CA1179262A - Homogeneous specific binding assay element and method for preventing premature reaction - Google Patents

Abstract

ABSTRACT

A homogeneous specific binding assay element, a method for its preparation, and a method for its use in determining a ligand in or the ligand binding capacity of a liquid sample are disclosed. The test element comprises a solid carrier incorporated with reagents for a homogeneous specific binding assay system which produces a detectable response, usually an electromagnetic radiation signal, that is a func-tion of the presence or amount of the ligand in or the ligand binding capacity of the sample. There is further disclosed a method for preparing a homo-geneous specific binding assay element for deter-mining a ligand in or the ligand binding capacity of a liquid sample by incorporating a carrier with a composition which includes a label conjugate, com-prising a label component coupled to a ligand moiety or a specific binding analog thereof, and a reagent reactive with the label conjugate, which method comprises (a) incorporating the carrier with the reagent reactive with the label conjugate in a first liquid and drying the carrier; and then (b) incorporating the carrier of (a) with the label conjugate in a liquid effective to prevent reaction with the reagent reactive with the label conjugate prior to contact of the element with the sample and drying the carrier.

Description

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~OMOGENEOUS SPECIFIC BINDING ASSAY ELEMENT
AND METHOD FOR PREVENTING PREMATURE REACTION

BACKGROUND OF THE INVENTION

1. FIE~D OF THE INVENTION

This invention relates to test devices or elements, their preparation and their use in determining a ligand in or the ligand binding capacity of a liquid sample based on a specific binding assay, e.g., immunoassay, principle. In particular, this inven-tion relates to solid state carrier elements incor-porated with homogeneous specific binding assay reagents.

2. BRIEF DESCRIPl'ION OF THE PRIOR ART

Test devices in the form of test strips and --similar solid state analytical elements have become commonplace in the analysis of various types of samples, particuarly biological fluids. Test strips designed for detecting clinically significant sub-stances in biological fluids, such as serum and urine, have been advan~ageous in the diagnosis of disease.
.

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Test strips of various types have been known and used for many years in a wide variety of fields, from the most familiar pH test paper devices to in vitro diagnostic devices for the detection of various urine 5 and blood components such as glucose, protein, occult blood and so forth (e.g., as described in U.S. Patents Nos. 3,164,534; 3,485,587; and 3,012,976). Reagent compositions found in such conventional test strips interact with the constituent or constituents to be 10 determined by direct chemical reaction and, for this and other reasons, have limited sensitivity, being applied to the detection of substances that are present in liquid samples at concentrations in the millimolar range or above.
On the other hand, the development of specific binding assay techniques has provided useful analy-tical methods for determining various organic sub-stances of diagnostic, medical, envirsnmental and industrial importance which appear in liquid mediums 20 at very low concentrations. Specific binding assays are based on the specific interaction between the ligand, i.e., the bindable analyte under determina-tion, and a binding partner therefor. Where one of the ligand and its binding partner is an antibody and 25 the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In conventional specific binding assay techn-iques, a sample of the liquid medium to be assayed is combined with various reagent compositions. Such 30 compositions include a label conjugate comprising a binding component incorporated with a label. The binding component in the label conjugate participates with other constituents, if any, of the reagent composition and with the ligand in the medium under 35 assay. This forms a binding reaction system in which 11~9Z6~

two species, a bound-species and a free-species, of the label conjugate are formed. In the bound-species, the binding component of the label conjugate is bound by a corresponding binding partner e.g., an antibody, whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the label conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand to be detected in the test sample.
Where the label conjugate in the bound-species is essentially indistinguishable in the presence of the label conjugate in the free-species by the means used to monitor the label, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to in the art as "heterogeneous". Where the bound-species and free-species forms of the label conjugate can be distinguished in the presence of each other, the separation s~ep can be avoided, and the assay is said to be "homogeneous".
The first discovered type of highly sensitive speci~ic binding assay was the radioimmunoassay which employs a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials and the necessity of a separation step, homogeneous assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacterio-phages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme activators and inhibitors, cycling reactants, organic and inorganic catalysts, 9~6~

