Label Of Secondary Antibody Research Articles

Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors

Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1pg/mL to 100ng/mL with a detection limit of 0.052pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01pg/mL to 100ng/mL and a lower detection limit of 0.0043pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields.

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au–SH or Au–NH3+ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65UmL−1 with a detection limit of 0.01UmL−1. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.

Ultrasensitive nonenzymatic immunosensor based on bimetallic gold–silver nanoclusters synthesized by simple mortar grinding route

A simple nonenzymatic immunosensor was developed for sensitive and selective determination of squamous cell carcinoma antigen (SCC-Ag) based on bimetallic gold–silver nanoclusters (AuAg NCs) and reduced graphene oxide-tetraethylene pentamine (rGO-TEPA). rGO-TEPA was used as substrate material to immobilize antibodies since rGO-TEPA has large surface area and excellent conductivity to enhance the electrochemical response signal. AuAg NCs synthesized through simple mortar grinding route were used as catalytic labels of secondary antibodies; meanwhile, AuAg NCs could efficiently enhance the electron transfer. Under the optimal conditions, the electrochemical immunoassay exhibited a wide linear range of 0.005–20ng/mL with a low detection limit of 1.3pg/mL for SCC-Ag. The proposed sensing strategy enriches the electrochemical immunoassays and has a promising application in bioassay analysis.

Ultrasensitive non-enzymatic and non-mediator electrochemical biosensor using nitrogen-doped graphene sheets for signal amplification and nanoporous alloy as carrier

A rapid, environmental friendly, and sensitive biosensor for detection of zearalenone (ZEN) was developed at glassy carbon electrode (GCE) using nanoporous bimetallic alloy as label of secondary antibody (Ab2). The biosensor was prepared by immobilizing nitrogen-doped graphene sheets (N-GS) captured primary antibody (Ab1) on GCE. The analytes were then bound to Ab1 for further capture of antibodies, labeled by the nanoporous bimetallic alloy, nanoporous PtCo alloy (NP-PtCo). NP-PtCo with three-dimensional bicontinuous spongy structures and various hollow channels was prepared by corrosion of PtCoAl alloy, and exhibited remarkably improved electrocatalytic activity toward H2O2. Within ZEN concentration range (0.005–25ngmL−1), the biosensor exhibited a highly sensitive response to ZEN with a detection limit of 2.1pgmL−1. Several factors that would affect the performance of the biosensor were studied, such as erosion time of NP-PtCo, concentration of NP-PtCo, pH of buffer solution, and concentration of N-GS. After H2O2 was injected into the buffer solution, the amperometric response of the biosensor can be used to detect ZEN by chronoamperometry. The developed biosensor shows good selectivity and sensitivity to ZEN and is promising in feed safety analysis.

Ultrasensitive multiplexed immunosensors for the simultaneous determination of endocrine disrupting compounds using Pt@SBA-15 as a non-enzymatic label.

The simultaneous determination of different analytes based on multiplexed immunosensors is attractive and promising in bioassays and environmental analysis. In this paper, a novel ultrasensitive multiplexed immunoassay method was developed for the determination of endocrine disrupting compounds (EDCs) based on disposable screen-printed carbon electrodes. Different capture antibodies were separately immobilized on the carbon electrodes to form an immunosensor array. Platinum nanoparticle-functionalized mesoporous silica nanoparticles (Pt@SBA-15) was used as catalytic labels of secondary antibodies. Due to the catalytic activities of the platinum nanoparticles and the large surface area of SBA-15, a strong electrical response towards the analytical antigens was achieved. The multiplexed immunoassay array enables the simultaneous determination of different analytes. Using diethylstilbestrol and estradiol as model analytes, this multiplexed immunoassay method showed wide linear ranges with detection limits down to 0.28 and 1.2 pg mL-1 for diethylstilbestrol and estradiol, respectively. The proposed sensing strategy enriches electrochemical immunoassays and has a promising application in bioassays and environmental analysis.

Streamlining Immunoassays with Immiscible Filtrations Assisted by Surface Tension

Immunoassays are utilized for a wide variety of clinical and biomedical research applications. In typical immunoassays, analytes are captured, labeled, and quantified on a single surface (e.g., the bottom of a well plate). In order to minimize the background, this type of assay must be washed multiple times between each of these steps to ensure residual reagents (e.g., unbound labeling antibody) are removed from the system. In this manuscript, the immunoassay is fundamentally reconfigured, such that each reagent is confined to its own well and no wash steps are required. Using immiscible filtration assisted by surface tension (IFAST), a technique developed for nucleic acid and whole cell purifications, immunoassays can be drastically simplified such that all reagent manipulation is performed at the start of the assay (i.e., no pipetting steps are necessary during the assay). Analytes are bound to paramagnetic particles via antibodies and drawn through oil barriers between four isolated compartments: (1) sample well, (2) primary antibody labeling well, (3) secondary antibody labeling well, and (4) readout buffer well. Using this technique, we have demonstrated repeatable detection of as little as 188 fg of protein. IFAST immunoassay functionality is demonstrated by detecting a well accepted prostate cancer biomarker, prostate specific antigen (PSA). Assay performance was assessed by measuring known concentrations of recombinant PSA protein. The assay was then used to measure PSA concentrations in conditioned media and human plasma samples.

