Enhanced Assay Formats

Reactions performed in a true homogeneous solution are expected to follow the law of mass action. In such cases, the binding reactions would reach equilibrium 

This study suggests that by increasing the concentration of the reactants in 

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A reduction in cutinase production should result in the PNB-1 mutant being less virulent. The pathogenesis of the two strains were evaluated in a pea stem bioassay developed by Kolattukudy and coworkers in which infection by Fusarium solani results in wound formation within three days on the epicotyl of pea sedlings (15). The virulence of T-8 had previously been shown to be reduced in this assay by the addition of inhibitors of cutinase or by rabbit anticutinase antibodies (15-18), indicating that cutinase played an important role in pathogenesis. When the cutinase-defective mutant was evaluated in the bioassay, the mutant exhibited a 55% reduction (p < 0.05) in virulence compared with the T-8 parental strain and the addition of purified cutinase at 1 mg/ml to the mutant enhanced wound formation to 80% of that of the parent (p > 0.5). These data further support the notion that the mutant was defective in cutinase. 

The use and variety of modifications that result in enhanced functionality has rapidly grown in line with the development of novel assay formats and methodologies. In general, these modifications are aimed at providing new signal generation or transduction mechanisms, sensitivity gains, or as... 

Immunoassays are inherently sensitive and specific. However, with continued need to develop increasingly sensitive assays to support preclinical and clinical studies, there have been ongoing efforts to enhance the capabilities of these techniques. Advances in critical binding reagents, detection systems, new assay formats and automation have resulted in improved immunoassay technology. 

Areas in which technological innovations have been accomplished include detection instruments [ranging from small portable luminometers (M3) to camera-type detectors (B32, K30) and charge-coupled devices (L3)], data analysis (E3, SI2), and assay formatting, e.g., immunofiltration (HI6). The so-called camera luminometers (B32) offer the joint advantages of ease of use, portability, and detectability without an electrical power supply. With fast film such as Polaroid 612 (20,000 ASA), exposure times can be as short as 5 sec. Examples of assays using enhanced chemiluminescence and Polaroid 612 film include a-fetoprotein (L9), albumin (L8), immunoglobulins and transferrin (J3), and ferritin (07). 

Several different assay formats, as already mentioned earlier, can generally be used such as ELISA, fluoroimmunoassay (FIA), dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), RIA, and SPR. For the quantification of proteins in biological matrices such as blood, plasma, serum or urine, most often FLISAs are validated. 

Since the mid 1970 s many different structures and classes of lanthanide chelates have been synthesised (Rgure 5). For high sensitivity assays the dissociation enhanced system is currently the best approach, however for screening assays where the sensitivity or detection limitation is not an issue the fluorescent chelates can be used to simplify the assay. Some of these chelates can be even used to develop homogeneous and non-separation assays. This assay format is essential for the measurement of a reaction where the components have only a weak binding affinity thus cannot withstand a wash or separation step. 

On this basis, the dissociation-enhanced lanthanide fluorescence immunoassay was developed. Mostly a two-site assay format is used whereby antibodies are immobilized at microtiter plates and after applying the sample antigen, europium-chelate-labeled antibodies bind to the sample. A commonly used chelating reagent is Nl-(p-isothiocyanato-benzyl)-diethylene triamine. After the addition of a S-diketone as a dissociation enhancer, at acidic pH, europium is released and fluorescence of Eu is measured. Other TRFIAs use stabilized lanthanide chelates or stable chelates. 

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