prosthetic groups, chemiluminescent reactants, and fluorescent molecules. Such homogeneous specific binding assay systems provide a detectable response, e.g., an electromagnetic radiation signal, such as chemiluminescence, fluorescence emission, or color change, realted to the present of amount of the ligand under assay in the liquid sample.
Commercially available test means for performing specific binding assays are usually in the form of test kits comprising a packaged combination of con-tainers holding solutions or rehydratable compositions of the reagents necessary for carying out the assay.
To perform the actual assay method, aliquots of such solutions must be manually or instrumentally dispensed into a reaction vessel with the sample. If manually dispensed, the assay consequently requires the time and skill of a technician, and if instrumentally dis-pensed, the assay consequently requires the expense and maintenance of dispensing apparatus.
Solid phase test devices have been applied to heterogeneous specific binding assays in attempts to overcome the inconveniences and disadvantages of the requisite separation step. A commonly used solid phase device of this type comprises a nonporous surface, such as the interior surface of a test tube or other vessel, to which antibody is affixed or coated by adsorption or covalent coupling. U.S.
Patents Nos. 3,826,619; 4,001,583; 4,017,597; and 4,105,410 relate to the use of antibody coated test tubes in radioimmunoassays. Solid phase test devices have also been used in heterogeneous enzyme immuno-assays ~U.S. Patents Nos. 4,016,043 and 4,147,752) and in heterogeneous fluorescent immunoassays (U.S.
Patents Nos. 4,025,310 and 4,056,724; and British Patent Spec. No. 1,552,374).

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The use of such heterogeneous specific binding assay test devices is exemplified by the method of U.S. Patent No. 4,135,884 relating to a so-called "gamma stick". The test device is incorporated with the antibody reagent and is brought into contact with the liquid sample and with the remaining reagents of the reaction system, principally the label conjugate.
After an incubation period, the solid phase device is physically removed from the reaction solution and the label measured either in the solution or on the test device.
Similar devices where the antibody reagent is entrapped in a matrix such as a gel or paper web are described in U.S. Patents Nos. 3,925,017; 3,970,429;
4,138,474; 3,966,897; 3,981,981 and 3,888,629 and in German OLS 2,241,646. Likewise, devices for use in hetergeneous specific binding assays wherein the antibody reagent is fixed to a matrix held in a flow-through column are know (U.S. Patents Nos. 4,C36,947;
4,039,652; 4,059,684; 4,153,675; and 4,166,102). The test device is usually incorporated with less than all of the necessary reagents for carrying out the assay and is merely a means for rendering more con-venient the necessary separation step.
Finally, heterogeneous specific binding assay test devices have been described wherein most or all of the necessary reagents are incorporated with the same carrier element, and wherein reagent/sample contacts and separation of the free- and bound-phases are accomplished by capillary migrations along the carrier element (U.S. Patents Nos. 3,641,235; 4,094,647 and 4,168,146). The devices described in such patents are generally considered difficult to manufacture and susceptible to irreproducibility due to the complex 1 1~7 nature of the many chemical and physical interactions that take place along the carrier element during performance of an assay.
The application of homogeneous specific binding 5 assay reagent systems to solid state test devices would provide great advantages to the routine user of such assay systems. The determination of ligands appearing in very low concentrations in liquid samples would be simplified to the steps of contacting the device with the sample and measuring, either by visual observation or by instrumental means, the resulting signal. Reagents would be provided in a solid form, with no need to store, dispense or mix liquid reagents as required when using the prior art test kits. Solid state devices would also be much more adaptable to automation than the prior art liquid systems.
The prior art lacXs a detailed teaching of how to apply homogeneous specific binding assay reagent systems to solid state test devices. British Patent Spec. No. 1,552,607, commonly assigned herewith, describes homogeneous specific binding assay systems employing various novel labels, including chemilumi-nescent labels, enzyme substrate labels and coenzyme labels. At page 23, lines 12 et seq of this patent there is the suggestion of incorporating the assay reagents with various carriers including liquid-holding vessels or insoluble, porous, and preferably absorbent, matrices, fleeces, or blocks; gels; and the like.
German OLS 2,537,275 describes a homogeneous specific binding assay reagent system and poses the possibility of using slides or strips incorporated with antibody in performing the assay. In this suggestion, the label conjugate would be first mixed with the sample and thereafter the antibody incorporated 1~7~'~6'~

