Competitive Format

Competitive formats, direct (Fig. 5.4a and 5.4b) or indirect (Fig. 5.4c and 5.4d) are commonly used when testing for low-molecular-weight compounds, which often possess a single-epitope region. In both competitive formats, the 

2nd Specific anti-analyte antibody labeled (Gold, latex, 

If the sample mixture contains the unlabeled analyte, it will preferentially occupy the target-specific binding sites at the test line in competition with the analyte conjugated with colored particles (direct) or the immobilized carrier protein (indirect). In both formats, a non-compliant (positive) sample is indicated by the absence of the test line or 

SAMPLE Analyte absent SAMPLE Analyte present 

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The most used EIA reagents conjugate a fluotophote such as fluorescein-isothiocyanate (EITC) or thodarnine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluotophotes for EIA and their spectral characteristics ate presented in Table 3. EIA assays ate available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color deterrnination, EIAs require a fluorometer, and thus ate primarily laboratory-based. 

The temperature is chosen to maximize reaction rate and to avoid the competitive formation of the tetrafluoride. Alternative preparation methods include the reaction between TiCl and gaseous HF,... 

When an aqueous solution of an arenediazonium salt is added to an alkaline buffer solution, an initial rapid reaction occurs. All experimental evidence is consistent with the hypothesis that only the (Z)-diazoate is formed. Theoretically, however, the competitive formation of the (ii)-isomer in very small quantities cannot be excluded... 

The synthesis of a-substituted phosphonates 89, via the electrophilic addition of phosphorylated C-radicals 88 (generated by reaction of BujSnH to the readily accessible a-phosphoryl sulfides (or selenides)) and electrophilic addition to electron rich alkenes, has been described [57] (Scheme 26). A large excess of alkene is necessary to minimize the competitive formation of the undesired compound 90 resulting from direct reduction of the initial radical 88. The ratio 89/90 has been measured for each example. The synthesis of the a-mono- or a,a-di-substituted (R or phosphonates 89 shows that the free radical approach... 

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. 

An asymmetric variant of this reaction was developed using chiral Pd complex 111 with either silanes or disiloxanes [66-68]. Both relative and absolute stereochemistries were controlled in this system and good yields (60-85%) were obtained after oxidation (Eq. 18). Formation of the silane-containing product was inhibited by the presence of water due to competitive formation of the palladium hydrides and silanols [68]. The use of disiloxanes as reductants, however, provided expedient oxidation to the alcohol products without decreasing the isolated yields enantioselectivity was 5-15% lower in this more robust system [66]. Benzhydryldimethylsilane proved to be a good compromise between high yield and facile oxidation [66]. Palladium com-... 

More recently, a new mode of cis-trans isomerization of a disilene has been suggested for the extremely hindered disilene 27. As will be detailed in Section VIII. B, 27 undergoes thermal dissociation into the corresponding silylenes. Monitoring the thermolysis of (Z)-27 at 50°C by H and 29Si NMR reveals a competitive formation of the isomerized ( >27 and benzosilacyclobutene 37, which is most likely formed by intramolecular insertion of silylene 36 into the C—H bond of the o-bis(trimethylsilyl)-methyl group (Scheme 3).22,59 This suggests the possible occurrence of cis-trans isomerization via a dissociation-association mechanism. 

Assuming that the metabolic pathways are similar in the biosynthesis of related isocyanoterpenes, these studies remain difficult, due in part to the competitive formation of other secondary metabolites. In addition to the common trio (-NC, NCS, -NHCHO) of the nitrogenous functions found attached to these skeletons, analogs such as -CN, -CNO, and -SCN foreshadow the complexity of identifying and selecting specific precursors to be targeted for incorporation into the family of marine isonitriles. 

To test our new signal reagent based on GZ-11 the detection system was applied to two competitive-format immunoassays. These two assays (for atrazine and clenbuterol) normally use chromogenic detection systems. While these colorimetric assays may be adequate for laboratory use, chemiluminescent detection offers potential advantages in sensitivity and on site screening applications [33],... 

In order to explain the competitive formation of the 1 1 and 1 2 adducts and the formation of the 2,6-octadienyl rather than the 1,6-oc-tadienyl chain, a mechanism was proposed (62, 69) in which the insertion of one mole of butadiene to the Pd—H bond gives the 77-methallyl complex (68) at first, from which 1-silylated 2-butene is formed. At moderate temperature and in the presence of a stabilizing ligand, further insertion of another molecule of butadiene takes place to give C5-substituted n-allyl complex 69. The reductive elimination of this complex gives the 1 2 adduct having 2,6-octadienyl chain. In the usual telomerization of the nucleophiles, the reaction of butadiene is not stepwise and the bis-n--allylic complex 20 is formed, from which the l, 6-octadienyl chain is liberated. 

