Capture assays

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

Tremaine LM, Joerg FA. 1989. Automated gas chromatography-electron-capture assay for the selective serotonin uptake blocker sertraline. J Chromatogr B 496 423. 

Rabin DU, Pakner-Crocker R, Mierz DV, Yeung KK. (1992) An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase. J Immunol Methods >6, 101-5. 

Plates for antibody-capture assays Where a specific antibody is available, this may be a better assay to use than step 8 above, because the antigen is less likely to be subject to denaturation... 

Cussac D, Newman-Tancredi A, Duqueyroix D, Pasteau V, Millan MJ. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays influence of receptor reserve and relationship to agonist-directed trafficking. Mol Pharmacol 2002 62 578-589. 

Newman-Tancredi A, Cussac D, Marini L, Millan MJ. Antibody capture assay reveals bell-shaped concentration-response isotherms for h5-HTlA receptor-mediated G alpha(i3) activation conformational selection by high-efficacy agonists, and relationship to trafficking of receptor signaling. Mol Pharmacol 2002 62 590-601. 

The assay data system should also manage and capture assay metadata including biological and pharmacological categories, technical parameters related to assay method and endpoints, project-related information, and other data relevant to each assay. Metadata vocabulary should be managed by... 

The HRE peptide was shown to minerahze a total of seven nanoclusters, ZnS, Au , Ag°, Pt°, Cu , Ti02, and Ag2S. All nanoparticles were formed by mixing appropriate ratios of metal to peptide to form a metal peptide precursor complex. To this solution, sulfide or reductant was added to form the appropriate encapsulated metal nanocluster. Condensation of titanium isopropoxide in the presence of HRE was used for synthesis of Ti02-(HRE) nanoparticles. Once formed, all of the nanoclusters were characterized using UV-vis, IR, powder XRD, transmission electron microscopy, and an antigen capture assay,... 

ELISA is also of limited application when the antigen is extremely diluted, such that other molecules interfere with the performance of the technique. One approach in this case is the so-called substrate-capture ELISA . Here, a substrate for a protease, for example, is bound to the plastic plate in order to absorb and concentrate the specific enzyme for detection by immune reaction. The first modification of a standard capture assay technique in which a metalloprotease substrate is used to capture the enzyme of interest was described by Wacher et al. in 1990, and is summarized in generalized form below. The substrate-capture ELISA greatly simplifies the mixture in which the enzyme is detected and removes potentially interfering substances, thus avoiding some of the difficulties... 

The main difference between the capture and the sandwich assays is how the label probe-target complex is immobilized on a solid phase. In capture assays, hybridization with the immobilized capture probe determines the kinetics and specificity/detectability/sensitivity characteristics of the assay. In sandwich assays, an immobilized molecule, which is not a nucleic acid (e.g., streptavidin, antibodies), serves to capture the hapten probe-target-label probe ternary complex (thus both probes need to be modified). The affinity matrix-hapten interaction thus determines the kinetics (usually 3-10 times faster than solid phase hybridization), the sensitivity and detectability... 

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.

Antibodies to DNA RNA, DNA DNA and RNA RNA hybrids have been described (Section 7.4.2) (Stollar and Stollar, 1970 Rudkin and Stollar, 1976 Pisetsky and Coster, 1982). Monoclonal antibodies to unusual nucleic acids have also been reported (Lee et al., 1989) as well as against RNA DNA hybrids (Bogulawski et al., 1986). In capture assays, with DNA capture probe linked to nylon beads, hybrids resulting from annealing of the RNA target with the capture probe can be detected with the specific antibody. 

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.

Direct capture assays are the most common format used in piezoelectric immunosensors. Primarily, because its simplistic format exploits the label-free advantages of piezoelectric transducers. 

---