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Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

Background: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins: peptides and large organic compounds. Objectives: To determine t

Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germ

Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity

Antibody diversity, a molecular feature which allows these proteins to specifically interact with a diverse set of targets, is created at the genetic level by a variety of means. These include germline gene segment recombination, junctional diversity and single basepair (bp) substitution. We here demonstrate that a human high affinity antibody specific for an exogenous protein antigen carry three

Mouse monoclonal antibodies against outer membrane proteins of a vaccine strain of Neisseria meningitidis B : 4:P1.15

Background. Neisseria meningitidis (Nm) is a Gram negative diplococcus causing bacterial meningitis and fulminant septicemia. In order to allow efficient characterization of infecting strains, antibody reagents for use as analytical tools have proven to be invaluable tools. Similarly, antibodies against relevant bacterial antigens may guide in the selection of components to be included in developi

Altered gene expression associated with apoptosis in a pre-B-leukemic cell line following cross-linking of MHC class I

The major histocompatibility complex class I (MHC-I) has recently been shown not only to present antigens to the immune system but also to mediate transmembrane signaling, resulting in activation, inactivation, or apoptosis. Such signaling has been observed in both normal and malignant cells of the B and T cell lineage. Cross-linking of MHC-I on the pre-B- acute-lymphocytic cell line KM-3 induces

Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques

An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying

Evaluation of novel control elements by construction of eukaryotic expression vectors

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

Low affinity, antibody binding of an Escherichia coli-derived component

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

Characteristics of human antibody repertoires following active immune responses in vivo

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

Immunoglobulin production induced by CD57+ GC-derived helper T cells in vitro requires addition of exogenous IL-2

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

Selection of binders from phage displayed antibody libraries using the BIAcore(TM) biosensor

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 immunization of naive human B cells yields high affinity immunuglobulin G antibodies as illustrated by phage display

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

Selection of phage displayed antibodies based on kinetic constants

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

Structural domains involved in human cytomegalovirus glycoprotein B-mediated cell-cell fusion

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

CD4+CD57+ T cells derived from peripheral blood do not support immunoglobulin production by B cells

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