Immunoassay formats

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

Figure 1 Schematic of the quasi-equihbria using heterologous haptens in coating antigen immunoassay formats. Ka represents the equilibrium constant for binding of antibody (Y) to target analyte (A). Kh is the equilibrium constant for the binding of antibody to hapten-protein conjugate (H-) immobihzed on a solid phase...

FIGURE 5.2 Schematic representation of (a) competitive and (b) non-competitive immunoassay formats. 

FIGURE 5.3 A schematic illustrating a competitive immunoassay format used for the detection of estradiol. (Reprinted from [11] with permission from Elsevier.)... 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

In a so-called competitive immunoassay format the antigen competes with a labeled antigen for a limited number of antibody-binding sites. It can be shown that in this case the ultimate sensitivity of the assay (when the [Ab] approaches zero) is dependent on the equilibrium constant K and the reliability of the signal measurement of the bound fraction at zero dose [15],... 

An example of an immunoassay format is shown in Figure 3. This immunoassay format relies on partial saturation of the solid-phase antibody (Ab) by the antigen (Ag) and on its competition with the labeled antigen (Ag-L) for the available antibody sites. At low antibody and tracer concentrations the sensitivity of... 

Figure 3 Layout of competitive solid-phase antibody immunoassay format in an incubation step antigen (Ag) and labeled antigen (Ag-L) compete for the solid-phase antibodybinding sites (Ab) after the solid-phase antibodies are washed, the activity of the bound labeled antigen is measured, and a calibration curve constructed. The measured signal is inversely proportional to the antigen concentration.

As occurred with the other antibiotics, commercial immunoassay formats, also available as kits for tetracyclines and penicillins such as the Parallux, the LacTek, or the Charm II, have also been placed on the market for the analysis of sulfonamides (see Table 4). Thus, the Parallux detects sulfamethazine and sulfadimethoxine in raw milk with a LOD of 10 pg L1. The Charm II detects almost all sulfonamides in honey and milk with a LOD in the range from 1 to 10 pg L, whereas LacTek is able to detect sulfamethazine. Moreover, the 5101SULlp and 5101SUDAlp tests reach LOD values for sulfamethazine and sulfadiazine of around 0.2 pg L 1 and they have been applied to the analysis of urine, milk, and plasma. These tests have proved to be efficient as a point of care for on-site applications on farms. Moreover, commercially available antibodies can be found from several sources such as Silver Lake Research, US Biological, Cortex Biochem. Inc., Accurate Chemical Scientific, Fitzgerald Industries International Inc., and Biotrend Chemikalien GmbH. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

As described above, the dendrimer-coupled antibody conjugates show uniquely enhanced properties compared to the classical double antibody systems. Important characteristics such as complete solubility in aqueous buffers, flexibility in immunoassay format, ability to improve assay sensitivity, consistent, reproducible manufacturing and favorable stability has driven the utilization of these dendrimer-based reagents in Stratus CS, the latest member of the Stratus family of immunochemistry analyzers. In this new analyzer system, the primary... 

The aim of this chapter is to discuss fluorescence concepts that are used in selected immunoassay applications. The primary focus is on fluorescence topics of recent interest that provide insight into the characteristic properties of antibodies and antigens in immunoassays, or that describe enhancements in immunoassay technologies. The basic reagents and instrumentation required for immunoassay purposes are discussed first, followed by a brief description of immunoassay formats. The principles that are utilized in various fluorescence immunoassay technologies are outlined with specific examples and their significance. Since it is beyond the scope of this chapter to review all of the applications of fluorescence immunoassays, apologies are extended to authors that this chapter fails to cite. A number of comprehensive treatments of fluorescence immunoassay (FIA) applications and related topics are available. 18 ... 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

This NRL sensor was used for fhe rapid detection of Campylobacter jejuni and small toxins, including several mycotoxins [ochratoxin A, fumonisin B, aflafoxin Bi, and deoxynivalenol (DON)] from food pro-ducfs (Ngundi et ah, 2005, 2006 Sapsford et ah, 2006). They used a sandwich immunoassay formaf fo detect C. jejuni in milk and yogurt and a competitive immunoassay format to detect the mycotoxins. 

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