Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.
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PMID:By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. 140 59

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.
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PMID:By-passing immunization. Human antibodies from V-gene libraries displayed on phage. 174 94