Fungsi Sel Imun Interleukin-4 (IL-4) pada Ikan Kerapu Tikus (Cromileptes altivelis) dengan Induksi Protein Imunogenik Vibrio harveyi <br><i>[The Function Of Interleukin-4 (IL-4) Immune Cells On Humpback Grouper (Cromileptes altivelis) By Induction Of Immunogenic Protein Of Vibrio harveyi ]<i>

Abstract Humpback grouper (Cromileptes altivelis) is a commodity to be cultivated because it has a high price, but the grouper highly susceptible to Vibriosis (Vibrio harveyi). The research objective was to see the function of immune cells of interleukin-4 (IL-4) are formed due to the induction of immunogenic proteins of Vibrio harveyi. Research procedures include Vibrio harveyi immunogenic protein isolation, SDS-PAGE electrophoresis, elektroelusi and dialysis, clinical exam of Vibrio harveyi immunogenic proteins, and immunohistochemical to look at immune cells (cells IL-4) with using a secondary antibody labeling of anti IL-4 biotin conjugate. The results showed that Vibrio harveyi protein found have immunogenic properties is 51.16 kDa. This protein is an adhesin protein. Protein 51.16 kDa was purified by electroelution and the protein will be used as a vaccine administered by intraperitoneal injection in grouper (Cromileptes altivelis) at a dose of 31.6 ug protein / 150 g of fish weight. In organ tissues of the brain, eyes and kidneys of induced fish by immunohistochemical examination, It was detected Immune cells (cells IL-4) using a secondary antibody labeling of anti IL-4 biotin conjugate. Tues formed IL-4 can be seen on the network grouper indicated by a change in tissue color with an inverted microscope examination of the golden brown color is formed on the IL-4 immune cells. It show the cross-reaction between 51.16 kDa immunogenic protein induced in fish with anti IL-4 biotin conjugate. The conclusions is the protein of Vibrio harveyi with molecular weight of 51.16 kDa is an immunogenic protein which able to induce formation of IL-4 in grouper.

Current status and future prospects in the search for protein biomarkers of immunosenescence

Complex adaptations including changes in cellular redox status, the production of high levels of pro-inflammatory cytokines and alterations in immunity occur as the result of aging of the immune system (immunosenescence). These events are thought to underlie the progression of chronic degenerative diseases of aging, such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. It is envisaged that identifying early biomarkers of immune aging would aid in identifying individuals at risk of age-related disease and would allow the discovery of novel intervention strategies. Proteomics has emerged as a rapidly expanding and innovative field, investigating protein expression, interaction and function at a global level. Several proteomic strategies, including use of mass spectrometry and non-mass spectrometry-based detection systems (including secondary antibody labeling with fluorescent tags) may be particularly advantageous in identifying biomarkers of immune health. Application of these approaches may identify factors that both contribute to (and define) age-dependent deregulation of the immune system.

Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold–streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody–25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.

Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella.

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.

Calcium activation of mussel gill abfrontal cilia

The mechanically-activated abfrontal cilia ofMytilus edulis gill are compared with the mechanically-inhibited lateral cilia using intact tissue, cell models, and isolated cilia preparations. 1. In contrast to lateral cilia, the abfrontal cilia of the mussel gill are mechanically activated to beat in normal seawater. Like the mechanically-induced inhibition of lateral ciliary beat, the activation of abfrontal cilia is dependent on the presence of extracellular Ca++. 2. Cilia of Triton-extracted, ATP-reactivated ‘models’ of abfrontal cells are quiescent at submicromolar Ca++ levels; however, when the Ca++ concentration reaches 20 μmol/l, the cilia are activated. In contrast, lateral cilia are active below micromolar levels of Ca++ and are inhibited above. 3. Trifluoperazine (TFP) at 50 μmol/l or calmidazolium at 20 μmol/l inhibit the Ca++ activation of abfrontal cilia, implying that calmodulin is involved in the activation of abfrontal ciliary movement. In contrast, TFP prevents the Ca++ -induced arrest of lateral cilia, implying that in this case calmodulin mediates Ca++ inhibition. 4. Mytilus lateral cilia can be selectively released by treating the gill with high salt and serotonin; the remaining classes of cilia can be released with EDTA-seawater treatment. 5. Polyacrylamide gel electrophoresis reveals that the respective axoneme and membrane fractions of the lateral and the remaining cilia are virtually identical, with the notable exception of a considerably greater amount of a very high molecular weight polypeptide in the membrane-matrix fraction of the lateral cilia. Both the lateral and the remaining cilia contain 3–4 μmol/l levels of calmodulin. 6. Estimated by anticalmodulin: ferritin secondary antibody labeling and by UV activation of bound TFP to produce a fluorescent derivative, the amount of calmodulin appears greater in abfrontal than in lateral cilia while laterofrontal cilia appear to contain little or none.