Reverse capture assays

Fig. 8.5. Reverse capture assays allow an improvement of detectability by a decrease in background staining. Two oligomers (labeled and polyadenylated) are hybridized with the target (I, II) and captured with paramagnetic beads (PMB) covered with oligo-dT hairs (III). Nonspecifically adsorbed oligomers are usually tighter bound than hybrids to dT-hairs, allowing a selective desorption of specific hybrids (IV). These can be recaptured (V), followed by the determination of the amount of label bound.

Fig. 8.6. Sensitivity and detectability depend on various factors. In example I (Thompson and Gillespie, 1987), the sensitivity is 1. Increasing the probe concentration does not improve sensitivity but deteriorates the detectability. In another example (van Gijlswijk et al., 1992), the sensitivity in a hybridization assay, using POase-catalyzed luminol reaction, was similar in the one-step and three-step methods but the detectability improved for the latter. Similarly, in reverse capture assays detectability improves after one or a few cycles while sensitivity decreases only slightly. In example II (Oser and Valet, 1988), simple adjustments in the (time-resolved fluorescence) procedure improved the detectability somewhat but the sensitivity increased about 100-fold for the well-strip method.

Several of the previously described methods are readily adapted for the purification of specific mRNAs. Most convenient is the system based on the use of paramagnetic beads (Table 3.16) in which the oligo(dT) should be replaced by specific oligomers. Alternatively, the (reverse) capture or sandwich assays described in Section 8.3 are well suited for hybrid selection. 

The reverse capture autoantibody microarray is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA) (see Fig. 1). The basic platform consists of a glass microarray slide arrayed with 1000 highly specific well-characterized monoclonal antibodies against 500 unique antigens. These antibodies are used to immobilize native proteins. Because the reagents used in the procedure are non-denaturing, antigens are... 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

A prominent advantage of this assay procedure is the feature that the complex of hapten and labeled antibody was captured on a solid phase (PMP) and separated from the reaction medium before signal determination. This additional step not only reduces interference due to biological specimens but also eliminates the tedious transfer of supernatant, which is essential in conventional immunometric assays. This immunometric assay provided somewhat improved specificity in terms of the cross-reactivities with T2 and reverse T3 (3,3, 5 -L-triiodothyronine). The authors speculated that the dissociation rate of the antibody-cross-reactant complex would be faster than that of an antibody-analyte complex thus the former binding would be preferentially substimted by T2 immobilized on CPG. 

In contrast to sandwich immunoassays, in which a capture antibody is immobilized on a substrate ( forward phase arrays ), in the so-called reverse phase protein arrays the complex mixture of analytes is immobilized on a substrate. The biological sample could be a cell lysate or from another source. Each spot then comprises the entire proteome of a biological system at a certain state or after a certain treatment. Each array contains a multitude of biological states. Several copies of such arrays are produced and each one is probed with a single antibody. Thus, each assay shows, in one experiment, the expression levels of a particular protein in all the different biological states represented on the array. Since the entire proteome is contained in each spot, essentially any protein can be analyzed in the context of different experimental foci. 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

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