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«SPECIFICATION PATENT EUROPEAN ® ® Date of publication of patent spécification: 08.01.86 (fï) Int. Cl.4: G 01 N 3 3 / 5 4 3 (H) Application ...»

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Europâisches P a t e n t a m t

à European Patent Office @ Publication number:


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® Date of publication of patent spécification: 08.01.86 (fï) Int. Cl.4: G 01 N 3 3 / 5 4 3 (H) Application number: 80303405.7 (S) Date offiling: 26.09.80 (H) Method for analysis for a member of an immunological pair using a test surface; kit and test surface material therefor.

® Proprietor: SYVA COMPANY (§) Priority: 26.12.79 US 106620 3181 Porter Drive Palo Alto, CA 94304 (US) (43) Date of publication of application:

22.07.81 Bulletin 81/29 (72) Inventor: Litman, DavidJay 7401 Rollingdell Drive Cupertino California (US) (§) Publication of the grant of the patent:

Inventor: Ullman, Edwin Fisher 08.01.86 Bulletin 86/02 135 Selby Lane Atherton California (US) (B) Designated Contracting States:


(7?) Representative: Harrison, David Christopher etal MEWBURN ELLIS & CO 2/3 Cursitor Street (§) References cited:

London EC4A1BQ(GB) EP-A-0 017908 GB-A-2 019 562 GB-A-2021262 US-A-3839153 (0 CM CM C0 Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall CL be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1 ) European patent convention).

m Courier Press, Leamington Spa, England.

There is continuing interest in developing new, simpler and more rapid techniques to measure the presence of an analyte in a sample suspected of containing an analyte. The analyte may be any of a wide variety of materials, such as drugs, naturally occurring physiological compounds, polutants, fine chemicals, contaminants, or the like. In many cases, speed is important for the measurement, particularly with certain physiologically active compounds. In other situations, convenience can be a major consideration.

One convenient technique which has found wide application is the use of a "dip stick." Having a solid rod orfilm which can be dipped in a sample and then subsequently processed to produce a signal based on the amount of analyte in the original sample can provide many conveniences. There is ample instrumentation to measure a signal, such as light absorption or fluorescence, of a compound bound to a solid surface. Also, the dip stick allows for convenient handling, transfers, separations, and the like.

In developing an assay, it is desirable that there be a minimum number of steps and transfers in performing the assay, as well as a minimum number of separate reagents. Therefore, while a dip stick adds a convenience to separations, the separations in themselves are undesirable. Furthermore, the fewer the reagents that have to be packaged, added, and formulated, the fewer the errors which will be introduced into the assay and the greater economies and convenience of the assay.

It is therefore desirable to develop new assay methods, particularly employing surfaces which may or may not be separated from the assay medium for measurement, where the signal may be developed without concern as to the presence of reagents in the solution affecting the observed signal on the surface.

Patents concerned with various immobilized reagents in different types of test strips include U.S.

Patent Nos. 3,993,451; 4,038,485; 4,046,514; 4,129,417; 4,133,639; and 4,160,008, and DE-A-2,636,244.

Patents disclosing a variety of methods involving separations of bound and unbound antigen include U.S.

Patent Nos. Re. 29,169; 3,949,064; 3,984,533; 3,985,867; 4,020,151; 4,039,652; 4,067,959*; 4,108,972;

4,145,406; and 4,168,146*.

GB-A-2019562 discloses an essentially homogeneous assay in which a signal is associated with a large number of particles which are dispersed throughout the assay medium, thereby providing the signal at a uniform level throughout the liquid medium. The purpose of the particles is to provide thereon a microenvironment for locally enhancing the rate of production of signal. In contrast, the present invention seeks to provide the signal at a surface which is distinctfrom the assay medium, for example on a dip-stick.

According to one aspect of the present invention there is provided a method for detecting.the presence of an analyte in a sample suspected of containing said analyte, where said analyte is a member of a immunological pair (mip) consisting of ligand and homologous antiligand;

said method involving the partitioning of a catalyst bound to a mip ("catalyst-bound-mip") between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; characterised in that said surface is a non-dispersed surface which retains a discrete existence in the assay medium and is discernible from the medium, the method further involving the change in concentration of a signal generating compound associated with said surface, said change in concentration being related to the amount of reaction product produced by said catalyst bound to said surface; by

a) combining in an aqueous assay medium,

1) said sample;

2) mip-bound-surface;

3) catalyst-bound-mip; and

4) the remaining members of the signal producing system, which system includes at least one catalyst including said catalyst-bound-mip, and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation, inhibition or destruction of a signal generating compound associated with said surface and capable of producing a detectable signal on said surface which is distinct from any signal produced in the bulk solution, with the proviso that said signal producing system is completed not later than about the time of addition of said catalyst-bound-mip to said surface, when said signal producing system consists essentially of said catalyst-bound-mip and its substrates;

b) waiting a sufficient time for at least a portion of said catalyst-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; and

c) determining the amount of detectable signal at said surface as a function of the presence of analyte in said sample.

The invention also provides an analytical test device comprising a bibulous base having one or more enzymatic or non-enzymatic catalysts covalently bound thereto and a member of an immunological pair bound thereto.

