nitrophenols and 3-(2--spiroadamantane)-4-methoxy-4-(3---phosphoryloxy)phenyl-1-2-dioxetane

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Topics: Adamantane; Bacterial Proteins; Biotin; Deoxyuracil Nucleotides; Digoxigenin; DNA, Bacterial; Enzymes, Immobilized; Hymecromone; Indicators and Reagents; Listeria monocytogenes; Nitrophenols; Organophosphorus Compounds; Polymerase Chain Reaction; Sensitivity and Specificity; Streptavidin; Substrate Specificity; Thymine Nucleotides

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.

Topics: Adamantane; Alkaline Phosphatase; Base Sequence; DNA Probes; Fluorescent Dyes; Hymecromone; Membranes, Artificial; Molecular Sequence Data; Nitrophenols; Nucleic Acid Hybridization; Organophosphorus Compounds

This communication presents four different assay systems for the determination of myeloperoxidase in body fluids. One is based on conventional chemiluminescence, two on luminescence-amplified enzyme measurement using either spiroadamantane-1,2-dioxetanes with alkaline phosphatase or luminol/peroxidase/4-iodophenol coupled with a peroxidase label. The assays covered the range 0-600 micrograms/l peroxidase. Established reference range for EDTA plasma from healthy volunteers gave an upper limit of 250 micrograms/l (95% confidence limits). Intra-assay coefficients of variation were less than 5% for all assays, as seen in compound precision profiles. Inter-assay variation was less than 10% throughout the whole concentration range. Kinetic curves for the light production were performed over a 2 hour period for the enhanced luminescence assays. Dynamic ranges of these assays were compared with the conventional colorimetric assay using 4-nitrophenyl phosphate--alkaline phosphatase. The assay was standardized using a commercially available myeloperoxidase preparation with defined enzymatic activity. The protein content was estimated and the laboratory standard compared and calibrated in mass units.

Topics: Adamantane; Alkaline Phosphatase; Animals; Cattle; Colorimetry; Immunoassay; Immunoenzyme Techniques; Iodobenzenes; Luminescent Measurements; Luminol; Microchemistry; Nitrophenols; Organophosphorus Compounds; Peroxidase; Peroxides; Phthalazines; Polystyrenes; Reference Values