Antibody-antigen reaction quantitative

To recover quantitative concentrations from an assay, it is necessary to establish standard response curves for a particular antibody-antigen reaction. As an example,... 

Biosensors either work as biocatalytic or as bioaffinity sensors. In biocat-alytic sensors, mostly enzymes are immobihzed at an electrode surface to act as selective catalysts. Enzymes catalyse slow reactions. The reaction rate, under appropriate conditions, is a quantitative measure for the substrate concentration. If the sample is one of the reactants, i.e. if the sample itself is the substrate, then chemical sensors can be created on this basis. The reaction rate can be measured in terms of an electrolysis current if amperometric biosensors are appHed. An alternative approach is to indicate the reaction product of the catalysed reaction selectively, as is done with potentiometric biosensors, hi bioaffinity sensors, as the second group, commonly very stable complexes with sample molecules are formed and bound strongly to the sensor surface. The extent of this complex formation is a quantitative measure of the sample concentration. It can be measured indirectly, since many properties of the electrode are changed by complex formation. In bioaffinity sensors, mostly the antibody-antigen reaction is utilized. 

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). 

Accordingly, when the system is working optimally the amount of hemolysis is quantitatively and inversely proportional to the amount of antigen added to the system. The hemolysis can be read spectro-photometrically (B9, M12), or the red blood cells can be tagged with Cr, and the amount of lysis determined by counting the amount of Cr which becomes soluble after lysis has occurred (B12). Not all antigen-antibody reactions will fix complement e.g., horse antisera do not, nor do univalent antibody-antigen systems (A12). 

Methods very similar to classical immunoassays in the sandwich format are easily implemented in flow systems (Fig. 2d). In this type of noncompetitive assays, again, antigen is captured and concentrated from an appropriate volume of sample on an immunosorbent (-Abi) column while nonantigenic components are eluted. Subsequent to the capture step, labeled second antibody (Abj-label) is introduced into the mobile phase and swept into the column, where it binds to the -Ab]-Ag complex to form -Ab -Ag-Ab2-label. Unbound Ab2-label is swept from the column, and when the label is an enzyme, antigen is quantitated indirectly by conducting an enzyme assay in the column. After substrate incubation, the reaction product is transported to a detector at the column terminus. Ag and Ab2-label can be introduced in the column sequentially or simultaneously. In some instances both modes led to similar sensitivity [55], and in other cases simultaneous injection produced a greater response than sequential injection [56]. The term sandwich has also been applied to the procedure carried out to quantitate Ab by capturing a complex Ab-Ag-label onto a protein G capillary column [57]. In this case detection is performed after elution. 

Figure 3. Diffusion analysis of freely diffusing molecules in solution. Fluorescently labeled molecules produce brief, intense photon bursts such as the ones shown in (a) on the photon detectors. Molecules labeled with different colors can be spectrally separated and detected on different detectors, which results in colocalized emission to both detectors in the case of molecular reaction events, such as e.g. antibody-antigen binding. Such binding events also change the diffusion time of the molecules through the excitation spot, which can be measured by the burst width or, more quantitatively by an autocorrelation analysis of an entire diffusion trace, which results in an average diffusion time as shown on (b).

Immunochemical methods are highly sensitive techniques for the detection and identification of protein targets by antigen-antibody specific reactions. Radial immunodilfusion and western blotting are among the most used techniques for the quantitative estimation of processed food and bioproduct proteins based on their immunoreactivity with specific antibodies. 

Turbidimetric Agglutination Immunoassays. Agglutination—precipitation immunoassays were among the first practical appHcations of the antigen—antibody reaction in diagnostic tests. These assays are not as widely used in the 1990s as EIA and FIA because they are either not quantitative enough or lack the sensitivity limits of RIA, EIA, and EIA. 

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... 

Precision is a quantitative measure of the random variation between repeated measurements from multiple sampling of the same homogenous sample under specified conditions.27 The weakness of the ELISA is its imprecision. The imprecision is related to the nature of the biological reaction — the reaction between antigen and antibody — and its inherent variability. Typically, the precision of an average ELISA is about 20% relative standard deviation, but can be as high as 30% in some circumstances. 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

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