Eastman-kodak Company

Annie L Wu

John Bruce Findlay

Richard C Sutton

Harold C Warren

Robert T Belly

Susan J Danielson

Brian A Snyder

Brent A Burdick

Gregory J McClune

Gary E Norton

Patricia M Scensny

Chung-yuan Chen

A target nucleic acid can be amplified and detected using a water-insoluble capture probe having a short oligonucleotide (15-20 nucleotides) covalently attached to a polymeric particle, and one or more secondary oligonucleotides. The capture probe and secondary oligonucleotides are complementary to the target nucleic acid, although the sequences of complementarity are different. Moreover, each of the secondary oligonucleotides has a length which is equal to or greater than the length of the capture probe oligonucleotide. The secondary oligonucleotides are hybridized to the target nucleic acid either before or during hybridization of the target to the capture probe. The result is improved hybridization efficiency of capture probe and target nucleic acid.

Detection of amplified nucleic acid using secondary capture

A biotinylated target nucleic acid can be isolated from a mixture of nucleic acids using a capture reagent comprising avidin. The targeted nucleic acid is complexed with avidin on a substrate and separated from uncomplexed materials. The complex is heated at above about 65.degree. C. for a suitable time to cleave the avidin-biotin bonds to release the desired targeted nucleic acid. Upon release, the targeted nucleic acid can be collected or detected in a suitable manner, such as with a complementary probe. This method is also useful for preparing single-stranded DNA for a number of uses.

Preparation and isolation of single-stranded biotinylated nucleic acids

Reagents have been prepared from water-insoluble polymeric particles to which are covalently attached avidin, biotin or an avidin or biotin derivative. The polymeric particles comprise a polymer on at least the outer surface which is derived from at least one ethylenically unsaturated monomer having a reactive activated 2-substituted ethylsulfonyl, vinylsulfonyl or active halogen atom. Covalent attachment of avidin, biotin or an avidin or biotin derivative is effected either directly or indirectly through these reactive groups. The resulting reagent is useful in analytical elements and various analytical methods including agglutination and sandwich assays. The immobilized avidin, biotin or derivative can be used to complex with the corresponding biotin or avidin molecule which may be conjugated to a compound of biological interest.

Avidin-and biotin immobilized reagents and methods of use

A specific binding immunoassay method for reducing the 'hook' effect for the measurement of C-Reactive Protein has been discovered for both solution and dry analytical elements comprising contacting a liquid sample containing C-reactive protein in the presence of calcium ions with (a) a first antibody Ab1 specific for C-reactive protein, Ab1 being immobilized on a water-insoluble substrate and (b) a labeled, unbound second antibody Ab2 specific for C-reactive protein to obtain a water-insoluble complex of Ab1, ligand, and Ab2; (2) separating the water-insoluble complex from the liquid sample and unreacted Ab2; and (3) measuring either the amount of Ab2 associated with said water-insoluble complex or the amount of unreacted Ab2 as an indication of the amount of C-reactive protein in the sample.

Use of calcium in immunoassay for measurement of C-reactive protein

Hydrolyzable substrates comprise blocked moieties which, when cleaved from the substrate during hydrolysis, provide electron transfer agents. The released electron transfer agents can be recycled between a reductant and a reducible compound that upon reduction provides a detectable species. Alternatively, they can be recycled between an oxidant and an oxidizable compound that upon oxidation provides a detectable species. These substrates are useful in analytical compositions, elements and methods for the determination of hydrolytic analytes, such as hydrolytic enzymes or biological cells containing such enzymes.

Hydrolyzable compounds which release electron transfer agents and

The interference of dehydroascorbic acid, an oxidation product of ascorbic acid, in analytical methods is reduced with the use of certain nitrogen-containing organic buffers. Dehydroascorbic acid tends to cause premature reduction of reducible compounds which are used to provide detectable species in the presence of an analyte. These organic buffers, as opposed to inorganic buffers, prevent premature reduction of the reducible compounds. An analytical method can be carried out in solution or in a dry analytical element.

Use of organic buffers to reduce dehydroascorbic acid interference in

Polymeric mordants are used with certain rigid fluorescent dyes or precursors thereof to increase fluorescence intensity. Compositions and analytical elements containing the mordants and dyes or dye precursors are useful in biological studies, such as cell and tissue staining and cell cytometry, and in analytical determinations for various analytes, e.g. living organisms.

Use of polymeric mordants to increase the intensity of rigid fluorescent

A multilayer analytical element for the determination of creatine kinase comprises a support having thereon, in order and in fluid contact, a registration layer and an isotropically porous spreading layer. The registration layer contains a binder material at a coverage of at least about 8 g/m.sup.2, which coverage improves the stability and keeping properties of the element in high humidity environments. This element is useful in an analytical method for determining total creatine kinase or any one of its isoenzymes, e.g. creatine kinase-MB.

Determination of total creatine kinase or an isoenzyme with a multilayer

A method for preparing a blush polymer coating composition containing an immunologically reative species comprises milling the species and the other materials used in the composition to uniformly disperse the species therein. This coating composition can be used to prepare analytical elements for determining analytes (e.g. creating kinase-MB) whereby the effects of potential interferents are immunochemically removed. By milling the immunologically reactive species, e.g. antisera, into the coating composition, the resulting element has improved stability resulting in improved keeping in high humidity environments.

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Penfield, NY, United States of America

Annie L Wu