By Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)
Antibodies are essential instruments for study, analysis, and remedy. Recombinant methods enable the amendment and development of approximately all antibody houses, reminiscent of affinity, valency, specificity, balance, serum half-life, effector features, and immunogenicity.
Antibody Engineering presents a accomplished toolbox overlaying the well-established fundamentals but additionally many interesting new suggestions. The protocols mirror the newest "hands on" wisdom of key laboratories during this nonetheless fast-moving box. beginners will enjoy the confirmed step by step protocols, which come with invaluable sensible recommendation; skilled antibody engineers will relish the recent principles and ways. The booklet is a useful source for all these engaged in antibody study and improvement.
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V. Schaefer et al. decreased phage titer. The size of the white pellet does not necessarily reflect a high or low phage titer. 4. Resuspend the phage pellet in 400 ml PBS (with 10% (v/v) glycerol). For complete resuspension, incubate the phage solution on an orbital shaker at 800 rpm for 15 min at 4 C. Pellet insoluble matter (cell debris) by centrifugation for 10 min at 11,000 g and 4 C and transfer the phage solution to a fresh tube. Use 100 ml phage solution per well in an ELISA assay to distinguish phages displaying functional scFv antibody from those which display nonfunctional or nonproductive antibody fragments.
L L L A A Q P A M A D Y K D . . pAK400scFv, pJB33scFv ® SD2 pelB signal sequence . . GA AGGAGA T A T A C A T A T GA A A T A CC T A T T GCC T A CGGC AGCC . . M T7g10 K Y L L P T A A . . b pAK100scFv ¬ VH EcoRI Sfi I myc tag . . CGGC C T CGGGGGC CG A A T T C G A GC A G A A GC T G A T C T C T G A GG A A G A C . . A S G ® gene III 250-406 A E F E Q K L I S E E D C T G T AGGG T GG T GGC T C T GG T T CCGG T G A T T T T GA T T A T GA A A AG . . L * G G G S G S G D F D Y E K . . pJB12scFv ¬ VH EcoRI Sfi I trypsin cleavage site .
2 l l l l l l l l 25 Materials 5 Â 106 cells from a growing or frozen hybridoma culture or spleen cells, respectively PCR primers (Figs. 3) and corresponding plasmids (Figs. , E. coli XL1-Blue) (available in electrocompetent/ chemocompetent form from Stratagene) Anti-M13 antibody HRP-conjugate (GE Healthcare; # 27-9421-01) PEG 6000 (Fluka) Sterile, RNase-free equipment: pipet tips, tubes, RNase-free ultra high purity (UHP) water, baked nondisposable glassware, and sterile, disposable plasticware Standard molecular biology equipment and reagents for: – Determining the isotype of mAbs (Roche IsoStrip Mouse Monoclonal Antibody Isotyping Kit) – Purifying RNA (Invitrogen TRIzol reagent and Qiagen RNeasy Mini Kit) – Performing a cDNA synthesis reaction (Qiagen QuantiTect Reverse Transcription Kit) – Performing PCR reactions – Purifying PCR products (Macherey Nagel PCR clean-up Gel Extraction Kit) – Cutting and gel-purifying DNA (Sigma-Aldrich GenElute Gel Extraction Kit) – Concentrating DNA (Amicon Microcon 30 for volumes less than 500 ml) – Ligating and transforming DNA – Growing bacteria and phages – Conducting an Enzyme Linked Immunosorbent Assay (ELISA) – Performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting.
Antibody Engineering by Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)