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Rabbit IgG (rabbit + igg)
Selected AbstractsMacroporous Silicon Microcavities for Macromolecule Detection,ADVANCED FUNCTIONAL MATERIALS, Issue 11 2005H. Ouyang Abstract Macroporous silicon microcavities for detection of large biological molecules have been fabricated from highly doped n-type silicon. Well-defined controllable pore sizes up to 120,nm have been obtained by systematically optimizing the etching parameters. The dependence of the sensor sensitivity on pore size is discussed. Excellent infiltration inside these macroporous silicon microcavities is demonstrated using 60,nm diameter latex spheres and rabbit IgG (150,kDa; 1Da,=,1,g,mol,1). The sensing performance of the device is tested using a biotin/streptavidin couple, and protein concentration down to 1,2,,M (equivalent to 0.3,ng,mm,2) could be detected. Simulations show that the sensitivity of the technique is currently approximately 1,2,% of a protein monolayer. [source] Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopyJOURNAL OF MOLECULAR RECOGNITION, Issue 6 2004Paul J. Cachia Abstract A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti- Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coil strand of the heterodimeric coiled-coil domain and individually captured on the biosensor chip through dimerization with the immobilized K-coil strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-antibody interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14,×,10,9 to 1.60,×,10,5,M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development. Copyright © 2004 John Wiley & Sons, Ltd. [source] Thiopropyl-agarose as a solid phase reducing agent for chemical modification of IgG and F(ab,)2BIOTECHNOLOGY PROGRESS, Issue 5 2008Natalia Ferraz Abstract Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid-phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab,)2 fragments, with mercaptohydroxypropylether-agarose (thiopropyl-agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS-PAGE analysis. Optimized reduction with thiopropyl-agarose introduced six thiol groups in the F(ab,)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab,)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl-agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 ,moles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl-agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates. [source] FK506-binding protein localizations in human penile innervationBJU INTERNATIONAL, Issue 5 2008Gwen Lagoda OBJECTIVE To determine if FK506-binding proteins (FKBPs) are localized to the autonomic nerve supply of the human penis because FK506 (an immunosuppressant drug) has been linked to enhanced nerve regeneration after nerve injury and neurodegenerative diseases by binding to FKBPs, a select group of immunophilins. MATERIALS AND METHODS Human lower genitourinary tract specimens were obtained and embedded in paraffin wax. The tissue was sectioned (10 µm) and processed for immunohistochemistry using antibodies for FKBPs 12, 38, 52, 65, 135 and neuronal nitric oxide synthase (nNOS). To confirm specificity of the antibody, we processed some tissue in the absence of primary antibody, with mouse or rabbit IgG, and with a blocking peptide for FKBP12. RESULTS In the pelvis, immunoreactivity for all the FKBPs and nNOS was localized to the periprostatic ganglia although FKBP12 was the only FKBP localized to nerve bundles in this location. In penile tissue, immunoreactivity for all five FKBPs and nNOS was localized to nerves, although immunoreactivity for FKBP38 was minimal. The FKBPs were also evident in epithelial, endothelial and smooth muscle cells of the prostate and penis. Negative controls did not produce staining. CONCLUSIONS Identification and localization of immunophilins to nerves coursing in prostate and penile tissue suggest a likely molecular basis to apply immunophilin ligand therapy to protect or regenerate cavernosal nerves. Our findings support the hypothesis that immunosuppressant drugs such as FK506, working via specific receptor mechanisms, are potentially useful to sustain erectile function in men after radical prostatectomy. [source] Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N -glycans as cross-reactive allergens in honeybee and yellow jacket venomCLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2004W. Hemmer Summary Background IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy. Objective To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot. Methods Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N -glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N -glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated. Results Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3. Conclusions The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization. [source] | ||||||||||