Immunoassays noncompetitive binding

Noncompetitive binding enzyme immunoassay. E is inhibited upon binding. 

A second form, the sandwich immunoassay, is based on noncompetitive binding and involves two separate Ag/Ab interactions (Wisdom, 1976). Typically Ab is attached to a solid support and exposed to a sample containing the Ag of interest as depicted in Fig. 1. The amount of Ag bound to the solid phase-Ab is determined following incubation with a second ligand-specific labeled Ab (Ab ). In contrast to a competitive assay, the amount of Ab-bound Ag, and therefore the amount of enzyme label, is proportional to the Ag concentration in solution. 

HI. Hara, T, Nakamura, K., Satomura, S., and Matsuura, S., Noncompetitive immunoassay of thyroxine using a liquid-phase binding assay. Anal. Chem. 66, 351—354 (1994). 

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). 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

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... 

Noncompetitive (also known as two-site, sandwich, labeled antibody, or immunometric methods) (see Chapter 9) require two antibodies capable of simultaneously binding PTH (Figure 49-14) (1) a capture antibody immobilized to a solid phase, and (2) a signal or reporter antibody labeled with a measurable substance or an enzyme changing the concentration (substrate or product) of a measurable substance. Unlike competitive immunoassays, with noncompetitive methods, both antibodies are added in excess, ensuring that aU analyte is measured. After formation of the ternary complex or sandwich, excess labeled antibody is removed by washing before quantification of complexes. Noncompetitive immunoassays provide increased sensitivity, specificity, reproducibility, and convenience. 

Table 1 The plethora of formats of immunoassay. Ab = antibody, Ag = antigen. Noncompetitive and competitive heterogenous binding assays where the probe is labelled with an enzyme are known as enzyme-linked immunosorbent assay (ELISA)...

Figure 5 Schematic representation of the most widely used noncompetitive biologieal immunoassay, the enzyme-linked immunosorbent assay (ELISA), (a) Antibody, selective for analyte, is immobilized on microtiter plate well surface (b) sample added (c) analyte in sample binds to antibody while other compounds in matrix remain in solution (d) sample solution containing nonbound molecules discarded and wells washed (e) analyte remains bound to antibody (f) second antibody nzyme conjugate added (g) conjugate binds to bound analyte (h) solution containing nonbound conjugate discarded and wells washed (i) conjugate remains bound 0 enzyme substrate added (k) substrate S converted to colored or fluorescent product P at a rate which is proportional to the amount of bound enzyme and hence to the concentration of analyte in the sample.

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 . Principle of a noncompetitive immunoassay AB 1 is a catcher antibody, and binds to the analyle. AB 2. should be able to hind a second (identical or different) antigenic epitope. AB 3 bind.s to foreign antibodies (of type AB2) and is covalently linked to an enzyme.

Immunoassay can be broadly categorized as competitive or noncompetitive. With noncompetitive methods, also called inrmunometric methods, the antibody is usually present in excess. In the competitive mode (Figure 4.26), antigens compete for a limited number of antibody binding sites. Initially, the... 

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