Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
  • G01N33/521—Single-layer analytical elements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

• This invention relates to an analytical element useful in determining an analyte or analytes in a sample, as well as apparatus containing this element. It also relates to the process of making said elements and apparatus, as well as methods of using these.
• Dry chemistry is a field which has expanded dramatically in recent years. Broadly stated, the field relates to the use of dry reagents, such as test strips, film strips, and so forth, for the analysis of liquid samples.
• test strips frequently involves the detection of a reaction between the analyte, or substance being determined, with some material which has been incorporated into the dry reagent or test strip, or the detection of a further reaction which can occur only when the first one occurs.
• the former include "diffusion assays” , such as those taught by Deutsch et al., U.S. Patent No. 4,235,601, where one determines formation of a reaction complex by its slower movement through a test strip, as compared to unreacted materials.
• Such tests are not as common as the latter type, however, which include such systems as "IEMA” assays, displacement or competition assays, and so forth.
• a complex may form, but it is not the complex formation that is determined; rather the complex which forms is between the analyte being determined and a binding partner, which either carries a reactive label or causes the release of a reactive label. This label then reacts with its own reaction partner to form a detectable substance, such as a color.
• a detectable substance such as a color.
• Yet another object of this invention is to provide a method for using the apparatus. How these and other aspects of the invention are accomplished will be seen from the disclosure which follows.
• compositions containing an integral array of an indicator system and a reactive system can be prepared, which does not undergo reaction prior to contact with a sample contained in a carrier solvent in which both the reactive system and the indicator system are soluble.
• the composition is prepared by incorporating one of the two systems, generally, but not exclusively the reactive system, into a carrier in which the system is insoluble and which prevents interaction between these systems.
• This mixture, slurry, or other formulation is combined with the indicator system. Any liquid carrier in the mixture is drawn off, by evaporation, or other means, to produce an integral array containing both the indicator system and the reactive system.
• the insoluble carrier may remain a part of the integral array. If it does so, it is chosen so that it is inert with respect to the different systems.
• indicator system any of the various reagents known to the art which are progenitors of substances which give detectable signals when reacted with other components of a test system. Generally, these are enzyme substrates which, when cleaved by an enzyme, produce a color. Other types of indicators may include, e.g., fluorescent systems.
• the indicator system should not be thought to be limited to substrates alone; by system is meant one or more components which are involved in a reaction.
• a first component may be, e.g., a substrate, and a second component, an enzyme.
• the substrate, in unreacted form may not react with the enzyme in the indicator system, but require reaction with some other substance to form a substrate which can react with the enzyme.
• This type of system is elaborated upon infra.
• the reactive system comprises a reagent or reagents which act in concert with the indicator system to form either a detectable substance or some product which is an intermediate prior to formation of the detectable signal.
• the reactive system can be nothing more than an enzyme which reacts with a substrate in an indicator system. It can also be, however, a substance which is necessary to activate one or more components of the indicator system, or a necessary reagent for activation of the reagent system. For example, if the indicator system contains a portion of an enzyme, the reactive system may contain the other portion. When the two are brought together, a complete, active enzyme forms which can then react with a substrate molecule.
• the reactive system contains a substance which is necessary to complete a color forming reagent.
• the indicator system may contain a colorless enzyme substrate system which, after reaction, forms a second substrate which is also colorless.
• the second substrate may react with the reactive component to form a colored product where the first one cannot.
• the reactive component requires an enzyme to carry out the color forming step of the reaction, one has an ideal opportunity to incorporate the test system into an analytical apparatus. If an enzyme is required to carry out the reaction of reactive system and indicator system product, then one can incorporate the second enzyme, in the form of a labeled analyte binding partner in. a test apparatus which also contains a test element as described supra.
• the manner in which the analytical element is formed calls for an integral disposition or array of both reactive and indicator systems.
• "Integral array” simply means that the components are disposed in a manner such that they are contacted at the same time by the sample being analyzed.
