ABSTRACT
An enzyme-labeled immunometric assay has been developed for measuring digoxin concentrations in serum or plasma. Unitized, compartmentalized reagents are used with an automated sample-processing instrument. The enzyme activity of the processed sample, which is directly proportional to the digoxin concentration, is measured by using a reagent strip and the Ames Seralyzer reflectance photometer. The test takes less than 15 min, and digoxin concentrations are calculated from a two-point calibration line stored in the instrument. Within-run CVs for controls at four concentrations ranged from 2.3% to 3.8%; between-run CVs were from 1.5% to 2.6%. Results obtained with clinical serum samples correlated well (r greater than 0.96) with those obtained by fluorescent polarization immunoassay (Abbott TDx) and RIA (Clinical Assays and NML). This rapid and convenient method for monitoring digoxin concentrations in serum or plasma is particularly well suited for decentralized sites such as emergency rooms, urgent-care centers, and physicians' offices.
Subject(s)
Digoxin/blood , Antibodies, Monoclonal , Autoanalysis , Cross Reactions , Digoxin/standards , Humans , Immunoenzyme Techniques , Photometry , Reagent Strips , Time Factors , beta-GalactosidaseABSTRACT
Preparation of F(ab')2 fragments from mouse monoclonal IgG by papain digestion can result in incorporation of traces of papain into antibody fragments by disulfide exchange. Such trace contamination can have detrimental effects on the integrity of these antibody fragments following reduction. Gel filtration and ion exchange chromatography procedures did not eliminate papain contamination from F(ab')2 preparations. The use of antibody specific for papain to remove this contamination from F(ab')2 preparations is described.
Subject(s)
Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin G/isolation & purification , Animals , Antibodies, Monoclonal/analysis , Disulfides , Mice , Molecular Weight , Papain/analysisABSTRACT
Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.
Subject(s)
Biotin , DNA , Escherichia coli/analysis , Immunoenzyme Techniques , Nucleic Acid Hybridization , RNA, Bacterial/analysis , RNA, Ribosomal, 23S/analysis , RNA, Ribosomal/analysis , Animals , Avidin/metabolism , Bacteriological Techniques , Biotin/metabolism , DNA/analysis , Leukocytes/analysis , RNA, Bacterial/urineABSTRACT
Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.
Subject(s)
Antibodies, Monoclonal/immunology , DNA/immunology , Nucleic Acid Hybridization , RNA/immunology , Animals , Antibody Specificity , DNA, Ribosomal/immunology , Escherichia coli , Immunoassay/methods , Immunosorbent Techniques , Mice , RNA, Ribosomal/immunologySubject(s)
Oligonucleotides/analysis , RNA, Transfer/analysis , Saccharomyces cerevisiae/analysis , Ammonium Sulfate , Base Sequence , Bromine , Chromatography , Chromatography, Thin Layer , Coliphages/enzymology , Countercurrent Distribution , Iodine , Lysine , Nucleosides/analysis , Oxidation-Reduction , Pancreas/enzymology , Pseudouridine/analysis , Quaternary Ammonium Compounds , RNA, Transfer/isolation & purification , Ribonucleases , Spectrophotometry, Ultraviolet , Thiouridine/analysisSubject(s)
Nucleotides/analysis , Oligonucleotides/analysis , RNA, Transfer/analysis , Saccharomyces cerevisiae/analysis , Base Sequence , Chemical Phenomena , Chemistry , Chromatography, DEAE-Cellulose , Coliphages/enzymology , Lysine , Models, Structural , Oligonucleotides/isolation & purification , Pancreas/enzymology , RNA, Messenger , RibonucleasesABSTRACT
The nucleotide sequence of one of the two major lysine transfer RNA's from bakers' yeast has been determined. Its structure is compared to that of a lysine tRNA from a haploid yeast. A total of 21 nucleotides differ in the two molecules. Only the T-psi-C-G (thymidine-pseudouridine-cytidine-guanosine) loop and its supporting stem are identical.