The invention further provides a kit for use in an immunoassay comprising in combination a member of an immunological pair and a catalyst or a precursor to a dye bound to an insoluble surface, an enzyme ('Patents of particular interest) bound to a member of an immunological pair, and a leuco dye which undergoes an enzymatically catalyzed reaction to produce an insoluble dye capable of binding to said surface.

A method is provided employing a relatively rigid insoluble, preferably bibulous, surface to which is conjugated a member of an immunological pair (abbreviated as "mip") the immunological pair consisting of ligand and a receptor which specifically binds to the ligand or their functional equivalent for the purposes of this invention. In addition to the surface, a signal producing system is provided which has as one member a catalyst, normally an enzyme, which is conjugated to a mip. Depending upon the amount of analyte present, the catalyst labeled mip will be partitioned between the bulk solution of the assay medium and the surface. The signal producing system provides a signal generating compound at the surface which generates a signal which is not significantly affected by any signal generating compound produced or present in the bulk solution. Therefore, the signal generating compound may be generated in the assay medium in the presence of unbound catalyst labeled mip. When the only catalyst in the signal producing system is the catqlyst-labeled-mip, various expedients can be employed to enhance the difference in the rate of formation of the signal generating compound at the surface as compared to the bulk solution, e.g.

enhancing the catalyst turnover rate at the surface. In addition to enhance the simplicity of this protocol, the last of the components of the signal generating system will be added at about the time of or before the addition of the catalyst bound to the mip.

Compositions are provided for performing the assay comprising combinations of the surface and various reagents in relative amounts for optimizing the sensitivity and accuracy of the assay.

The subject assay provides for a convenient method for detecting and measuring a wide variety of analytes in a simple, efficient, reproducible manner, which can employ visual inspection or conventional equipment for measuring a spectrophotometric property of a product bound to a surface.

In accordance with the subject invention, an assay method and compositions are provided for measuring a wide variety of analytes, where the analyte is a member of an immunological pair (mip), the pair consisting of a ligand and a receptor (antiligand) which specifically binds to the ligand, or their functional equivalent for the purposes of the assay. The assay method has two essential elements: a surface to which is conjugated a mip; and a signal producing system which results in a signal generating compound associated with the surface, producing a detectable signal in an amount related to the amount of analyte in the assay medium. Preferably, the signal producing system will effect a two or more step conversion involving one or more compounds to produce, block or destroy the signal generating compound, where the rate of change in the concentration of the signal generating compound is related to the average distance between two molecules on the surface. The molecules may be the same or different.

The immunological binding at the surface allows for localized enhanced concentrations of compounds of the signal producing system at the surface. Also, one may employ a scavenger as a third component which acts to inhibit the operation at the signal producing system in the bulk solution by scavenging an intermediate, catalyst or signal generating compound in the bulk solution.

The surface may be any convenient structure which substantially retains its form and may be separable from or part of the container. The manner of binding of the mip to the surface is not a critical aspect of this invention, so long as a sufficient amount of the mip is exposed to allow for binding to its homologous partner.

The signal producing system has at least two members: A catalyst, normally an enzyme, conjugated to a mip; and a solute which undergoes a reaction with a substance bound to the surface, and thereby directly or indirectly enhances or inhibits the production of a detectable signal. The association of a member of the signal producing system with the surface may be as a result of insolubilization, complexation with a compound on the surface or interaction, including reaction, with a compound on the surface.

Where an intermediate material is produced by the signal producing system in soluble form, both in the bulk solution and at the surface, a scavenger can advantageously be employed, so as to substantially minimize the interaction of the intermediate material produced in the bulk solution with the surface.

A wide variety of different systems may be employed for altering the degree of production of the product at the surface as compared to the bulk solution and for inhibiting intermediates or product produced in or migrating into the bulk solution from interacting with the surface. Depending upon the particular protocols, various additions, incubation steps, and reagents will be employed.

By providing for the production of a detectable signal generating material on the surface that is related to the amount of analyte in a sample, one can relate the signal level detected from the surface to the amount of analyte in the solution. By employing standards having known amounts of analyte under the same or substantially the same conditions as with an unknown, one can quantitate the detected signal level with the amount of analyte in the sample.

In accordance with the subject invention, the method is performed without requiring a separation of bound and unbound catalyst-bound-mip, nor requiring a separation of analyte from the remainder of the sample, although the latter may be desirable. This provides substantial advantages in the convenience of the protocol and in avoiding the difficulties in achieving a clean separation.

The subject invention achieves a precise, specific and sensitive technique for detecting and measuring ligands and ligand receptors. The method provides for the preferential production, inhibition of production or destruction of a compound at a rigid surface, which compound is involved with the generation of a signal at the surface. The signal generating compound associated with the surface will be of a sufficient depth on or in the surface to provide a measurable signal.

For a large number of analytes, the concentration range of interest will fall between 100 pg to one pg ml. For many analytes, the concentration range of interest will vary from about two-fold to 100-fold so per that a quantitative determination will require the ability to distinguish small differences in the concentration of the analyte in the assay medium. Immunoassays are predicated on detecting the complexation between ligand and receptor, where one or both may be labeled. The lower the concentration of the analyte, the fewer the number of complexes which are formed. Therefore, in order to be able to accurately determine the number of labeled complexes which are formed, either the label must provide a signal which can be efficiently counted at an extremely low level of events, e.g. radioactive emission, or the complex must permit amplification or multiplication, e.g. fluorescence or a catalyzed reaction.

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