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A Glacet

Publications and source records attributed to A Glacet.

8 recordsLinked to original sources
PubMed2001

The rescue by phage display of human Fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes.

Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5' primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.

Amino Acid Sequence↗
PubMed2000

Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties.

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.

Amino Acid Sequence↗
PubMed1999

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments.

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.

Acetyl-CoA Carboxylase↗
PubMed1997

Human recombinant anti-Rh(D) monoclonal antibodies: improvement of biological properties by in vitro class-switch.

Therapeutic use of human monoclonal antibodies has so far been hampered by their poor availability. Recent developments in recombinant DNA technologies are expected to fill this gap; the variable and constant sequences of antibodies can be selected independently and then subsequently joined to express whole antibodies. We assessed here the potential of this methodology to obtain novel anti-D antibodies with improved biological characteristics. The sequences coding for heavy and light chains of two anti-Rh(D) monoclonal antibodies (one IgG1 and one IgG3) were isolated and co-expressed into murine myeloma cell P3X63.Ag8.653, either directly (parental antibodies) or after exchange of constant heavy chain sequences (class-switched antibodies). Parental antibodies produced either by transfectomas or by hybridomas behaved similarly in analysis of biochemical, binding and effector properties. Class-switched antibodies displayed altered functional properties over parental antibodies. Of particular interest, one of them showed improved phagocytosis potencies over both parental antibodies. Additionally, these results indicate that functional properties do not always simply reflect the addition of properties of constant and variable parts but that interactions between constant and variable regions may interfere.

Amino Acid Sequence↗
PubMed1996

Use of the DAF assay to assess the functional properties of polyclonal and monoclonal RhD antibodies.

The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

[Dye lasers: experimental histopathologic study of the effects of various wavelengths on the macular region].

Pathological experimental study of dye laser photocoagulation simulating the confluent treatment of subretinal new vessels in the macular area of a Macacas Cynomolgus monkey. 38 days after treatment the lesions become similar on the choriocapillaris and retinal pigment epithelium whatever the wavelength; the choriocapillaris is always occluded and the pigment epithelium destroyed. The internal limiting membrane is ondulated only with yellow and orange colors. These results are compared to those of similar experimental studies.

Animals↗