Filtyp
Proteomic experiments can be difficult to handle because of the large amount of data in different formats that is generated. Samples need to be managed and generated, data needs to be integrated with samples and annotation information. A laboratory information management system (LIMS) can be used to overcome some of the data handling problems. In this chapter, we discuss the role of a LIMS in the
We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antib
Glycoprotein B (gB, gpUL55) is the major antigen for the induction of neutralizing antibodies against human cytomegalovirus (HCMV), making it an attractive molecule for active and passive immunoprophylaxis. The region between aa 552 and 635 of HCMV gB (termed AD-1) has been identified as the immunodominant target for the humoral immune response following natural infection. AD-1 represents a comple
A novel technology in the area of antibody engineering has been developed which allows for the creation of new types of antibody molecules. It is called complementarity-determining region (CDR) implantation and permits the random combination of CDR sequences formed in vivo into a single master framework. Thus, totally new gene combinations can be produced and used in selection processes. The resul
Experiments were carried out to analyze the function of cysteine residues at amino acid positions 506 (cI), 550 (cII), 573 (cIII), and 610 (cIV), in dimerization and/or disulfide linkage of human cytomegalovirus (HCMV) glycoprotein B (gB). Single c-codons or pairs were substituted in the gB sequence of constructs which were used for transfection and selection of stable transfectants. Analysis of g
Working with phage display, there are a number of ways for characterizing the phage and the molecules that they display. BIAcore(TM) biosensor and Origen Analyzer provide novel detection systems permitting sensitive and flexible analyses. Such analytical approaches are of critical importance to allow early selection of appropriate clones for further evaluation. In this review we describe the appli
IgM antibodies are often of low affinity (dissociation constant (K(d)) > 10-5 M) and therefore they are usually neglected as tools in, e.g., immunoassays. Previous studies have shown that low affinity biological interactions can be studied and exploited in affinity chromatography, biosensor technology and capillary electrophoresis. In this study we have demonstrated that IgM can be a useful ligand
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
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
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
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
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
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
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