More recently, a new and straightforward one-pot approach was reported by Bu et al. <2003JOC5415>. This reaction involves the cyclocondensation of l,2-dipyridin-2-yl-ethane-l,2-dione 120 and arylaldehydes, in the presence of ammonium acetate. Imidazo[l,5- ]pyridines 121 were obtained in reasonable yields, but competitive formation of imidazoles 122 was observed. The amount of ammonium acetate used in this reaction was also shown to strongly influence the yield of the cyclization (Scheme 38). 

Methods for the A-acylation of similar heterocycles, such as simple thiazolidinethiones, have been reported since 1977, namely acyl chlorides in miscellaneous conditions,586 or carboxylic acids under DCC-activation.60,61 However the easiest and most effective method involves acyl chlorides or carboxylic anhydrides in the presence of an amine.47 Applying that procedure on carbohydrate scaffolds Rollin and co-workers62 reported the synthesis of diverse /V-acylated OZTs. The reactions were performed with good yields and the /V-selective acylation was ascertained by NMR— namely the thiocarbonyl 13C chemical shift (Scheme 41). Thanks to the dual nature of the carbanion drifting in the reaction,596 60 no competitive formation of the thioester, as mentioned by Plusquellec el al. in the case of benzothiazole, was observed. 

The method outlined here competes well with the method developed earlier by Danheiser, et al.618 Its superiority is based on the fact that phenyl ester enolates give almost the same results as the S-phenyl thiolester enolates. However, handling the malodorous benzenethiol for the preparation of the active acid derivative and during workup of the p-lactone can be avoided. In addition, phenol is much cheaper than benzenethiol. The method is well suited for the preparation of p-lactones from symmetrical and unsymmetrical ketones. In addition to 3,3-dimethyM-oxaspiro[3.5]nonan-2-one, ( )-3-ethyl-1-oxaspiro[3.5]nonan-2-one and (3R, 4R )- and (3R, 4S )-4-isopropyl-4-methyl-3-octyl-2-oxetanone were prepared by this procedure in high yields (Notes 11 and 12). In the case of unsymmetrical ketones the less sterically crowded diasteroisomer is formed preferentially. With aldehydes as the carbonyl component the yields are unsatisfactory, because of the competitive formation of 1,3-dioxan-4-ones.6... 

Bis(pyridinium)iodonium tetrafluoroborate [I(Py)2BF4] reacts readily with alkenes to afford 1,2-disubstituted products arising from addition of iodine and pyridine. Synthetically more important is, however, the reaction of unsaturated systems with I(Py)2BF4 in the presence of nucleophiles, which provides a general method for vicinal iodofunction-alization of alkenes. In this regard, the addition of a stoichiometric amount of tetrafluo-roboric acid to the reaction medium is often required to avoid the competitive formation of products resulting from pyridine acting as a nucleophile. 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

Recently, a novel immunoassay has been developed for the quantitative determination of polybrominated biphenyls using indirect competitive format. The new method was optimized concenung the coating conjugate and antibody concentration, incubation time and temperature, the tolerance to organic solvents and so on. Under optimized conditions, PBB15 can be determined in the concentration range of 0.01-100 pg/L with a detection hmit of 0.02 pg/L. The cross-reactivities of the assays were below 8%. While water samples could be analyzed directly [94]. 

The competitive formation of the silyloxytelluride detected during the progress of the reaction, and of the silylated TEMPO formed by adding the radical scavenger TEMPO, suggests the following reaction pathway ... 

The major problems with industrial-scale applications of this procedure are the use of expensive reagents, the required chromatographic separation of the side products and the competitive formation of oxazolines. The use of carbamate protecting groups avoided the oxazoline problem and is usually preferred, since the resulting protected 3-amino-substituted /3-lactams 150 can later be deprotected and reacylated. 

Bi(0Tf)3xH20 has been found to catalyze carbonyl-Diels-Alder reactions in water involving glyoxylic acid as the dienophile (Scheme 14) [72]. In contrast to other Lewis acids (such as Sn(OTf)2, Yb(OTf)3, Nd(OTf)3, Ce(OTf)3 and Sc(OTf)3), the strong catalytic power of Bi(0Tf)3-xH20 made it necessary to reduce its amount as well as the reaction temperature. Bi(0Tf)3-xH20 enhanced the reaction rate but with some dienes the reaction led to competitive formation of the ene reaction product. 

Scheme 2.2 Competitive formation of alkenyl-vinylidene vs. allenylidene group.

The competitive formation of dinudear complexes containing halide bridging systems has also been reported (representative examples are shown in Figure 2.5) [21]. 

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