• the systems can be in a single layer uniformly distributed throughout, or in, apparently separable layers which are in physical contact with each other.
• the actual form of the integral array is not an essential feature of the invention. What is important is that they be configured such that reaction between the two does not take place until desired, i.e., by contact with the sample.
• this carrier substance is water soluble.
• the insoluble carrier precludes any reaction taking place, because both reaction system and indicator system must be in solution for the reaction to take place. This does not happen until the sample contacts the integral array.
• the analytical element integral array can include other materials.
• it may include a soluble matrix, such as a soluble polymer, which facilitates entry into the solution phase once the mutual solvent sample reaches the element.
• a particularly desirable embodiment also includes an inert material, such as titanium dioxide. The Ti0 2 does not react with any of the components of the element or test apparatus, but alters the optical properties of the matrix in which it is incorporated, thus rendering the signal more easily observable.
• the reactive system contains a first component which reacts with a second component which is either a part of the indicator system or some other part of the analytical element. These two components form a reaction product which then reacts with a third component, which is part of the reactive system, leading to formation of a detectable signal.
• a peroxidase substrate as the reaction product, where the reactive system contains a peroxidase and a peroxide source.
• the manner of producing the element will vary, and there is nothing critical about the manner in which it is formed.
• a carrier such as a fleece, filter paper, or film
• one of the systems such as the indicator systems
• the second system such as the reactive system
• Any liquid vehicle is then removed, such as by evaporation, and an analytical element containing the integral array which is desired remains.
• the analytical element can be sized, cut, and incorporated into a test strip in the same way as any other component is incorporated into a test strip. Those skilled in the art are familiar with such procedures, and they will not be repeated here.
• One preferred system for use in test systems involves the indicator system 1-naphthol- ⁇ -galactopyranoside and p-galactosidase, and the reactive system peroxidase and sodium perborate.
• the naphthol galactoside is colorless, and, upon reaction with B-galactosidase, releases 1-naphthol, which is also colorless.
• This product reacts with an oxidant, e.g., NaBO-, catalyzed by a peroxidase, to form a blue colored product.
• the problem results from the instability of peroxidase when co-impregnated with the oxidant, so that they cannot both be impregnated into reagent paper.
• the following composition overcomes this problem.
• a mixture of sodium perborate and TiO- was combined in a pulverizer for 10 minutes. 50 g of NaBO, was used for each 7.5 gm of TiO-. Following pulverization and mixing, a 2.5 g aliquot of the mixture was added to 8 ml of a 5% solution of Klucel ⁇ in acetone. Klucel is a registered trademark (Hercules) for a cellulose ether polymer.
• a substrate paper was prepared by impregnating a piece of Schleicher & Schuell 3512 paper with 10 mg/ml of 1-naphthol-p-galactopyranoside in methanol, and allowed to dry. This paper was then impregnated with 100 ⁇ g/ml of horseradish peroxidase in 10 mmol/L Na,B0, and allowed to dry. The Klucel mixture was applied to the substrate paper and allowed to dry.
• the element was tested for reaction by applying 25 ul of a 1 ⁇ g/mL solution of p-galactosidase.
• the formation of a blue color indicated the reaction of the substrate with the ⁇ -galactosidase to release 1-naphthol, which subsequently reacted with peroxidase and NaBO- to produce a blue dye.
• Test strips were prepared containing the element described supra. Briefly, the test strip contained, in this embodiment, a first part, which was a wick of Whatman 54 filter paper (0.6 by 2.0 cm) for introduction of sample. This wick contacted a conjugate pad (0.6 x 0.6 cm of filter paper) containing 0.16 U of a monoclonal antibody to phenobarbital conjugated to B-galactosidase. The conjugate zone contacted a matrix (0.6 x 2.0 cm of filter paper) which contained 24 ⁇ g of a phenobarbital- protein conjugate immobilized to the paper. This matrix contacted the analytical element described supra.

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• 1989
  • 1989-02-14 EP EP19890902994 patent/EP0432155A4/en not_active Withdrawn
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