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Selection of Recombinant Antibodies 2536. If the bacteria have reached OD 600approx 0.5 nm before they are needed, store theculture at 4°C to maintain the F pili on the E. coli cells. If used for titration, aM13K07 positive control is advised.7. It is advisable to conduct control titerings. To control the PBS, PEG solutionsuse 10 µL of this solution to “infect” bacteria with this solution and also plateout noninfected XL1-blue MRF′ to control the bacteria. It is recommended toclean the working place each time with virus-inactivating solutions (e.g.,Barrycidal 36, BIO-HIT, Germany) and to use filter tips for pipeting.8. Antibody phage binding unspecifically are usually enriched during panning.These unspecific binding usually results from misfolded or incomplete antibodies.They often bind to BSA, streptavidin, and plastic surfaces.9. Use the polyclonal antibody phage ELISA to select the suitable panning round forpicking.10. It is recommended to pick only 92 clones. Use the wells H3, H6, H9, and H12 forcontrols. H3 and H6 are negative controls—these wells will not be inoculated andnot used for the following ELISA with soluble antibodies. The wells H9 and H12are inoculated with a clone containing a phagemid encoding a known antibodyfragment. Therefore, the wells H9 and H12 are coated with the corresponding antigen.11. The appropriate IPTG concentration for induction depends on the vector design. Aconcentration of 50 µM was well suited for vectors with a Lac promoter like pSEX81(18), pIT2 (35), and pHENIX (36) and pHAL14 (18). The method for the productionof soluble antibodies works with vectors with (e.g., pHAL 14) and without (e.g.,pSEX81) an amber stop codon between antibody fragment and gIII. If the vector hasno amber stop codon the antibody::pIII fusion protein will be produced (37).AcknowledgmentsWe gratefully acknowledge the financial support by the German ministry ofeducation and research (Bundesministerium Für Bildung und Forschung[BMBF], Standard Method Protocol [SMP] “Antibody Factory” in the NGFN2program) and of the German Research Foundation (Deutsche Forschungsgeneins-chaf [DFG], SFB 578).References1. Von Behring, E. and Kitasato, S. (1890) Über das Zustandekommen derDiphtherie-Immunität und der Tetanus-Immunität bei Thieren. Deutsche Med.Wochenzeitschr. 16, 1113, 1114.2. Köhler, G. and Milstein, C. (1975) Continous cultures of fused cells secretingantibody of predefined specificity. Nature 256, 495–497.3. Winter, G. and Milstein, C. (1991) Man-made antibodies. Nature 349, 293–299.4. Courtenay-Luck, N. S., Epenetos, A. A., Moore, R., et al. (1986) Development ofprimary and secondary immune responses to mouse monoclonal antibodies used inthe diagnosis and therapy of malignant neoplasms. Cancer Res. 46, 6489–6493.5. Studnicka, G. M., Soares, S., Better, M., Williams, R. E., Nadell, R., and Horwitz,A. H. (1994) Human-engineered monoclonal antibodies retain full specific binding