This preliminary study was performed to prove the feasibility of a direct capacitive DNA biosensor for detection of nucleic acids. Two different methods for immobilization of the oligonucleotide probes were used. The ®rst type of sensor was composed of a gold rod with a self-assembled monolayer of a 26-base long oligonucleotide probe, modi®ed with an SH-group at the 5 H -end. Coverage studies showed that only around 20% of the surface was covered, probably due to the bulky nature of the probes. Hybridization studies performed in a¯ow-through cell showed selectivity towards a DNA sample containing single stranded fragments of cytomegalo virus (CMV) possessing a complementary sequence. As few as 25 molecules could be detected at sample concentrations of 0.2 attomolar with an injection volume of 250 mL. Controls with fragments of double-stranded CMV and single-stranded hepatitis B virus and tyrosinase mRNA gave all lower responses. The other type of sensor was modi®ed by covalent immobilization of a phosphorylated 8-base long oligonucleotide probe to a self-assembled monolayer of cysteamine. This biosensor also showed selectivity against single stranded fragments of CMV and also in this case as few as 25 molecules could be detected.

SinaryA novel quantitative ruminometric immunoassay (LIA)

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In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 μg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340–365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311–335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.