test device contacted with the reaction mixture.
After a suitable incubation time, it is proposed that the test device would be rinsed with buffer, dried, and then the signal (fluorescence) measured. Thus, this German OLS poses a test device and assay method much like those already known for heterogeneous specific binding assay techniques wherein the test device is immersed in the liquid reaction mixture, incubated, thereafter removed, washed, and finally read. Additionally, the proposed test device does not incorporate all of the binding assay reagents with the carrier element. Specifically, only the antibody is proposed to be incorporated with the carrier element with the label conjugate being sep-arately added to the sample under assay prior to contact with the proposed test device.
U.S. Patent No. 4,447,526 discloses a method for determining the presence of a ligand in, or the ligand binding capacity of, a liquid test sample com-prises the steps of adding to said liquid sample a label conjugate comprising said ligand, or a binding analogue thereof, chemically bound to a label, con-tacting said sample with a test device comprising a carrier matrix incorporated with reagents which, when combined with said label conjugate, produce a homogen-eous specific binding assay system which produces a detectable response which is a function of the presence of said ligand or said ligand binding capacity, thereby producing said response, and measuring said response.
Copending Canadian patent application No. 381,675, filed on July 14, 1981 and commonly assigned herewith, discloses a homogeneous specific binding assay device, a method for its preparation, and a method for its use '"' -L3 1 il7~Z6Z

in determining a ligand in, or the ligand binding capacity of, a liquid sample. This includes, for example, a test device for determining a ligand in or the ligand binding capacity oE a liquid sample, comprising (a) reagents for a homogeneous specific binding assay system which produces a detectable response that is a function of the presence of the ligand in or the ligand binding capacity of the sample, and (b) a solid carrier member incorporated with said reagents.
Copending Canadian patent application No. 400, 764, filed on April 8, 1982 and commonly assigned herewith discloses a homogeneous specific binding assay device for use in determining a ligand in a liquid sample, comprising (a) a reagent composition including a complex of (i) a label conjugate com-prising a label component coupled to said ligand or a specific binding analogue thereof, and (ii) a specific binding partner for said ligand, said label providing a detectable response, or interacting with a detectant system to provide a detectable response, which is different when the label conjugate is bound by said binding partner compared to when it is not so bound and (b) a carrier incorporated with said complex.

SUMMARY OF THE INVENTION

The present invention provides a homogeneous specific binding assay test element, a method for its preparation, and a method for its use in determining a ligand in or the ligand binding capacity of a liquid sample. The test element comprises (a) reagents for a homogeneous specific binding assay system which produces a detectable response that is a function of the presence, in a qualitative or quanti-tative sense, of the ligand in or the ligand binding 1~79~iZ
g capacity of the liquid sample under assay, and (b) a solid carrier incorporated with the reagents. Par-ticularly, the invention provides a method for pre-paring a homogeneous specific binding assay element for determining a ligand in or the ligand binding capacity of a liquid sample by incorporating a car-rier with a composition which includes a label con-jugate, comprising a label component coupled to a ligand moiety or a specific binding analogue thereof, and a reagent reactive with the label conjugate, which method comprises (a) incorporating the carrier with the reagent reactive with the label conjugate in a first liquid and drying the carrier; and then (b) incorporating the carrier of (a) with the label conjugate in a liquid effective to prevent its reaction with the reagent reactive with the label component prior to contact of the element with the sample and drying the carrier.
By precisely following the steps of the above defined method it is possible to incorporate all the reagents necessary for a specific binding assay into an integral single layer element.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. la is a schematic of the prosthetic group label immunoassay as described on pages 12 and 13.
Fig. lb is a schematic of the substrate-labeled immunofluorescent assay as described on pages 13 to 15.
Fig. lc is a schematic of the quenching immuno-fluorescent assay as described on pages 15 and 16.
Fig. 2 is a schematic of the conjugate prepara-tion in Example I.
Fig. 3 is a graph of data from Example I.
Fig. 4 is a graph of data from Example II.
Fig. 5 is a graph of data from Example III.

