Ab-Ag-complex

Homogeneous immunoassay is conducted entirely in solutions. In this format, separation of the labeled antibody (Ab) and the antibody-antigen (Ab-Ag) complex is required, and this is usually achieved using CE separations. For instance, separation of Cy5-BSA from unreacted Cy5 and the complex (formed from Cy5-BSA and anti-BSA) was achieved in a flow-through sampling chip [567]. 

A robust assay for theophylline was established within the clinical range of 10-20 pg/mL [330]. Since the Ab-Ag complex can be mobilized, no additional internal standard was needed [330], as in an immunoassay study for cortisol [1006],... 

RIA procedure Traditionally, a specific antibody (Ab) is incubated in the presence of labelled antigen (Ag ) to allow Ab to be saturated, that is, form a complex with Ag (Ab-Ag complex). This Ag is the labelled equivalent of the antigen (Ag) to be measured in the sample, and after Ag is added there is a competitive interaction where some Ag may be displaced from the Ab-Ag complex and replaced by Ag (i.e formation of Ab-Ag). The amoimt of Ag that is displaced will reflect the amount of Ag in the sample. As noted earlier, a standard curve is constructed where known amounts of standard antigen (Ag) are incubated with the same amount of Ab, where Ag displaces Ag from the Ab-Ag complex, thus allowing parallels to be drawn between the displacements which form Ab-Ag (sample) and Ab-Ag (standard). This process is illustrated in Figure 10.6. 

Separation for measurement The basis of measurement depends on the ability to separate the unbound or free" Ag from the Ab-Ag complex, which itself relies on differences in properties between the two components while maintaining (that is not disrupting) the Ab-Ag complex (Figure 10.6). There are a number of reagents used for separation including charcoal, hydroxyapatite, ammonium sulfate and polyethylene glycol, but the method that will be considered here relies on the use of dextran-coated activated charcoal (see Figure 10.7). With inherent... 

Fignre 8-32. Conceptual operation of the radioimmunoassay, RIA. The soluble Ab-Ag complex is both a product of the first step and a reactant in the second step of the assay. 

An Ab will interact with its Ag and form the usual Ab-Ag complexes. It is easy to see that if a polyclonal Ab were used for recognition with its Ag, the final electropherogram of the mixture could become quite complicated. For the above reasons, Nielsen et al. used the monoclonal Ab specific for... 

However, in the absence of true identification of these complexes, it is not 100% possible to identify each of the complex peaks (20,59). Indeed, in none of the existing CE immunoaffinity studies reported have any Ab-Ag complexes been identified by light scattering or mass spectrometric methods (60a,b). This would require, for example, a size exclusion chromatographic (SEC) separation of a particular complex and then on-line characterization with isolation and reinjection under CZE conditions (60c). 

Multiple labeled Ags (or Abs) lead to multiple peaks or, at times, a broad peak in the CE electropherograms. These species can then form more than one Ab-Ag complex, which complicates the analysis, due to perhaps insufficient resolution of the labeled Ags and the Ab-Ag complexes. Sometimes chemical modifications to the Ag (or Ab) are necessary to facilitate the separation by changing the final mass/charge ratio (74-76). 

The principle of direct immunoassays is that Abs are first tagged with FL labels and then mixed with the Ag sample of interest. The Ab should be in excess. After incubation, the mixture is injected into the capillary, and the amount of Ag can be determined by the Ab-Ag complex signal. Competitive immunoassays are usually preferred for small Ags because the separation of the Ab-Ag complex from the Ab can be very difficult for small Ags. As above, Shimura and Karger reported a direct immunoassay of hGH (61). Due to the focusing effect of CIEF (Fig. 4) and the high sensitivity of CE-laser-induced fluorescence (LIF), a detection limit of 0.1 ng/ml was achieved. Similarly, Chen demonstrated the direct immunoassay of IgG with a detection limit of 6 X1(T10 (74). 

Fig. 5.1. Molecular recognition of antibody (Ab) and antigen (Ag) resulting in an Ab-Ag-complex and unbound Ag.

Since the first appearance of immunoassays in about 1960 many methods have been used to partition and separate the Ab-Ag complex from other components of the reaction mixture. These include salting out with (NH4)2S04 or Na2S04, protein dena-turation/precipitation by solvent (e.g. CH3OH, C2H5OH, acetone) or precipitation by polyethylene glycol, followed by collection of the precipitated Ab-Ag by filtration or centrifugation. Nowdays the method of choice involves immobilisation of the Ab (for... 

In + [53]. The Ag was made by coupling In + to Ag molecules using the bifunctional chelate diethylenetriamine penta-acetic acid (DTPA). Because In + is not found in human tissue, interference from the clinical sample itself is avoided. The assay protocol used is outlined in Fig. 5. Following competitive equilibration of Ag and Ag with Ab (adsorbed to insolubilized protein A), the Ab-Ag complex was separated by centrifugation. The In " label of Ag was then released by acidification and determined by differential pulse anodic stripping voltammetry (DP-ASV). The... 

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. 

Capillary zone electrophoresis (CZE), the most widely used CE mode, is also the mode most frequently used for performing CE immunodetection. The first assay of immunoaffinity capillary electrophoresis described was carried out with CZE in noncompetitive format (Fig. 8a) [79,80]. This format has been used to determine a protein in a matrix as complex as human serum by incubating the sample with specific antibody for 1 hr before the introduction onto the CE column [81]. The size of the immunocomplex peak was seen to increase with incubation time. One drawback of this method is the long incubation time needed because of the slow reaction kinetics of formation of Ab-Ag complex. To prevent noticeable complex dissociation during the analysis, conditions to achieve a separation time shorter than 3 min were chosen. 

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