Enzyme-tracer format

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... 

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. 

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. 

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. 

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.

Early isotope tracer experiments by David Shemin permitted the elucidation of the formation of the immediate precursor of the porphyrin needed for the cytochromes and for hemoglobin. These studies indicated that the glycine methylene carbon and nitrogen were incorporated along with both carbons of acetate. Subsequent enzymatic studies in both bacteria and animals revealed a condensation reaction between succinyl-CoA and glycine to yield 5-amino-levulinate and C02 (presumably by way of an enzyme-bound /3-keto acid, a-amino-/3-ketoadipate) (fig. 22.13). 

The MIP-ILAs based on a direct format will be described elsewhere. ILAs based on indirect methods will be reviewed in this chapter. Like immunoassays, the first MIP-ILA, reported in 1993 by the Mosbach group [25] for the analysis of theophylline and diazepan, was based on a radioactive tracer. Since this work, many other indirect MIP-ILAs have been developed using non-isotopic tracers such as fluorophores, enzymes, or electroactive probes. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

Similarly, Crespi-Perellino et al. (13,15), using cell cultures of A. altissima and providing L-, D-, and D,L-[w(7Av/cz c- C lryptophan as the precursor, carried out tracer experiments and proved the biosynthetic pathway to canthin-6-one alkaloids to be as follows (Scheme 7) tryptophan )8-carboline-l -propionic acid — 4,5-dihydrocanlhin-6-one (29) canthin-6-one (1)—> l-hydroxycanthin-6-one (10) l-methoxycanthin-6-one (11) —> l-methoxycanthin-6-one 3-oxide (12). In the biosynthetic pathway to canthin-6-one alkaloids, oxidation proceeds stepwise. The hydroxyl group at position 1 of canthin-6-one is methylated, and 11 is readily formed this formation is considered to be a transmethylation promoted by a specific enzyme. 

Haptens and anti-hapten antibodies are reagents which can be used in a wide variety of assay formats. One fundamental division among immunoassay formats is based on the type of label or tracer which is ultimately detected. Two of the most common types of label are radioisotopes and enzymes, used respectively in radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is the older of the two and remains very important and widely used in clinical and research situations. However, the use of radioisotopes carries with it possible health hazards, the need for special handling precautions, and mandatory regulatory oversight. Because EIA avoids these problems, it is gaining popularity rapidly among users of immunoassays. 

Histidine ammonia lyase (HAL, histidinase, histidine-a-deaminase, E.C. 4.3.1.3) is capable of abstracting ammonia from L-histidine (17), resulting in the formation of urocanoic acid [Scheme 12.6-4, (6)], an intermediate in the metabolism of l-histidine,n). HAL has also been identified as a key enzyme in the synthesis of secondary metabolites such as Nikkomycin in Streptomyces tendae,ul The mechanism of the enzyme has been investigated and seems to proceed via the carbanion intermediate111, 13]. Synthetic applications of HAL are difficult to achieve, particularly as the enzyme is sensitive to oxygen1131. The utility of HAL is limited to niche applications such as the synthesis of radiolabeled urocanic acids as tracers of histidine metabolism1"1. 

Since, as discussed above, chorismate (4) is the branch point of the pathway and SCO4506 (MqnA) is the enzyme responsible for the formation of futalosine (8), attempts were made to demonstrate the reaction in a cell-free system. Structural examination of futalosine indicated that the nucleoside moiety is likely derived from inosine (36). Isotopic tracer experiments established that the C-6 and C-7 positions of futalosine (8) between chorismate (4) and inosine (36) are likely derived from a C2 unit derived from either pyruvate or PEP. A recombinant enzyme from T. thermophilus (TTHAO803) was tested for the formation of futalosine (8). However, no futalosine was formed, but instead w-hydroxybenzoate and 3-(l-carboxyvinyloxy) benzoate were formed from chorismate, both in the presence and in the absence of flavin mononucleotide (FMN). 

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