Analytical methods hybridization assays

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

Matthews, J A., Batki, A., Hynds, C., and Kricka, L. J. (1985) Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays. Analytical Biochem 151,205-209... 

The most significant limitation of immunochemical methods is inadequate sensitivity such that preamplification by PCR is required. Further improvements in solid phases, reporter enzymes, substrates, and hybridization probes will extend the range of analytes amenable to assay without amplification. Solid phases designed specifically for DNA immunoassays are needed. Enzyme engineering for thermostability. 

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. 

Only limited development of new methodologies has taken place for immunochemical analysis of nucleic acids. Most published methods rely on modifications to classical DNA probe hybridization or immunoassay methods, with considerable blending of the two. For example, some methods employ immobilized oligonucleotide probes to capture the analyte DNA followed by immunoenzymatic detection. Other methods use immunocapture followed by detection with an enzyme-labeled DNA probe. Distinctly new methodologies mostly impact on assay formats (e.g., DNA microarrays and in situ hybridization) and detection reagents (e.g., chemiluminescent enzyme substrates). 

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