Enzyme tracer

Neuroanatomists have taken advantage of the phenomenon of fast retrograde transport to locate remote nerve cell bodies in the CNS of an experimental animal that are connected to an identified axonal fiber tract whose origin is uncertain. The tracer material [purified horseradish peroxidase (HRP) enzyme] is injected in the region of the axon terminals, where it is taken up by endocytosis and then is carried by retrograde axonal transport over a period of several hours to days back to the nerve cell body. The animal is sacrificed, and the enzyme tracer is localized by staining thin sections of the brain for peroxidase activity. 

Figure 18 Flow chart of the automated on-line flow injection immunoassay (FllA). Six steps are involved in each cycle (1) addition of antibody and incubation (2) addition of analyte (or standard) and incubation (3) addition of enzyme-tracer and incubation (4) addition of substrate and incubation (5) downstream measurement of fluorescence (6) regeneration of affinity column...

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. 

The preparation and characterization of the anti-triazine antibody and the enzyme tracer coupled to horseradish peroxidase was previously described in detail [1],... 

Mix 50 pL of sulfapyridine standard solutions (from 1.92 to 30,000 nmol L 1) with 50 pL of the anti-sulfonamide class-specific antibodies modified magnetic bead suspensions (15 pg of MB-Tosyl) followed by the addition of 50 pL of enzyme tracer 0.075 pgmL-1 [3]. 

In most cases, competitive immunoassays for the analysis of small molecules are carried out in microtitre plates. The majority of these assays use an enzyme as label, thus leading to the term enzyme immunoassay, and the most commonly used is the ELISA (enzyme-linked immunosorbent assay) that has a heterogeneous format (separation of bound and unbound). This format can be set up either in the enzyme-tracer format (Figure 3.3.1 A) or in the coating antigen format (Figure 3.3. IB). The result shows a... 

It is essential that test-kits are used for the intended purpose in a specific matrix. Thus test-kits can limit the applications in which the technology can be applied. In some cases, it will be necessary to set up an in-house assay format for which it will be necessary to buy the separate individual immunoreagents, such as antibodies, enzyme-tracer or analyte-derivative protein conjugates. In some cases this is difficult because the quality and supply are not always guaranteed. A recent list of antibody manufacturers, suppliers and services is given by Blow, 2007. 

Table 3.3.2 Standard operating procedures for ELISAs - enzyme-tracer format...

The rest of the quantitative results received for pesticides within the 2nd and 3rd PT campaign by SMETs were gained by ELISAs by one participant. In the 2nd SWIFT-WFD PT campaign the participant used the enzyme-tracer format of the... 

In the 3rd PT campaign, for isoproturon and diuron determination in both RM15 and RM17 the ELISAs were used in the coating antigen format which is usually less sensitive compared to the enzyme-tracer format. 

The common fluorogenic substrates used for three widely used enzyme tracers are />hydroxyphenylacetic acid for HRP, 4-methylumbelliferyl phosphate for alkaline phosphatase, and 4-methylumbelliferyl-P-D-galacto-pyranoside for p-galactosidase. 

The DL (or least detectable dose) is the smallest concentration of the analyte that produces a signal which can be significantly distinguished from zero for a given sample matrix with a stated degree of confidence. Very often a dose is selected which inhibits 10-20% of enzyme tracer from binding with the Ab or the dose calculated after subtraction of two or three times the standard deviation from the mean measurements of the zero dose signal. ... 

Precision and accuracy of lA are important properties which deserve special attention. The quality and stability of the employed material (microtiter plates, pipettes) and reagents (e.g. Abs, enzyme tracer or buffers), play a crucial role. ° The long-term stability of reagents has to be ensured, e.g. by freeze-drying of Abs and, if necessary, addition of stabilizing components to the test reagents such as the enzyme tracer. 

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