Repeated immobilization stress reduces the gene expression of the type 1 and 2 IP3 receptors in stellate ganglia

Inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by phosphoinositid hydrolysis and triggers IP(3) receptor (IP(3)R) mediated calcium release from intracellular pools. To determine whether immobilization stress affects the gene expression and protein level of IP(3)R in stellate ganglia, animals were immobilized once for 2h and/or for 7 days, 2h daily. After decapitation, stellate ganglia were extirpated and the gene expression of IP(3) receptors was evaluated. Protein levels of IP(3) receptor were measured by Western blot analysis using the antibody against IP(3) receptor. In the present work, we clearly show that type 1 and 2 IP(3) receptors, but not the type 3 IP(3) receptor, are expressed in stellate ganglia. Both types, type 1 and 2 IP(3) receptors, are not significantly affected by single 2h immobilization stress on mRNA and protein level. However, gene expression of both these types is significantly reduced by repeated immobilization stress for 7 days, 2h daily. The IP(3) receptor protein is reduced as well. Physiological relevance of our observations remains to be elucidated.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Enhancing immunoassay detection of antigens with multimeric protein Gs

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.     

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.     

Cell-based immobilization strategy for sensitive piezoelectric immunoassay of total prostate specific antigen

A novel yeast cell-based strategy for the immobilization of antibodies using an amine-terminated self-assembly film has been proposed. A quartz crystal microbalance sensor was according fabricated by coupling with anti-prostate specific antigen (anti-PSA) for PSA immunoassay. The crystal was modified with cysteamine to deposit yeast cells, on which anti-PSA antibodies were immobilized. The surface topologies of the as-prepared crystals were characterized by use of scanning electron microscopy. In contrast to the traditional glutaraldehyde (GLU) approach, the yeast cells could allow antibody molecules bound with higher bioactivity and achieve better immunoreaction capability. Results indicate that immunoassay prepared using the developed yeast cell-binding procedure exhibits increased analytical performance compared with that produced using the GLU cross-linking procedure. A PSA serum concentration in the range of 5.0-604.0ngml(-1) can be determined by this new system.     

脂肪酶的固定化及其性质研究

采用吸附与交联相结合的方法国定化脂肪酶,研究了脂肪酶固定化的工艺条件,并考察了固定化脂肪酶的催化性能和稳定性。试验结果表明,WA20树脂固定化脂肪酶的最适条件是：酶液pH7．0、给酶量300IU／g树脂、固定时间8h,所得固定化脂肪酶的活力约为165IU／g树脂;固定化酶稳定性较高,在冰箱内贮存6个月活力没有下降,操作半衰期约为750h,而未用戌二醛文联的固定化脂肪酶操作半衰期仅约290h;固定化脂肪酶催化橄榄油水解的最适条件是：PH8．0、温度55℃、底物浓度60％（V／V）、搅拌转速500r／m。     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin–avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

Antibody-antigen complex formation with immobilized immunoglobulins.

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.     

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

抗体固定化方法研究进展

在免疫分析和生物芯片中,抗原-抗体特异性结合被广泛应用,其中抗体的固定化是研发高效诊断和分离工具的关键环节。生物分子工程、材料化学与交联剂化学的进步极大地促进了抗体固定化技术的发展。 抗体可以通过物理吸附、共价偶联和亲和相互作用固定到不同类型的固相表面。 抗体固定化的目标是以一种正确的空间取向将抗体固定到固相表面,在完全保留抗体构象和活性的同时最大化抗原的结合能力,这对固相化抗体的分析性能至关重要。 对固定抗体到固相载体表面的各种最新方法进行了阐述,包括物理吸附法,通过羧基、氨基、巯基、糖基和点击化学的共价结合法以及基于生物亲和作用的固定法,并对固定化抗体的表征方法进行了归纳,最后对抗体固定化方法的发展方向进行了展望。     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Schiff base copper(II) chelate as a tool for intermolecular cross-linking and immobilization of protein

A new method for intermolecular cross-linking or bridging of protein has been proposed. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde, alpha-amino acid residue and copper(II). Reliability of the process as a tool for protein cross-linking was evaluated by chromatographic procedures. Behavior of salicylaldehyde in a column packed with Sepharose attached alpha-amino acid residue showed that salicylaldehyde was bound tightly to the gel in the presence of copper(II) ion and was eluted by the addition of EDTA. The association was shown strong enough to be applied for the purpose of cross-linking of proteins. It was also proved that BSA salicylaldehyde conjugate was immobilized specifically to the column, and the process was reversed by the addition of EDTA as well. The method is proposed to be useful not only for immobilization of enzyme but also for cross-linking of proteins since the method is free from unexpected random coupling products which are unavoidable with bifunctional cross-linking reagents.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.     

Effect of two distinct stressors on gene expression of the type 1 IP3 receptors

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers, which triggers calcium release from intracellular pools via IP3 receptors. Previously we have shown that single immobilization stress increased gene expression of both, the type 1 and type 2 IP3 receptors (IP3R1 and IP3R2, respectively). In this study we evaluated whether long-term exposure to softer stressor (cold exposure to 4 degrees C) can affect the response to single immobilization stress. We examined modulation of the type 1 IP3 receptor gene expression by each stressor separately, and then in their combination. Rats were immobilized for 30 min and 120 min and were decapitated immediately or 3 h after immobilization. Cold stress was performed by exposure of animals to 4 degrees C temperature for 1, 7 and 28 days. To determine the effect of both stressors in combination, animals exposed to cold for 28 days were afterwards exposed to immobilization for 120 min and decapitated 3 h after the end of stressful stimulus. Our results verify that single immobilization increases the IP3R1 gene expression in left atria of rat heart, while cold stress elevates the level of gene expression only after the exposure to cold for 7 days. The exposure to cold for 28 days did not increase the gene expression of the type 1 IP3 receptor compared to control. Application of both stressors (28 days of cold exposure followed by 120 min of immobilization with subsequent 3 h rest) showed the tendency of increased IP3R1 gene expression compared to absolute, nonstressed control, but level of the type 1 IP3 receptor mRNA was significantly lower compared to mRNA levels of solely immobilized animals. Thus, cold exposure affects the response of the gene expression of the type 1 IP3 receptor to immobilization stress.