Heterobifunctional cross-linkers containing 4,9-dioxa-1,12-dodecanediamine spacers.

A series of heterobifunctional linker arms has been prepared by functionalization of (tert-butoxycarbonyl)-4,9-dioxa-1,12-dodecanediamine [tBOC-HN(CH2)3 O(CH2)4 O(CH2)3 NH2] with anhydrides or acid chloride.

Monoadduct forming photochemical reagents for labeling nucleic acids for hybridization.

We describe the synthesis of three angelicin derivatives which can be used for labeling nucleic acids with biotin. These compounds were used to label nucleic acids in the presence of lysed cell constituents. The resulting labelled nucleic acids show hybridization to a genus specific probe for E. coli. The relative comparison of sensitivity indicates that a polyamine linker is better than a polyethylene oxide linker between the biotin and angelicin moieties.

Rapid identification of microorganisms by nucleic acid hybridization after labeling the test sample.

A novel method for rapidly identifying microorganisms has been developed. This method employs a monoadduct-forming furocoumarin derivative, which can photochemically label nucleic acids. The labeled nucleic acid can, in turn, be hybridized simultaneously to a panel of immobilized probe DNAs arrayed as dots on a solid support such as nitrocellulose. This procedure offers several advantages over more conventional hybridization techniques in that sample nucleic acids can be photolabeled without substantial sample preparation and that identification can be achieved by a single, rapid hybridization reaction.

Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses.

New dip-and-read test for determining leukocytes in urine.

A new reagent strip for the determination of leukocytes in urine (LEUKOSTIX; Ames) is described. The test is based on the esterase activity in leukocytes as a marker. Upon contact between the reagent matrix and a urine containing leukocytes, an amino acid ester is hydrolyzed by the esterase to its corresponding alcohol. The free alcohol then couples with a diazonium salt to produce a purple azo dye. The relative concentration of leukocytes in the urine is obtained by visually comparing the strip reaction with a color chart. Performance of the strip was evaluated in a clinical study involving eight different sites and 867 urine specimens. The comparison method was sediment microscopy; specimens containing five cells or more per high-power field were considered to be positive. Sensitivity was 76.3%, specificity 80.8%. Performance was comparable with that of the CHEMSTRIP LN (Boehringer-Mannheim Diagnostics, Inc.) leukocyte test, which we evaluated concurrently.

A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

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.

A colorimetric immunoassay based on an enzyme inhibitor method.

A competitive binding immunoassay was developed using an enzyme inhibitor for labeling the analyte. Thyroxine was labeled by covalent coupling of the alpha-amino group to the gamma-carboxyl of the glutamyl residue of methotrexate. This thyroxine-methotrexate conjugate was a potent inhibitor of dihydrofolate reductase. When antibody was bound to the thyroxine moiety, the inhibitor was inactivated. Thus, a competitive binding immunoassay for thyroxine was demonstrated based on colorimetric measurement of dihydrofolate reductase activity.

Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format.

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.