Fc Region (fc + region)

Distribution by Scientific Domains

Selected Abstracts

CE-based noncompetitive immunoassay for immunoglobulin G in bovine colostrum products

Jin Zhao
Abstract A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2,min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n,=,7). A linear relationship was established for the IgG concentration in the range of 1,5,mg/L with a linear correlation coefficient (r,=,0.9917). The LOD was 0.1,mg/L (S/N,=,3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved. [source]

Multifunctional Dendrimer-Templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection

Hye Jung Han
Abstract Dendrimers, with their well-defined globular shape and high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. The synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol-functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich-type enzyme-linked immunosorbent assay (ELISA) to detect IL-6 and IL-1,, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13,pg,mL,1 for IL-6 luminol detection and 1.15,pg,mL,1 for IL-1, TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from a normal (nonpregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors, and field-effect biosensors. [source]

Enzyme Replacement Therapy for Murine Hypophosphatasia,

José Luis Millán PhD
Abstract Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5,-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6 -dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2,/,), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, ,CT, and histomorphometry. Results:Akp2,/, mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2,/, mice. [source]

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27,MHz quartz-crystal microbalance

Hideyuki Mitomo
Abstract Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x,=,1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27,MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (,mmax), association constants (Ka), association and dissociation rate constants (k1 and k,1, respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region

Katalin Uray
Abstract The IgG binding Fc, receptors (Fc,Rs) play a key role in defence against pathogens by linking humoral and cell-mediated immune responses. Impaired expression and/or function of Fc,R may result in the development of pathological autoimmunity. Considering the functions of Fc,Rs, they are potential target molecules for drug design to aim at developing novel anti-inflammatory and immunomodulatory therapies. Previous data mostly obtained by X-ray analysis of ligand,receptor complexes indicate the profound role of the CH2 domain in binding to various Fc,Rs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity Fc,Rs, like Fc,RIIb and Fc,RIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231,298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble Fc,RIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble Fc,RIIb, as well as to Fc,Rs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg255,Ser267 sequence of IgG1 is implicated in the binding to Fc,RIIb. In addition we found that peptides corresponding to the Arg255,Ser267, Lys288,Ser298 or Pro230,Val240 when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNF,, a pro-inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating Fc,Rs, and mediate Fc,R dependent function. Peptide Arg255,Ser267 can also be considered as a lead for further functional studies. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Directed attachment of antibodies to kinesin-powered molecular shuttles

Amanda Carroll-Portillo
Abstract Biomolecular motors, such as kinesin, have been used to shuttle a range of biological and synthetic cargo in microfluidic architectures. A critical gap in this technology is the ability to controllably link macromolecular cargo on microtubule (MT) shuttles without forming extraneous byproducts that may potentially limit their application. Here we present a generalized approach for functionalizing MTs with antibodies in which covalent bonds are formed between the carbohydrate in Fc region of polyclonal antibodies and the positively charged amino acids on the MT surface using the crosslinker succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH). Antibody-functionalized MTs (Ab-MTs) produced through this approach maintained motility characteristics and antigenic selectivity, and did not produce undesirable byproducts common to other approaches. We also demonstrate and characterize the application of these Ab-MTs for capturing and transporting bacterial and viral antigens. While this approach cannot be applied to monoclonal antibodies, which lack a carbohydrate moiety, it may be used for selectively functionalizing MT shuttles with a variety of carbohydrate-containing cargoes. Biotechnol. Bioeng. 2009; 104: 1182,1188. © 2009 Wiley Periodicals, Inc. [source]

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of Fc,RI with Fc,RIIb

S. J. Wigginton
Summary Background IgE binds to mast cells and basophils via its high-affinity receptor, Fc,RI, and cross-linking of Fc,RI-bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross-linking of Fc,RI with Fc,RIIb, an ITIM-containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti-idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to Fc,RI-bound IgE, via its Fab regions, and the negative regulatory receptor, Fc,RIIb, via its Fc region. Objective To assess the ability of human 2G10 to inhibit anti-IgE and allergen-driven basophil degranulation through cross-linking of Fc,RI-bound IgE with Fc,RIIb. Methods 2G10 was assessed for its ability to bind to Fc,RIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti-IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL-4 expression intracellularly, using flow cytometery. Results Human 2G10 was able to bind to Fc,RIIb on transfected cells and on purified basophils, and induce a dose-dependent inhibition of both anti-IgE and Der p 1-driven degranulation of basophils. Conclusion The inhibition of basophil degranulation by the human IgG1 anti-idiotype 2G10 highlights the therapeutic potential of IgE-reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor Fc,RIIb. [source]