A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct f luorescence detection with capillary electrophoresis, or the DNA͞PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.The ability to rapidly identify specific sequences in DNA samples is becoming more important as links between phenotypes and genotypes are established. Two areas in which the correlation between phenotype and genotype is becoming increasingly well understood are oncology and genetic disorders (1-3). Rapid analysis methods for the presence or absence of a specific sequence will further help establish correlations between genotypes and phenotypes. Moreover, the rapidly expanding database of sequence information will lead to increased needs for genetic analysis methods in areas such as studies of gene expression, pathogen detection, and diagnostics.Southern hybridization (4) is a technique often used to analyze genetic loci, using fragments of defined length derived from either PCR or restriction enzyme digestion. Southern hybridization derives its great utility from the ability to provide both size and sequence information. Sequence detection is supplemented with information regarding the genetic context. While Southern hybridization is a core technology in molecular biology, it has disadvantages that limit its application for screening large numbers of DNA samples. The Southern technique is a laborious multistep procedure, and sequence discrimination between closely related sequences is often difficult to obtain on a routine basis.In our continued studies of the properties of peptide nucleic acid (PNA) (5, 6), a DNA mimic with a neutral peptide-like backbone, we have been working with labeled PNA oligomers in hybridization procedures. The hybridization of PNA oligomers to complementary DNA is essentially independent of the ionic strength due to the neutral backbone of PNA. Thus a PNA oligomer will hybridize to a complementary DNA oligomer under conditions where DNA͞DNA hybridization is strongly disfavored, i.e., at low ionic strength (6). Also, since the backbone is neutral, the electrophoretic mobility of PNA oligomers will be determined by their size, and the charge (if any) contributed by the label. As such, they will generally migrate much more slowly than DNA in an electric field (6-8).In an attempt to dramatically simplify the ...

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