Hybridization 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). 

The analytical sensitivities of the different quantitation methods have been compared using serial dilutions of patients specimens (Butterworth et al., 1996) and the Eurohep HBV DNA standards (Zaaijer et al., 1994). In both cases, bDNA was shown to be about 10-fold more sensitive than the liquid hybridization (Abbott) and the hybrid capture (Digene) assays. Using the Eurohep HB V standards, the detection limits were 2.5 X 106 genomes/ml for bDNA and 2.5 X 107 genomes/ml for both liquid hybridization (LH) and hybrid capture (HC) assays. 

Amagliani, G., Omiccioli, E., del Campo, A., Bruce, I. ]., Brandi, G., and Magnani, M. (2006). Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples. /. Appl. Microbiol. 100, 375-383. 

Mazzulli T, Drew L, Yen-Lieberman B, Jekic-McMuUen D, Kohn DJ, Isada CM, et al. Multicenter comparison of the Digene Hybrid Capture CMV DNA assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia. J Clin Microbiol 1999 37 958-63. 

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.

Immunoassays for nucleic acids usually employ a solid phase on which the analyte is immobilized either before or after hybridization. Two general configurations, immunocapture or hybridization capture (enzyme-linked oligonucleotide-sorbent assay or ELOSA), are possible (Figure 1). Careful design and optimization of each component can yield a high-performance assay. Typical components for a microwell PCR immunoassay are provided in Table 1. 

Probes that hybridize to the target and also to either preamplifier or amplifier molecules are termed label extenders. The locations of the capture and label extender probes used in the hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV-1 assays are shown in Figs. 3,4, and 5, respectively. All target probes are designed to hybridize to the most conserved regions of the genomes. For HBV, the... 

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

All DNA hybridization assays are subject to cross-hybridization, in which an oligonucleotide that is not a perfect sequence match hybridizes with the capture probe. The cross-hybridization in the OFRR was investigated using samples of oligonucleotides with either 0-, 1-, 2-, 5-, or 25-base mismatches when compared with the 25 base-pair biorecognition capture probe. The resulting resonant mode spectral shifts for the respective mismatch are plotted in Fig. 14.7b. The measurements show a difference of 1.3 pm and 2.8 pm for one and two base-pair... 

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