Read e-book online Antibody Engineering PDF

By Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)

ISBN-10: 3642011438

ISBN-13: 9783642011436

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.

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Antibody Engineering by Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)


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