Solution assay formats

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay 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. 

A buffered stock solution of the complex was prespotted on the tab. The dry tab with preimmobilized E5-Ab complex was then utilized to perform the assay. b At the time of assay a buffered stock solution of the complex was spotted on the tab either by itself or as a mixture with the analyte depending on the specific assay format. 

In nearly all cases studied, the amount of primary antibody required in the E5-Ab complex to perform an assay has been found to be substantially less than that required for the double antibody immune complex format. This was found to be the case (Table 19.1) when the E5-Ab complex was either directly immobilized on the solid phase, to imitate the double-antibody immune complex format, or utilized in a solution phase format [12],... 

Heterogeneous fluorescence immunoassays have many different assay formats, but all possess one unifying feature the free fluorophore remaining in solution after binding of some of the fluorophore to antibody must be removed before quantification can be achieved. Again, both negative and positive reading assay formats have been employed. 

In this pull-down assay, the enzymatic reaction is carried out completely in solution. Samples taken from the reaction mixture are then transferred to a SAM-modified MALDI target, on which the remaining substrate and the reaction product are selectively immobilized. Subsequent to the extraction of the analytes, the target is rinsed, treated with matrix, and MALDI-MS analysis is carried out. A major advantage of this assay scheme is that the inherent danger of negative influences on the reaction kinetics, which may be caused by immobilization of the substrate as in standard SAMDI-MS-based assay formats, is circumvented. Additionally, by selective extraction of the analytes of interest and removal of the other... 

Figure 8.1 Assay formats in BIA can be performed in a solid phase (A) or in solution-based (B-D) assay format. Applications are given in the figure. For details see text.

It should be noted, however, that mixture-based hbraries are not limited to the solution state, but have also been screened while bound to a sohd support, such as plastic pins,P cellulose membranes,or resin beads, This assay format is favorable for direct-binding assays involving soluble acceptors. 

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. 

This assay format is well suited to the screening of test compounds for potential antiviral activity. The assay provides a minimal assessment of antiviral activity by measuring the levels of HBV virion release from the cells, as well as providing a measurement of cytotoxicity (see Subheading 3.2.2.), Two rows of cells will be required for each compound, plus four rows for the assay controls (two for untreated, and two for positive antiviral control (e.g., 3TC). After treating for 9 d, the media are harvested from the antiviral plates and transferred to 96-well U-bottomed plates. They are then centrifuged, and supernatant is transferred to tubes for dot-blot hybridization analysis of HBV virion DNA. The medium is aspirated off of the toxicity plates and discarded. Toxicity plates are then incubated with neutral red dye (methylthiouracil [MTT] can also be used if preferred), washed with DPBS, developed with an acetic acid/ ethanol solution, and assayed in a plate reader. 

These requirements have lead to the following analytical solutions. Assays for low amounts of compounds must typically have highly sensitive detection schemes. Rapid throughput often relies on robotic methods. The large number of compounds has led to parallel high-density analysis formats, such as 96-well plates. To handle the large amounts of data, special software, databases, and visualization methods have been developed. 

Beside solution assays, AMPPD and AMPGD can be used for detection of nucleic acids on membranes since the AMP D anion is hydrophobic and will attach to the membrane in some fashion in the direct vicinity upon formation. This allows its application in assays where resolution is required such as Northern and Southern hybridization. 

---