Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (Phi = 16 nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K-a: approximate to 10(15) M-1) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass, concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Real-time immunoassay of ferritin using surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used for the first time to determine the concentration of ferritin in both HBS-EP buffer and serum. The monoclonal antibody was immobilized on the carboxymethyl dextran-modified gold surface by an amine coupling method. The interaction of antibody with antigen was monitored in real-time. The signal was enhanced by sandwich amplification strategy to improve the sensitivity and specificity of the immunoassay, especially in serum. The linear range of the assay in serum is over 30-200 ng ml with the detection limit of 28 ng ml(-1). The sensitivity, specificity, and reproducibility of the assay are satisfactory. The analyte and enhancement antibody-binding surface could be regenerated by pH 2.0 glycine-HCl buffer and the same antibody-immobilized surface could be used for more than 50 cycles of ferritin binding and regeneration.

Kinetic characteristics of the interaction between morphine and its antibody by SPR competitive immunoassay

It is impossible for surface plasmon resonance to measure directly the binding kinetics between a low-molecular-weight analyte interacting and its immobilized binding partner. Solution competition method was applied to the kinetic study of the interaction between morphine and its antibody. The affinity constant between the antibody of morphine and morphine-BSA immobilized on the sensor chip was also obtained. The result showed that the affinity of polyclonal antibody is stronger than that of monoclonal antibody. And it also indicated that the protein combined with the analyte affected the binding of antibody to antigen.

Energy landscape theory, funnels, specificity, and optimal criterion of biomolecular binding

We study the nature of biomolecular binding. We found that in general there exists several thermodynamic phases: a native binding phase, a non-native phase, and a glass or local trapping phase. The quantitative optimal criterion for the binding specificity is found to be the maximization of the ratio of the binding transition temperature versus the trapping transition temperature, or equivalently the ratio of the energy gap of binding between the native state and the average non-native states versus the dispersion or variance of the non-native states. This leads to a funneled binding energy landscape.

Studies on the interaction of porphyrin with antibody with fluorescent spectrum and UV-Vis differential spectra

After meso-tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin combined with McAb 1F2, there was a significant hyperchromic effect, indicating that the combination of porphyrin and antibody is rigid and compact, aromatic amino acids exist at the combining sites of antigen in antibody. These aromatic amino acids are Trys and Trps, but the numbers of Trp are more than that found for Trys. The stochiometric ratio of porphyrin to 1F2 is 1:1, the disassociation constant was determined as(2.084+/-0.216) x 10(-10) mol/L by a method of fluorescence quenching, showing that both have a high affinity.

Computer simulation of human iodothyronine deiodinase single-chain antibody

Iodothyronine plays a major role in growth, basic metabolism and organ formation. It has an extremely limited source in the body. In this thesis, we designed iodothyronine(T4) as hapten. Then a single chain antibody displayed on phange was obtained from a human phage displaying a single chain antibody library. The specific genes of E3 was subcloned in P-5E vector. According to its amino acid sequences, we simulate its three dimention structure by computer. It has never been reported in PDB.

Studied on the interactions of antibody-porphyrin by circular dichroism and ultraviolet-visible spectrophotometry

Circular dichoism and UV-vis measurements were used to study the interaction between porphyrin and monoclonal antibodies ( McAbs). McAbs-porphyrin complex formation is usually accompanied by significant bathochromic shift and hyperchromicity changes of the absorption maxima in the porphyrin soret band region. Induced CD spectra in the same region (350 similar to 450 nm) were detected upon complex formation. They follow Lamb-Beer's law and exhibit isosbestic behavior. Both the UV-Vis and induced CD spectra of the antibody: porphyrin complex remain unchanged over a broad pH range ( pH 6 similar to 11), indicating remarkable stability of the complex and reflecting the dominant role of hydrophobic interaction between the hapten benzophenone and the antibody combining site.

Amplified immunoassay of human IgG using real-time biomolecular interaction analysis (BIA) technology

An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.

Preparation of rare earth ion induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational change of calmodulin induced by metal ions. Bovine calmodulin was firstly modified by 2,4-dinitrofluorobenzene to improve its immunogenicity, then, the derived protein was saturated with trivalent europium ions and injected to Balb/c mice as antigen. After four times of immunization, a corresponding antibody was detected and its titer in serum was determined as 1 : 12 000. By fusing of the spleen cells with hybridoma cells, a europium induced conformation-specific anti-calmodulin monoclonal antibody cell strain named as 2C3 was produced successfully. The molecular recognition ability of antibody to apocalmodulin and holocalmodulin showed a significant difference, indicating that this antibody could be applied to the studies of different effects of metal ions on the conformational change of calmodulin and its interaction with target molecules.

Studies on enzymological properties of antibody elicited by meso-tetra(alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin

meso-Tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene) porphyrin was used as a complete antigen to elicit monoclonal antibody 1F2 through the immunization and cell fusion techniques. McAb 1F2 obtained was demonstrated very pure by HPLC and MALDI/TOFMS. The retention time of McAb 1F2 was 2. 63 min. The subtype of McAb 1F2 was IgG2a. The relative molecular weight was 156 678. 8. When the McAb 1F2-porphyrin was formed, the maximal absorption of the porphyrin soret region had a redshift from 408 to 416 nm and hyperchromical effect, showing that the antigen-antibody combination was rigid and intense, and the abzyme constancy was high. But compared with HRP, the activity of the abzyme was only 4. 687 5 U/mg and 1. 899 % of that of HRP. Its K-m was 20. 29 mmol/L, k(cat) 396. 82 min(-1), k(cat)/K-m. 1. 955 7 X 10(4) L . mol(-1) . min(-1).

Preparation of europium induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

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.

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 mug/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HC1 buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.

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.