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- 9a -Fig. 6 is a schematic of the conjugate preparation in Example IV.
Fig. 7 is a graph of data from Example IV.
Fig. 8 is a schematic of the conjugate preparation in Example V.
Fig. 9 is a graph of data from Example V.
Fig. 10 is a schematic of the conjugate preparation in Example VI.
Fig. 11 is a graph of data from Example VI.
Fig. 12 is a graph of data from Example VII.
Fig. 13 is a graph of data from Example VIII.
Fig. 14 is a graph of data from Example IX.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a test device for use in carrying out homogeneous specific binding assays, e.g., immunoassays, having all of the con-venience features of conventional analytical test 1~'7~

strips and other test elements of similar design. As in the case of such conventional devices, the present invention provides a solid carrier, usually a matrix of one sort or another, incorporated with all of the reagents necessary to perform a given assay whereby the user has only the task of bringing the test device into contact with the sample to be tested and measuring the resulting response. Where the entire process is automated, an instrument for performing the same manipulations can have a much simpler design than one having to deal with conventional liquid chemistry systems now used for performing homogeneous specific binding assays.

1. HOMOGENEOUS SPECIFIC ~INDING ASSAYS

Reagents for any homogeneous specific binding assay system may be incorporated in the present test device. In general, homogeneous specific binding assay techniques are based on the special interaction between (1) a conjugate of a binding component and a label and (2) a binding partner to the binding com-ponent in the conjugate, whereby a characteristic of the label is different when the label conjugate is bound by the binding partner compared to when such conjugate is not so bound. The affected charac-teristic of the label may be of any measurable nature,for instance, a chemical or physical quality of the label. In some cases, the affected characteristic is a chemical reactivity in a predetermined reaction which involves the formation or breaking of chemical bonds, covalent or noncovalent. In other cases, the affected characteristics is a physical characteristic of the label which can be measured without chemical reaction.

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In the majority of cases, the present test device will incorporate homogeneous specific binding assay reagents which respond to the ligand or its binding capacity in the sample in an immunochemical manner. That is, there will be an antigen-antibody or hapten-antibody relationship between reagents and/or the ligand or its binding capacity in the sample.
Such assays there-fore are termed immunoassays and the special interaction between the label conjugate and its binding partner is an immunochemical binding.
Thus, in such instances, the binding component of the label conjugate is an antigen, hapten or antibody (or a fragment thereof) and the binding partner is its corresponding immunochemical binding partner. How-ever, it is well understood in the art that otherbinding interactions between the label conjugate and the binding partner serve as the basis of homogeneous specific binding assays, including the binding inter-actions between hormones, vitamins, metabolites, and pharmacological agents, and their respective receptors and binding substances.
Where the sample is being assayed to determine the presence or amount of a particular ligand therein, the reagents for the homo~eneous specific binding assay technique comprise, in the usual case, (1) a label conjugate composed of the ligand, or a binding analog thereof, chemically coupled to the label, (2) a binding partner for the ligand, e.g., an antibody or fragment thereof, a natural receptor protein, and the like, and (3) any ancillary reagents necessary for measuring the labeling substance in the label con-jugate. A limiting amount of the binding substance is introduced so that any ligand in the sample will compete with the label conjugate for binding to the binding partner. The distribution of the label 117~Z~;Z

between the bound-species and -the free-species will therefore determine the magnitude of the detectable response from the label, which in turn will be a function of the presence of the ligand. Another scheme for determining a ligand is presented where the label conjugate is composed of a labeled binding-partner of the ligand and, upon binding to the ligand, the label is affected in terms of its detectable response. Where ligand binding capacity of the sample is under assay, the label conjugate will be composed of the ligand, or a binding analog thereof, chemically coupled to the label whereby the capacity of the sample to bind the label conjugate, such as due to the presence of a binding partner of the ligand in the sample, detérmines the effect made on the detectable signal from the label.
Several different homogeneous specific binding assay systems are known in the art, and the following are examples, without limiting the scope of the pre-sent invention, of some such systems contemplated foruse in the present test device. The following systems are listed according to the nature of the label used.

(a) Enzyme Prosthet~c Group LabeZs In this system, where the label is a prosthetic group of an enzyme, the ability of a catalytically inactive apoenzyme to combine with the prosthetic group label to form an active enzyme (holoenzyme) is affected by binding of the label conjugate with its binding partner. Resulting holoenzyme activity is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in commonly assigned, copending application Serial No. 45,423, filed June .. . . . . .. . . . . . . .. . . . .

