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Antigen Binding (antigen + binding)
Selected AbstractsAn investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticlesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2005James Chesko Abstract This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2510-2519, 2005 [source] Polymorphism and signature of selection in the MHC class I genes of the three-spined stickleback Gasterosteus aculeatusJOURNAL OF FISH BIOLOGY, Issue 2006H. Schaschl The role and intensity of positive selection maintaining the polymorphism of major histocompatibility complex (MHC) class I genes in the three-spined stickleback Gasterosteus aculeatus was investigated. The highly polymorphic set of MHC class I genes found was organized in a single linkage group. Between 5 and 14 sequence variants per individual were identified by single-stranded conformation polymorphism (SSCP) analysis. Segregation analysis studied in 10 three-spined stickleback families followed the expected pattern of Mendelian inheritance. The gamete fusion in three-spined stickleback thus seems to be random with respect to the MHC class I genes. The DNA sequence analyses showed that the expressed MHC class I loci are under strong selection pressure, possibly mediated by parasites. Codons that were revealed to be under positive selection are potentially important in antigen binding. MHC class I sequences did not form significant supported clusters within a phylogenetic tree. Analogous to MHC class II genes, it was not possible to assign the class I sequences to a specific locus, suggesting that the class I genes may have been generated by recent gene duplication. [source] Improving the affinity of antigens for mutated antibodies by use of statistical molecular designJOURNAL OF PEPTIDE SCIENCE, Issue 7 2008Ilona Mandrika Abstract We demonstrate the use of statistical molecular design (SMD) in the selection of peptide libraries aimed to systematically investigate antigen-antibody binding spaces. Earlier, we derived two novel antibodies by mutating the complementarity-determining region of the anti-p24 (HIV-1) single chain Fv antibody, CB4-1 that had lost their affinity for a p24 epitope-homologous peptide by 8- and 60-fold. The present study was devoted to explore how peptide libraries can be designed under experimental design criteria for effective screening of peptide antigens. Several small peptide,antigen libraries were selected using SMD principles and their activities were evaluated by their binding to SPOT-synthesized peptide membranes and by fluorescence polarization (FP). The approach was able to reveal the most critical residues required for antigen binding, and finally to increase the binding activity by proper modifications of amino acids in the peptide antigen. A model of the active peptide binding pocket formed by the mutated scFv and the antigen was compatible with the information gained from the experimental data. Our results suggest that SMD approaches can be used to explore peptide antigen features essential for their interactions with antibodies. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3PROTEIN SCIENCE, Issue 9 2006Mingyue He Abstract The crystal structures of the Fab, fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 104 -fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position. [source] Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologiesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2007Meg Duroux Abstract The present work shows how UV ,light-induced molecular immobilisation' (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using ,LIMI,' arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1,,m in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies. [source] Kinetics of antigen binding to antibody microspots: Strong limitation by mass transport to the surfacePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Wlad Kusnezow Dr. Abstract It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology. [source] Formatted anti,tumor necrosis factor , VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 6 2006Ken Coppieters Objective The advent of tumor necrosis factor (TNF),blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin VH domains, as TNF antagonists in a mouse model of RA. Methods Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti,serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. Results The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. Conclusion These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively. [source] | ||||||||||