Assay formats labelled

Sandwich assays rely on secondary antibodies binding to the target antigen or antibody after the primary biorecognihon reachon. In a compehhve assay format, labeled and nonlabeled anhbodies (or anhgens) compete for the binding sites of... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Another popular assay format for kinase assays is the Lanthascreen. This format is a variation on the LANCE assay, but employs Tb as the cryptate. In this format N-terminally fluorescently tagged peptide substrate (acceptor) is phosphorylated by the kinase. Next, a phophospecific antibody which is labeled with terbium binds specifically to the phosphorylated product, placing the donor and acceptor in close proximity, generating a signal [25]. 

A3. Arnold, L. J., Jr., Hammond, P. W., Wiese, W. A., and Nelson, N. C., Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1588-1594(1989). 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

The binding of the antigen and antibody can be affected by several factors, including the conjugated label chosen for detection and the method used to conjugate the label, as well as the assay format itself. The selectivity of the ELISA can be affected by the assay format. In an ELISA with a two-site sandwich format, independent epitopes are bound by different antibodies.26 The specificity comes from multiple site recognition. Polyclonal antibodies can react with many epitopes on a complex antigen surface.24... 

The ELISA techniques offer advantages of longer shelf life of the labelled reagents and elimination of the use of radioisotopes which require expensive scintillation/gamma counters and special disposal needs (38, 56). They are also more sensitive than the RIA s. ranging from pg/ml to pg/ml depending on the size of the molecules, affinity of the antibody and the assay format used (48). 

Immunoassays can be classified according to different criteria. The particular type selected has a strong influence on the assay performance with regard to precision and sensitivity. The main criteria include [3, 22, 23] (1) labeled or unlabeled assay formats, with different type of labels (2) competitive or non-competitive immunoassays, and (3) homogeneous or heterogeneous immunoassays. These classifications can also be extended to MIP-ILAs (Fig. 2) ... 

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). 

The definition of the assay strategy and readout determines the requirements for the labeling of the substrate (see 2.3 covering assay formats). 

While the capture on DNA chips of fluorophore-labelled targets, and the extension of arrayed primers with fluorophore-labelled nucleotides has been widely used for some time, it is only more recently that assay formats have developed that utilize immobilized nucleic acids already modified with fluorophores. Fundamental analyses of surface monolayer structures and chemistries can be readily performed by immobilizing such modified oligonucleotides into SAM structures [105,106], but it is those interactions that can be monitored using fluorescence quenching or fluorescence resonance energy transfer (FRET) that have gained the most attention. 

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