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A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3' enhancer (3'E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a
This investigation describes the detection of a component in Escherichia coli capable of binding a large proportion of human antibody variable domains including otherwise highly monospecific antibodies induced by an in vivo antibody response. This interaction is of low affinity, but cross-linking of IgG molecules by, e.g. anti-immunoglobulin preparations, provides a sufficient degree of multivalen
Possibilities to develop human monoclonal antibody specificities have recently been much facilitated by improvements of human hybridoma technology but even more so by the emerging phage-display technique. However, until recently very little has been known about the characteristics at the molecular level of the induced, T cell-dependent human antibody response, frequently targeted by these techniqu
Germinal centers (GC) are well-defined areas in lymphoid organs were B cells proliferate and differentiate in response to T-cell-dependent antigens. The GC comprises B cells, follicular dendritic cells, tangible body macrophages, and a low number of CD4+ T cells. A large portion of these T cells expresses CD57. We have examined the ability of the CD4+CD57+ GC T cells to become activated and to tak
In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore(TM) biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were elu
In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro im
The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment
A novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing authentic or mutagenized human cytomegalovirus glycoprotein B (HCMV gB; gpUL55). Compared to transfectants expressing authentic HCMV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hd1; a
A small subpopulation of CD4+ T cells found in peripheral blood coexpresses the CD57+ marker normally found on, e.g., NK cells. It is known that this population occurs in a higher frequency in certain diseases. The same antigen has also been shown to be expressed on CD4+ T cells derived from germinal centers. The localization of this cell population to specialized lymphoid structures suggests that
The lower matrix protein (pp65) is a major product of many laboratory strains of cytomegalovirus (CMV). It is thus an integral part of many CMV serological assays based on native antigen. Recombinant fragments of pp65 have previously been investigated for their usefulness in more-defined assays. The latter antigens have, however, failed to develop a positive response with serum samples derived fro
A new simple, reproducible and sensitive ELISA that uses trichloroacetic acid (TCA) for the coating of lipooligosaccharide (LOS), smooth lipopolysaccharide (LPS) or lipid A to the solid phase has been developed. The experimental parameters (temperature of coating, time of coating, antigen concentration and TCA concentration) were evaluated by a complete factorial design (24). As a result of the ev
The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120
Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable reg
We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and
Glycoprotein B (gB, gpUL55) is a major antigen for the induction of neutralizing antibodies against human cytomegalovirus, making it an attractive antigen for active and passive immunoprophylaxis. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure which requires a minimal linear amino acid sequence of more than 75 amino acids (aa 552-635) for antibody binding. We
Flow cytometry has been utilized to evaluate the stability of antibody production by unstable subclones of a human × human × mouse heterohybridoma. Heterogeneity of cell-associated immunoglobulin heavy chain expression was demonstrated in different subclones and an increased frequency of cells containing low levels of heavy chain was found to correlate with low antibody productivity. However, the
The human antibody response to the conserved neutralization-related site on the gp 116 of human cytomegalovirus (HCMV) was investigated in healthy blood donors by the use of synthetic peptides. Anti-HCMV positive sera investigated in ELISA gave a reactivity of 48-56% with the peptide T7-13 (amino acids (aa) 67-86). Though epitope mapping revealed several individual fine specificities within this r
