic strategy is described for the generation of libraries comprising hapten-selective antibody genes against a group of structurally related low molecular weight target molecules. Hapten antibody libraries are frequently suffering from high background levels of irrelevant antibody genes as a consequence of the immunization, where the small non-immunogenic target molecule is coupled to a large immunogenic carrier protein. In order to elevate the percentage of hapten-specific genes in the library, B cells harboring antibody genes against the group of triazine herbicides were enriched from 21 individual splenocyte populations by means of immunomagnetic separation (IMS). IMS utilizes the specific binding of membrane-associated immunoglobulin receptors on the B cell surface to hapten-coated paramagnetic beads. The variable genes of the specifically enriched subpopulation were cloned into a phagemid vector. The corresponding library yielded up to 75% triazine binding antibody clones after three rounds of phage selection. At least half of these antibodies (abs) were displaceable by triazines resulting in quantitative assays with nanomolar sensitivities. In contrast, no displaceable clone was obtained at the same selection level in a control library, where IMS was omitted. Due to the elevated percentage of relevant antibody genes, the library can be utilized either for the direct isolation of functional antibodies against various triazine herbicides or as group-specific gene source for evolutionary antibody optimization.
Chair of Cell Biology, Center of Life Sciences Weihenstephan, Technische Universität München, Alte Akademie 12, Freising, Germany. kramer@wzw.tum.de
Karl
K
Kramer
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002138:Calibration; D018076:DNA, Complementary / Q000138:chemical synthesis; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006241:Haptens / Q000737:chemistry / Q000276:immunology*; D006540:Herbicides / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D018189:Immunomagnetic Separation / Q000379:methods; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014227:Triazines / Q000737:chemistry / Q000276:immunology*
D000906:Antibodies; D018076:DNA, Complementary; D006241:Haptens; D006540:Herbicides; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014227:Triazines