Homogeneous immunoassays

Applied Biochemistry and Biotechnology -     

Electrochemical immunoassays

Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests. Moreover, for opaque or optically dense matrices electrochemical methods are superior. Potentiometric, capacitive and amperometric transducers have been applied for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range and low detection limit, amperometric transducers are preferred. Competitive and non-competitive amperometric immunoassays have been developed with redox compounds or enzymes as labels. This review will give an overview of the most frequently applied principles in electrochemical immunoassays. The potential of an indirect competitive amperometric immunoassay for the determination of creatinine within nanomolar range and the circumvention of the most serious problem in electrochemical immunoassays, namely regeneration, will be discussed.     

Amperometmc immunoassays

When an electrode is placed in an electrolyte solution an electrical double layer is formed at the surface which functions electrically as a capacitor. An applied oscillating potential causes a certain current flow depending on the capacitance of this double layer. Carbon electrodes were prepared with immobilized antibodies (or antigens). When a specific antigen (or antibody) is added to the solutions, an antigen/antibody complex is formed at the electrode surface, which perturbs the electrical double layer and results in a current change. Dose-response curves can be obtained by measuring these current changes. Under the proper conditions this dose response is specific in the presence of non-specific proteins (e.g. serum). The method has been demonstrated, and dose-response curves obtained, for IgG, anti-IgG, anti-ferritin and S. Aureus cells. No labelled tag is required with this method.     

Electrochemical immunoassays

Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests. Moreover, for opaque or optically dense matrices electrochemical methods are superior. Potentiometric, capacitive and amperometric transducers have been applied for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range and low detection limit, amperometric transducers are preferred. Competitive and noncompetitive amperometric immunoassays have been developed with redox compounds or enzymes as labels. This review will give an overview of the most frequently applied principles in electrochemical immunoassays. The potential of an indirect competitive amperometric immunoassay for the determination of creatinine within nanomolar range and the circumvention of the most serious problem in electrochemical immunoassays, namely regeneration, will be discussed.     

化学发光免疫技术

化学发光免疫技术是化学发光与免疫测定结合起来的一种高效检测手段,化学发光是在特定化学反应中产生的光辐射。化学发光免疫技术可根据抗原抗体标记物的不同而分为发光物免疫测定、发光酶免疫测定和发光辅助因子免疫测定三大类型。     

Anti-metatype antibodies in immunoassays

In this review anti-metatype antibodies are described invoking new principles in immunoassay development. Anti-metatype antibodies are immunological reagents specific for the conformation of the liganded antibody active site which do not interact with bound ligand or unliganded antibody. Relationships between anti-metatype antibody reactivity and the ligand-induced conformational state of monoclonal antibodies are reviewed with emphasis on the fluorescein hapten as a small molecule model system. One characteristic result of the interaction of anti-metatype antibodies with liganded antibodies is a significant delay in the dissociation rate (k₂) of the ligand bound within the primary immune complex. The latter is an important consideration for assay development. Polyclonal and monoclonal anti-metatype antibody reagents are characterized in terms of their differential effects on the ligand dissociation rate. Anti-metatype antibody reactivity is further discussed in terms of protein-protein specificity patterns and relative interactions with idiotype-family members, structural derivatives, and site-specific mutants. Incorporation of principles inherent in the anti-metatype concept and their application to assay development are summarized.Abbreviations D₂O deuterium oxide - Fab 50 kd antibody fragment containing V_HC_H1 + V_LC_L domains - FITC(I) fluorescein isothiocyanate (isomer I) - Fv 26 kd fragment of the antibody molecule containing the variable domains of the H and L chains - Ig immunoglobulin - IgG immunoglobulin G with a mol. wt. of 150 kd. - IgM immunoglobulin M with a mol. wt. of 10⁶d - Id idiotype - Ka antibody affinity (k₁/k₂) in M^–1 - k₁ second order rate of ligand association in M^–1s^–1 - k₂ first order rate of ligand dissociation in s^–1 - K_D dissociation constant or the reciprocal of the affinity constant (1/Ka) - Mab monoclonal antibody - Met metatype - NMR nuclear magnetic resonance - SCA single chain Fv derivative containing a synthetic linker between the two variable domains - V_H variable domain of the antibody H chain - V_L variable domain of the antibody L chain     

NAD+ labeled immunoassays

Among the large number of labels which have been used in immunochemistry, NAD⁺, the coenzyme of many dehydrogenases, has been the label of choice. It allows the design of homogeneous immunoassays for haptens and proteins and it is detected by chemical amplification using enzymatic cycling procedures.     