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4, 1979 (corresponding to published British Patent Spec. No. 2,023,607). A particularly preferred prosthetic group-label assay scheme employs flavin adenine dinucleotide (FAD) as the label and apo-glucose oxidase as the apoenzyme. Resulting glucoseoxidase activity is measurable by a colorimetric detectant system comprising glucose, peroxidase, and an indicator system which produces a color change in response to hydrogen peroxide.
In such preferred assay scheme, the FAD-labeled conjugate is preferably of the formula:
NH R-L

<N ~N
Riboflavin (Phos ~ Ribose wherein Riboflavin-~Phos ~ Ribose represents the riboflavin-pyrophosphate-ribose residue in FAD, R is a linking groupS and L is the binding component, e.g., the ligand or analog thereof. A schematic representation of this type of assay is shown in Fig.
la of the drawings.

(b) E?~zyme Substrate ~abeZs In this system, the label is selected so that the label conjugate is a substrate for an enzyme and the ability of the enzyme to act on the substrate-label conjugate is affected, either in a positive or negative sense, by binding of the label conjugate with its binding partner. Action of the enzyme on the substrate-label con~ugate produces a product that is distinguishable in some feature, usually a chemical or physical feature such as chemical reactivity in an ~ 1 7~

indicator reaction or such as a photometric character, e.g., fluorescence or light absorption (color).
Assay systems of this type are described in commonly assigned, copending application Serial No. 894,836, filed April 10, 1~78 (corresponding to published German OLS 2,618,511) and U.S. patent No. 4,279,992, and in AnaZ. Chem. ~8:1933 (1976), AnaZ. Biochem.
77:55 (1977) and CZin. Chem. 23:1402 (1977). A
particularly preferred substrate-label assay scheme lC employs a label conjugate of the structure R-L

wherein R is a linking group and L is the binding component, e.g., the ligand or analogue thereof, whereby the ability of the enzyme ~-galactosidase to cleave the conjugate yielding a product distin-guishable by its fluorescence is inhibited by bind-ing of the conjugate with its binding partner.
An important application of this technique is in aminoglycoside antibiotic assays wherein the binding component is the antibiotic under assay or a binding analogue thereof. A schematic representation of the principles of this type of homogeneous immuno-assay for a drug is shown in Figure lb of the drawings.
In assays where antibody is used as the binding partner it has been found that other aminoglycoside antibiotics can cross-react with the antibody for the antibiotic under assay. Thus, such other antibiotics qualify as binding analogues and may be used to form the label conjugate. Further, the antibody qualifies as a reagent for use in assays for the cross-reacting ~ ~....

antibiotic. For example, in an assay for gentamicin it has been found that, with an appropriate antiserum, the binding component in the label conjugate can be gentamicin itself or sisomicin which cross-reacts.
Thus, gentamicin antiserum and a labeled sisomicin conjugate could be used in an assay for gentamicin.
Specificity problems are not encountered in clinical situations because it would be known what antibiotic was adminstered and only one aminoglycoside anti-biotic is administered at a time.
The ~-galactosyl-umbelliferone-label conjugates formed are of the formula:

~ ~ R - L

wherein R is a linking group terminating in an amino-linking group, preferably carbonyl; L is an amino-glycoside antibiotic coupled by a covalent bond tothe linking group R through a primary amino group therein; and n is an integer from 1 to the total number of primary amino groups in the selected anti-biotic.

~) Coenzyme label s The labsl conjugate in this system is composed in its label portion, by a coenzyme-active function-ality. The ability of the coenzyme label to par-ticipate in an enzymatic reaction is affected by ~7~6~

binding of the label conjugate with its binding partner. The rate of the resulting enzymatic reaction is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in commonly assigned, copending application Serial No. 894,836, filed April 10, 1978 (corresponding to published German OLS
2,618,511); and in AnaZ. Bioehem. 72:271 (1976), AnaZ. Biochem. 72:283 (1976) and AnaZ. Bioehem. 76:95 (1976).

(d) Enzyme ModuZator LabeZs The label conjugate in this system is composed, in its label portion, of an enzyme modulating func-tionality such as an enzyme inhibitor or stimulator.
The ability of the modulator label to modulate the activity of an enzyme is affected by binding of the label conjugate with its binding partner. The rate of the resulting enzymatic reaction is measurable by conventional detectant systems to yield an ultimately detectable signal. Assay systems of this type are described in commonly owned U.S. Patent No. 4,134,792.

