By Roland E. Kontermann, Stefan Dübel
Curiosity in recombinant antibody applied sciences has speedily elevated as a result of wide selection of attainable purposes in remedy and prognosis, particularly in melanoma remedy. the potential for producing human antibodies that aren't obtainable through traditional polyclonal or monoclonal ways has compelled the improvement of antibody engineering applied sciences even more.
This handbook provides a complete selection of special, step by step protocols supplied by means of specialists within the box. All simple equipment wanted in antibody engineering - not just ways to generate recombinant antibodies, but additionally protocols for research and their use - and lately built and rising applied sciences are lined. specifically, protocols at the following subject matters are provided:
Hybridoma immortalisation iteration and screening of antibody gene libraries from human donors, mice and rabbits Antibody choice on immunotubes, cells, tissues; proximity and step-back choices construction of human monoclonal antibodies to poisonous or hugely pathogenic brokers with no immunisation Improvment of antibody binding Antibody humanisation Genetic fusions for the construction of multifunctional antibody derivatives Radiolabelled recombinant antibodies Bispecific antibodies Antibody - enzyme fusions Intracellular antibodies selection of affinity and specificity machine research of antibody series and constitution Epitope research by means of a number of phage exhibit structures and peptide spot membranes Eukaryotic (plant, baculovirus, yeast, mammalian cells) and prokaryotic creation platforms for recombinant antibodies Purification platforms Xenograft mice rising applied sciences
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Additional resources for Antibody Engineering
11. Double digest the purified PCR product with the appropriate restric- tion endonucleases. Note: Calculate the amount of required enzyme carefully. Overdigestion may reduce the ligation efficiency. 12. Gel purify the digested PCR fragment. We recommend spin column kit systems. Alternatively, agarase digestion of low meting point agarose can be employed. 47 48 FRANK BREITLING K et al. , PvuII) Bi3 5¥-GAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTG Bi3b 5¥-AGGT (C/G) (A/C) AACTGCAG (C/G) AGTC (A/T) GG Bi3c 5¥-AGGT (C/G) (A/C) AGCTGCAG (C/G) AGTC (A/T) GG Bi3d 5¥-AGGT (C/G) CAGCTGCAG (C/G)AGTC (A/T)GG y chain CHI domain: (HindI I I) Bi4 5¥-CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT Reamplification primers for the introduction of other restriction sites heavy chain FRI region: Bi3f K chain constant domain: Bi5c K chain FRI region: 5¥- CAGCCGGCCATGGCGCAGGT (C/G) CAGCTGCAG (C/G) AG NcoI PvuII,PstI 5¥- GAAGATGGATCCAGCGGCCGCAGCATCAGC BamBI Not I BiBb 5¥- AATTTTCAGAAGCACGCGTAGATATC(G/T)TG(A/C)T(G/C)ACCCAA(T/A)CTCCA MluI EcoRV 3 Construction of scFv from Hybridoma by Two-Step Cloning Cloning and colony screening 1.
V) 2ii 00( 2 ~ " -;:; "0 c: KDIR HwII!! - • • • • • tet-cassette Sfi 1 c: ;:; • • Sfil £roRl HIndI!! Y ~ • • • • IIw11!! Ikallne phosphatase - Sftl £CoRI /llndI!! I It' b' l Sftl £CaRl 'tl Q. U Q. HwII!! Vol £CoRI ,tI :l u -=::> c • • HIndI!! g u '" • ~ 6 hIS ~ ", .. 0<1 6his - ! Sfi! '" c: "c: . HIndI!! Sfil SF! , p'IB '-;;;yc;r gill ,tI SF! _ I HwII!! Sfil £CoR! 2 ~ Sfi I Sfi I tetA tetR Sfi I 2 Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly Fig. 4. 5) can be used either for phage display (pAK 100 and pJB12) by the strategy outlined in Fig.
163:45-57 Dall'Acqua W, Carter P (1998) Antibody engineering. Curr Opin Struct Bioi 8:443-450 Dziegiel M, Nielson LK, Anderson PS, Blancher A, Dickmeiss E, Engberg J (1995) Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D. J Immmunol Meth 182:7-19 Freund C, Ross A, Pliickthun A, Holak TA (1994) Structural and dynamic properties of the Fv fragment and the single-chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy.
Antibody Engineering by Roland E. Kontermann, Stefan Dübel