Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples.
ABSTRACT Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.
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ABSTRACT: A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.Journal of Histochemistry and Cytochemistry 07/1998; 46(6):771-7. · 2.26 Impact Factor
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ABSTRACT: The biotinyl-tyramide protocol recently introduced for sensitive light microscopic immunocytochemistry was applied to electron microscopy and revealed various tissue antigens with high resolution. The protocol consists of an indirect method in which thin tissue sections are incubated successively within a specific primary antibody, followed by a biotinylated secondary antibody, streptavidin-HRP, and then finally with biotinyl-tyramide. The reaction product appears as a dense filamentous material that is deposited over particular cellular compartments. The labeling obtained for the antigens tested, amylase and heat-shock protein 70 in pancreatic acinar cells, insulin in pancreatic beta-cells, and carbamoyl phosphate synthetase and catalase in liver tissue, was found to be highly specific, with the labeling for each antigen confined to its particular cellular compartment. Background levels and nonspecific deposition of the staining were negligible. The use of biotinyl-tyramide therefore appears to be an alternative sensitive technique for immunoelectron microscopy.Journal of Histochemistry and Cytochemistry 12/1997; 45(11):1449-54. · 2.26 Impact Factor
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ABSTRACT: To evaluate the occurrence and clinical role of active parvovirus B19 infection in solid organ and bone marrow transplant recipients, 256 serum samples from 212 transplant patients were investigated retrospectively by competitive polymerase chain reaction. Sera were drawn during the transplantation period and up to 6 months after transplantation during a nonepidemic 1-year period. Three patients were found positive for B19 DNA; only one liver transplant patient had a clinically overt B19 infection. Overall, the rate of active parvovirus B19 infection in transplant subjects was low (1.42%), probably due to the high number of actively or passively immunized subjects among transplant recipients; this may also account for the asymptomatic infections observed.European Journal of Clinical Microbiology 11/1999; 18(11):811-3. · 3.02 Impact Factor