Assay formats competitive

Asphalt chemicals, ethyleneamines application, 8 500t, 506 Asphalt emulsifier amine oxides, 2 473 fatty acid amides, 2 458 Asphalt emulsions, 10 131 Asphaltenes, in petroleum vacuum residua, 18 589-590 Asphyxiants, 21 836 Aspirating aerators, 26 165-169 compressed, 26 168-169 propeller driven, 26 168 submersible, 26 169, 170t subsurface, 26 168 Aspiratory, 11 236-237 Aspirin, 4 103-104, 104t, 701 22 17-21. See also Acetylsalicylic acid as trade name, 22 19 for cancer prevention, 2 826 Aspirin resistance, 4 104 ASP oil recovery process, 23 532-533 Assay format, competitive, 14 142 Assay limits, in Investigational New Drug Applications, 18 692 Assays, for silver, 22 650. See also... 

Numerical data are a result of a literature search performed in the CAS database using the software SciFinder Scholar 2000. This search could not cover aU immunoassay publications, but was limited only to the ones in which the assay format (competitive or non-competitive) was specified. We consider, however, that the probability for the assay format to be mentioned is the same for both competitive and non-competitive immunoassays. 

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

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. 

Fig. 37 (a) QD-based sensing of cocaine by the formation of a cocaine-aptamer supramolecular structure that triggers FRET and (b) time-dependent luminescence spectra of the system in the presence of cocaine. The inset shows a calibration curve for variable concentrations of cocaine and a fixed so observation time of 15 min. (c) Schematic of the FRET-based TNT sensor and (d) increase of the QD luminescence upon addition of TNT in the competitive assay format. (Reprinted with permission from [220, 221], Copyright 2009 Royal Society of Chemistry and 2005 American Chemical Society)... 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-... 

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) ... 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

The ELISA tests are performed using different incubation steps, depending on the assay format, namely sandwich, competitive or inhibition immunoassays that correspond to the way the analyte is captured and further revealed, which is generally dictated by the nature of the analyte. 

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). 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

Esch, M. B., Baeumner, A. J., and Durst, R. A. (2001). Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format. Anal. Chem. 73 3162-3167. 

In an indirect competitive immunoassay, the competitor is commonly immobilized on a solid surface while the antibody and the analyte are added in the adjacent solution. After the competition, the fractions that are unbound to the solid surface are removed and the bound primary antibody is usually measured by the addition of a labelled secondary antibody (see Fig. 9.7). Even though this assay format is not as frequently used as the direct competitive immunoassay, it provides an advantage when analysing complex samples, i.e., the label (usually an enz5rme) does not come into direct contact with the sample matrix, so that interferences with the detection step are minimized. 

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