Noncompetitive immunoassay antibody-antigen complex

Noncompetitive Immunoassays. In a typical noncompetitive assay for an antigen, a capture antibody is first passively adsorbed or covalently bound to the surface of a solid phase. Various sequences in which the capture antibody can be attached are shown in Box 9-2. The simplest involves direct attachment to the solid phase. However, this can lead to some loss of antibody binding capacity because of steric factors or attachment of the antibody via its Fab region. To protect the binding properties of the antibody, more complex sequences have been devised. For example, the solid support can be coated with an antispecies antibody, and then... 

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. 

Using an antibody specifically recognizing the antigen-antibody complex, more direct noncompetitive hapten immunoassays, which can be regarded as semi two-site immunometric assay, could be established (S3). Figure 14 depicts two typical procedures of noncompetitive assays using anti-metatype antibodies, which are based on principle C in Fig. 4. 

Currently, one of the most sensitive EIA methods is the immune complex transfer immunoassay, which is a heterogeneous and noncompetitive EIA. This system can measure zeptomole per assay levels of protein antigens and antibodies as well as attomole per assay levels of several haptens. In this method, the primary immune reaction occurs on a solid support, after which the immune complexes are specifically... 

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