Labelling and detection of antibody or antigen

As a POC (point of care) device semi-quantitative flow-immunoassay methods for the detection of antibodies or antigens had been developed and are well establish in e.g. pregnancy tests. This format is also known as strip test, one step strip test, immunochromatographic test, rapid flow diagnostic, rapid immunoassay or lateral flow immunoassay (LFI). The label is anyway a colored colloidal particle. Additionally to metal colloids, carbon (black) and silica or latex (various colors) are in use. 

One of the most important contributions of immunochemistry to daily practice is the development of immunoanalysis. The interest in immunochemical methods increased when sensitive labels for detecting the complex antigen-antibody were introduced. The labeling of antibodies or antigens with radionuclides increased the sensitivity of immunochemical methods so remarkably that the determination of specific compounds at ng levels is quite common and under certain circumstances even much lower concentrations may be detected. 

Immunoassays are based on the ability of the immune system to produce a virtually unlimited variety of antibodies each with a high affinity for foreign compounds (immunogens like viruses, bacteria, proteins, and haptens). Analytically, this phenomenon can be exploited by detection of this immunoreaction using labeled antibodies or antigens (i.e., compounds that can be bound by antibodies). The equilibrium between antibody (Ab), antigen (Ag), and immune complex (Ab-Ag) may be expressed as ... 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species and accordingly can be visualized with differently labeled species-specific secondary antibodies (see Sect. 8.1). However, quite often the appropriate combination of primary antibodies from different host species is not available. A general problem relates to the fact that the available primary antibodies may originate only from one species either rabbit or mouse. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies (Ino 2004). 

Non-labelled immunosensors rely on various principles (Fig. 3.27.A). Either the antibody or the antigen is immobilized on the solid matrix to form a sensing device. The solid matrix should be sensitive enough at the surface to detect immunocomplex formation. Electrode, membrane, piezoelectric and optically active surfaces may in principle be used to construct non-labelled immunosensors. The antigen or antibody to be determined is dissolved in a solution and reacted with the complementary matrix-bound antibody or antigen to form an immunocomplex that alters the physical e.g. the electrode potential or intrinsic piezofrequency) or optical properties of the... 

Most electrochemical immunosensors use antibodies or antigens labelled with an enzyme that generates an electroactive product which can be detected at the electrochemical transducer surface. The combination of high enzyme activity and selectivity with the sensitive methods of electrochemical detection provides a basis for the development of immunosensors. Horse radish peroxidase (HRP) and alkaline phosphatase (AP) are popular enzyme labels and can be used with a variety of substrates. 

Electrochemical immunosensors based on screen-printed electrodes have recently been applied to the detection of environmental pollutants such as PCBs, PAHs, pesticides [17-20] and of important molecules in clinical and food field [21-23]. In this case, the screen-printed electrodes are both the solid-phase for the immunoassay and the electrochemical transducers antibody or antigen molecules are directly immobilised at the sensor surface (transducer) and one of these species is enzyme-labelled in order to generate an electroactive product which can be detected at the screen-printed electrode surface. 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

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