Chemiluminescence from polytetrafluoroethylene

The chemiluminescence emission from commercial samples of polytetrafluoroethylene, PTFE (Halon G-80 and Halon G-10), was studied at temperatures between 25 and 172°C. The emission intensity decreased on extended heating, removal of the surface layer, solvent extraction, or changing from an atmosphere of oxygen to an inert atmosphere. Films of PTFE deposited from the vapor phase showed comparatively little chemiluminescence under these conditions. Ozone-induced chemiluminescence was observed at 37°C from G-10 but not from G-80, or from films deposited from the vapor phase. The chemiluminescence intensity from ozone and G-10 was not reduced by solvent extraction.     

Liquid-Phase Chemiluminescence Reactors

Three types of flow reactors for chemiluminescence analysis are considered in this article. Their features are analyzed, and reasonable fields of their application are outlined.     

Lanthanide-based time-resolved luminescence immunoassays

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized.     

Magnetic microbead-based electrochemical immunoassays

This review provides a summary of recent works concerning electrochemical immunoassays using magnetic microbeads as a solid phase. Recent research activity has led to innovative and powerful detection strategies that have been resulted in sensitive electrochemical detection. Coupling of magnetic microbeads with highly sensitive electrochemical detection provides a useful analytical method for environmental evaluation and clinical diagnostics, etc. The huge surface area and high dispersion capability of magnetic microbeads strongly contributes towards the development of new sensitive, rapid, user-friendly, and miniaturized electrochemical immunoassay systems. Moreover, the immunocomplexes formed on the magnetic microbead surface can be easily detected without pretreatment steps such as preconcentration or purification, which are normally required for standard methods. The discussion in this review is organized in two main subjects that include magnetic-microbead-based assays using enzyme labels and nanoparticle tags. Figure SEM image of Dynabeads M-280 (12% γ-Fe₂O₃ in polystyrene, diameter is 2.8 μm)

Chemiluminescent tags in immunoassays

Although radioimmunoassay procedures have a number of advantages they do pose problems, especially with regard to the disposal of radioactive waste. Alternative methods, such as chemiluminescence, enzyme, fluorescence, or spin immunoassays, have been tested with the aim of replacing radioactive labels without loss of sensitivity, precision and accuracy.     

Fluorescence polarization immunoassays for pesticides

Fluorescence polarization immunoassay methods for the detection of pesticides and their metabolites or degradation products are reviewed. Advantages and limitations for application to pesticide detection in environmental and food samples are discussed. The influence of the structure of fluorescent-labeled tracers and the affinity and specificity of antibodies on analytical performance is examined. The methods are simple, readily automated, and rapid (total time for assay of a water sample is about 1 min) with sensitivity of 1 - 10 ng/ml pesticide in 0.01 - 0.1 ml sample.     

Electrothermal stirring for heterogeneous immunoassays

A technique is proposed to enhance microfluidic immuno-sensors, for example, immunoassays, in which a ligand immobilized on a microchannel wall specifically binds analyte flowing through the channel. These sensors can be limited in both response time and sensitivity by the diffusion of analyte to the sensing surface. In certain applications, the sensitivity and response of these heterogeneous immunoassays may be improved by using AC electrokinetically-driven microscale fluid motion to enhance antigen motion towards immobilized ligands. Specifically, the electrothermal effect is used to micro-stir analyte near the binding surface. Numerical simulations of antigen in a microchannel flow subjected to the electrothermal effect show that 6 V(rms) applied to electrodes near a binding region can increase binding in the first few minutes by a factor of seven. The effectiveness of electrothermal stirring is a strong function of the Damk?hler number. The greatest binding enhancement is possible for high Damk?hler numbers, where the reaction is limited by diffusion. Based on these results, the utility of this technique for diffusion-limited microfluidic sensor applications is demonstrated.     

Analytical Applications of Chemiluminescence

Most organic compounds react very slowly with atmospheric oxygen at room temperature. The weak chemiluminescence accompanying this autoxidation can be amplified and measured by sensitive instruments. Emission of light from many materials can also be induced mechanically and photochemically. The intensity-time curves are so characteristic that they can be used to establish whether the composition and history of two samples are identical. Moreover, numerous physical properties can be correlated with emission and thus measured.     

Chemiluminescence immunoassay for chloramphenicol

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87–100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1–10 ng/ml, and it displayed good linearity.