Document 0836
 DOCN  M9590836
 TI    Two-phase approach for the expression of high-affinity human anti-human
       immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.
 DT    9509
 AU    Takeda S; Dorfman NA; Robert-Guroff M; Notkins AL; Rando RF; Laboratory
       of Oral Medicine, National Institute of Dental; Research, National
       Institutes of Health, Bethesda, Maryland; 20892, USA.
 SO    Hybridoma. 1995 Feb;14(1):9-18. Unique Identifier : AIDSLINE
       GENBANK/L01413
 AB    We describe here a two-phase approach for the development of
       high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial
       expression system. The first phase of this technique involves the
       generation of human hybridoma cell lines producing high-affinity
       antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood
       lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and
       from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One
       MAb (T15G1), derived from the blood of the seropositive donor, was
       specific for HIV-1 gp41, recognized gp41 on the surface of
       HIV-1-infected cells and bound this antigen with an apparent
       dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed
       from the immunized volunteer, was specific for HIV-1 gp120 with a
       dissociation constant on the order of 8 x 10(-10) M, but was unable to
       recognize cell surface antigen. In the second phase of this technique
       the Fab domains of these two MAbs were molecularly cloned into a
       bacterial expression vector. mRNA was isolated from the M7B5 and T15G1
       hybridoma cell lines and used as a template for the production of cDNA.
       The cDNA was amplified using the polymerase chain reaction (PCR)
       technique, and then fused, in frame, into a bacterial expression vector.
       The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as
       dicistronic messages in bacteria using the IPTG-inducible lactose
       promoter (LacZ). DNA sequencing was used to define the gamma chain
       isotypes and the VH and VL chain gene usage. The binding specificities
       of rFabM7B5 and rFabT15G1 were indistinguishable from their respective
       intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)
 DE    Amino Acid Sequence  Antibodies, Monoclonal  Antibody Affinity  Base
       Sequence  Cell Transformation, Viral  Cloning, Molecular  DNA
       Primers/GENETICS  Escherichia coli/*GENETICS  Genetic Engineering
       Genetic Vectors  Herpesvirus 4, Human  Human  Hybridomas/IMMUNOLOGY  HIV
       Antibodies/*GENETICS  HIV-1/*IMMUNOLOGY  Immunoglobulins, Fab/*GENETICS
       Immunoglobulins, Heavy-Chain/GENETICS  Immunoglobulins,
       Light-Chain/GENETICS  Molecular Sequence Data  JOURNAL ARTICLE

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       protected by Copyright Law (Title 17, U.S.Code).