Assay formats limited reagent

Common immunochemical assay formats to select from include the 96-well microtiter plates, dipsticks, coated test tubes, and membrane-based flow through devices. If the end-user is a trained technician working in a well-equipped laboratory and needs to detect and tentatively identify, for example, antimicrobial residues in hundreds of meat samples per day, a multiwell or other high-through-put format should be chosen. If, on the other hand, the end user is a quality control inspector at a milk factory who has limited time to find out whether the penicillin residues in the milk waiting to be unloaded exceed a certain level, the same reagents used in the first instance may require a more user-friendly format such as dipstick or membrane-based flow through device. 

The double-antibody sandwich technique is applicable to large molecules. Small analytes have difficulty forming the double-antibody sandwich immunocomplex. The smallest analytes reported that have been used in a double sandwich assay are peptides of around 10 amino acid residues (158). The double-antibody sandwich technique measures the occupied antibodies using an excess-reagent assay protocol. A limited-reagent assay protocol can also be designed, as shown in Fig. 4. In this format, the antibodies are immobilized onto the solid phase,... 

Fig. 4 Antibody immobilization in a limited-reagent assay format. The antibody is immobilized onto the solid-phase support. Labeled antigen and sample are introduced, and they compete with one another to form immunocomplex (Ab-Ag or Ab-AgE) with the limited antibody sites on the solid-phase support. After washing, the substrate is added to produce the detecting product. The antibody unoccupied by the sample analyte is measured as the concentration increases, the signal responses decreases.

To detect the assay product, it is usually necessary to use a label, which is attached either to the antibody or the antigen. This label can be fluorescent, luminescent, radioactive, an enzyme or an electrochemically active group. Immunoassay reactions can be performed in a large variety of formats, in solution or on a solid support, with limited reagent or an excess of reagent. These formats are discussed in more detail in the following sections, after a description of antibody and antigen structure and immunocomplex formation. 

Only limited development of new methodologies has taken place for immunochemical analysis of nucleic acids. Most published methods rely on modifications to classical DNA probe hybridization or immunoassay methods, with considerable blending of the two. For example, some methods employ immobilized oligonucleotide probes to capture the analyte DNA followed by immunoenzymatic detection. Other methods use immunocapture followed by detection with an enzyme-labeled DNA probe. Distinctly new methodologies mostly impact on assay formats (e.g., DNA microarrays and in situ hybridization) and detection reagents (e.g., chemiluminescent enzyme substrates). 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

The only rate-limiting factor in a coupled assay should be the concentration of the initial and linking products and all other reagents should be in excess. The role of the auxiliary and indicator enzymes is essentially that of a substrate assay system and under optimum assay conditions the rate of the indicator reaction should be equal to the rate of formation of the initial product. The indicator reaction must be capable of matching the different test reaction rates and its velocity can be defined by the Michaelis-Menten equation in the usual way ... 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

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