Layer-by-layer assembly of multilayer films composed of streptavidin and biotinylated antibody by real-time biomolecular interaction analysis

Layer-by-layer assembly of multilayer films of streptavidin and biotinylated antibody was completed on the streptavidin coated surface. Real-time biomolecular interaction analysis (BIA) based on surface plasmon resonance technique was used to monitor the multilayer assembly in solution continuously. The results indicate that the uniform multilayer film can be fabricated successfully based on the strong interaction between streptavidin and biotin. The mean surface mass concentration of each adsorption layer is 1. 32 ng/mm(2) for biotinylated antibody, 2. 93 ng/mm(2) for streptavidin, according to the correlation of SPR response with surface concentration.

Photoelectrochemical reduction of CO2 mediated with methylviologen at roughened silver electrodes

The photoelectrocatalytic effect for the reduction of CO2 mediated with methylviologen (MV) was studied at mercury, polished silver and roughened silver electrodes using electrochemical and surface-enhanced Raman scattering (SERS) techniques. A large photoelectrocatalytic effect for the reduction of CO2 in the presence of MV was observed at the roughened silver electrode, whereas there was only a very small photoelectrocatalytic current at a more negative potential on mercury and polished silver electrodes. The SERS spectra of MV in the presence and absence of CO2, along with the electrochemical results, demonstrate that the surface adsorbed complexes, MV+ -Ag and MV0-Ag, played a role as the mediator for photoinduced electron transfer to CO2 in the solution. The results also suggest that the surface plasmon resonance of the nanoscale silver particle contributes to the overall photoelectrocatalytic effect on a roughened silver electrode.

Seed-mediated growth of large, monodisperse core-shell gold-silver nanoparticles with Ag-like optical properties

Large, monodisperse core-shell Au-Ag nanoparticles with Ag-like optical properties have been prepared by the seeding growth method in micellar media.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Enzymatic and kinetic properties of selenium-containing catalytic antibody with cGPX activity

Three selenium-containing catalytic antibodies mHB4, mHB5 and mHB7 which acted as mimics of cytosolic glutathione peroxidase(cGPX), were prepared by chemically introducing selenium into monoclonal antibodies HB4, HB5 and HB7. HB4. HB5 and HB7 were raised against a GSH derivative GSH-S-DNP dibenzyl ester, The cGPX activity of mHB4, mHB5, mHB7 were 170, 1 867, 32 U/mu mol, respectively. The cGPX activity of mHB5 was 0, 32 fold of natural rabbit liver cGPX and 1. 51 fold of m4A4. About two atoms of selenium existed in each of mHB5 molecule determined by inductively-coupled plasma/mass spectroscopy (ICP-MS), The optimal activity of mHB5 was at between pH 8. 4 and 8, 8, The reaction catalyzed by mHB5 involved a Ping-Pong mechanism. At pH 7. 0 and 37 degreesC, the apparent second-order rate constants for reaction of mHB5 with H2O2 and t-ROOH were as followed: k(+1) (H2O2) = 9. 71 x 10(6) L/(mol min), k(+1)(t-ROOH) = 5. 99 x 10(5) L/(mol.min). Rate accelerations (k(cat)/K-m/k(uncat)) 9. 8 x 10(6) and 3.7 x 10(5) fold those of the uncatalytic reaction were observed.

Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor

The biosensor based on surface plasmon resonance(SPR) technology is a very useful tool to study the interaction between biomolecles. The main advantages of this technique is to "visualize" macromolecular interactions directly in real time, and in a label-free mode rather than indirect methods like enzyme-linked immunosorbent assays (ELISAs). We immobilize human serum albumin (HSA) to the carboxymethyldextran-modified sensor chip surface covalently to detect the activity of anti-HSA in serum, and regenerate the surface with .1 mol/L phosphoric acid. The results show that SPR biosensor can detect the activity of anti-HSA in real-time quickly and the sensor chip can be used over 100 cycles.

Studies of soluble human catalytic antibody with glutathione peroxidase activity

By screening the phage-displayed human single chain antibody library, we have got the specific single chain antibody bound to GSH-S-DNP butyl ester as the hapten. The tertiary structure of the protein was analyzed with the aid of computer, and the results showed the CDR3 region located on the surface of the antibody. The soluble antibody was expressed in E. coli. and the active site serine was converted into selenocysteine with the chemical modifying method, which resulted in the catalytic antibody with GPx activity of 80 U/mu mol. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody was studied.

Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions

Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha (2a) and anti-interferon-alpha (2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible). Copyright (C) 2001 John Wiley & Sons, Ltd.

Hydrogen peroxide biosensor with enzyme entrapped within electrodeposited polypyrrole based on mediated sol-gel derived composite carbon electrode

A novel amperometric biosensor for the detection of hydrogen peroxide was described. The biosensor was constructed by electrodepositing HRP/PPy membrane on the surface of ferrocenecarboxylic acid mediated sol-gel derived composite carbon electrode. The biosensor gave response to hydrogen peroxide in a few seconds with detection limit of 5.0 x 10(-5) M (based on signal:noise = 3). Linear range was upto 0.2 mM. The biosensor exhibited a good stability. (C) 2001 Elsevier Science B.V. All rights reserved.

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic

Conversion of thyroxine (T-4) to 3,5,3'-triiodothyronine is an essential first step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronine. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against T4 into selenocysteines, can catalyze the deiodination of T4 with dithiothreitol (DTT) as cosubstrate. The mimic enzyme Se-4C5 exhibited a much greater deiodinase activity than model compound ebselen and another selenium-containing antibody Se-Hp4 against GSH. The coupling of selenocysteine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymatic activity. To probe the catalytic mechanism of the catalytic antibody, detailed kinetic studies were carried out in this paper. Investigations into the deiodinative reaction revealed the relationship between the initial velocity and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, involving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with K-m values for T-4 and DTT of approximately 0.8 muM and 1.8 muM, respectively, and a V-m value of 270 pmol per mg of protein per min. The activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU) with a K-i value of similar to 120 muM at 2.0 muM T-4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that PTU was a competitive inhibitor for DTT.

Molecular component structures mediated formation of self-assemblies

Molecular recognition directed self-assemblies from complementary molecular components, melamine and barbituric acid derivatives were studied by means of NMR, fluorescence, and TEM. It was found that both the process of the self-assembly and the morphologies of the resulted self-assemblies could be mediated by modifying the structures of the molecular components used. The effect of the structures of the molecular components on the formation of the self-assemblies was discussed in terms of intermolecular interactions.

A selenium-containing catalytic antibody with type I deiodinase activity

Acting as a mimic of type I deiodinase (DI), a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T-4) into selenocysteines, can catalyze the deiodination of T-4 to 3,5,3'-triiodothyronine (T-3) with dithiothreitol (DTT) as cosubstrate. Investigations into the deiodinative reaction by Se-4C5 revealed the relationship between the initial velocity and substrate concentration was subjected to Michaelis-Menten equation and the reaction mechanism was ping-pong one. The kinetic properties of the catalytic antibody were a little similar to those of DI, with K-m values for T-4 and DTT of approximately 0.8 muM and 1.8 mM, respectively, and V-m value of 270 pmol per mg protein per min. The activity could be sensitively inhibited by PTU with a K-i value of approximately 120 muM at 2.0 muM of T-4 concentration, revealing that PTU was a competitive inhibitor for DTT, (C) 2001 Academic Press.

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

Functionalized inorganic-organic composite material derivated by sol-gel for construction of mediated amperometric hydrogen peroxide biosensor

A novel functionalized inorganic-organic hybrid material with cation exchange property was prepared by sol-gel method. The H2O2 biosensor was fabricated by simply dipping the horseradish peroxidase-containing functionalized membrane modified electrode into Meldola's blue (MDB) solution. MDB was adsorbed and firmly immobilized within the membrane. The electrochemical behavior of MDB incorporated in the membrane was more reversible compared with that of the solution species and suitable as mediator for the horseradish peroxidase. The response time was less than 25 s. Linear range is up to 0.6 mM (COH. coeff. 0.9998) with detection Limit of 9 x 10(-7) M. High sensitivity of 75 nA mu M cm(-2) was obtained due to high MDB-loading. The biosensor exhibited a good stability. (C) 1999 Elsevier Science B.V. All rights reserved.