(e) ~nzyme Labe Z6 In this system, the label is an enzyme and the activity of the enzyme label is affected by binding of the label conjugate with its binding partner.
Resulting enzyme activity is measurable by conven-tional detectant systems to yield an ultimately detectable signal. Assay systems of this ~ype are described in U.S. Patents Nos. 3,817,837 and 4,043,872.

1~7~3Z62 (f) QuenchabZe FZuorescent LabeZs The label conjugate in this system is composed in its label portion, of a fluor the fluorescence of which is quenched in some measurable degree when the label conjugate is bound by its binding partner, usually a protein such as an antibody. The fluores-cent label is measured directly, with its fluores-cence being the detectable signal. Assay systems of this type are described in U.S. Patents Nos. 4,160,016 and in J. CZin. Path. 30:526 (1977). A schematic representation of the principles of this type of assay is shown in Fig. lc of the drawings.

(g) ChemicaZZy-E~cited FZuorescent ~abeZs In this system, the label is again a fluor, however, the ability of the fluor label to be chemi-cally excited to an energy state at which it fluoresces is affected by binding of the label conjugate with its binding partner. Chemical excitation of the label is usually accomplished by exposure of the fluor label to a high energy compound formed in situ. Assay systems of this type are described in commonly-owned Canadian Patent No. 1,133,392.

(h) DoubZe Antibody Steric Hindrance ~abeZs Another assay system is the double antibody immunoassay system described in U.S. Patents Nos.

3,935,074 and 3,998,943. The label conjugate com-prises two epitopes, one of which participates in the immunological reaction with the ligand and anti-ligand antibody and the other of which is bindable by Z~iZ

a second antibody, with the restriction that the two antibodies are hindered from binding to the label con-jugate simultaneously. The second epitope can bc a fluorescent substance the fluorescence of which is quenched by the second antibody binding, or may participate in an ancillary competitive binding reaction with a labeled form of the second epitope for binding to the second antibody. Various detec-tant systems are possible in such a system as des-cribed in the aforementioned patents. Related assaysystems are described in U.S. Patents Nos. 4,130,462 and 4,161,515 and in British Patent Spec. No. 1,560,852.

(i ) Energy Trans f er Labe Zs In this system, the label is one member of an energy transfer donor-acceptor pair and the binding partner is conjugated with the other of such pair.
Thus, when the label conjugate is bound by its binding partner, the energy expression of the donor component of the pair is altered by transferance to the acceptor component. Usually, the donor is a fluor and the acceptor is a quencher therefor, which quencher may or may not be a fluor as well. In such embodiment, the detectable signal is fluorescence, but other detectant systems are possible also. Such assay systems are described in U.S. Patents Nos. 3,996,345;

4,174,384; and 4,199,559 and in British Patent Spec.
No. 2,018,424.

1~79Z6~, ( j) Other Lc~be Zs Other homogeneous specific binding assay systems described in the art which can be used in the present invention include the use of labels such as:
(i) nonenzymic catalysts, such as electron transfer agents (see U.S. Patent No.
4,160,645);
(ii) nonenzymic chemiluminescers (see commonly owned, copending application Serial No.
894,836 which corresponds to pubiished German OLS 2,618,511);
(iii) "channeling" labels (see British Patent Spec. No. 2,018,986);
(iv) "particle" labels (see British Patent Spec. No. 2,019,562); and (v) labeled liposome particles (see U.S.
Patent No. 4,193,983).

2. LIGAND

The present assay may be applied to the detection of any ligand for which there is a specific binding partner and, conversely, to the detection of the capacity of a liquid medium to bind a ligand (usually due to the presence of a binding partner for the ligand in the medium). The ligand usually is a peptide, polypeptide, protein, carbohydrate, glyco-protein, steroid, or other organic molecule for which a specific binding partner exists in biological systems or can be synthesized. The ligand, in func-tional terms, is usually selected from the group comprising antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances. Usually, the , 117~;32~;~
- 2~ -ligand is an immunologically-active polypeptide or protein of molecular weight between 1,000 and 10,000,000, such as an antibody or antigenic polypeptide or protein, or a hapten of molecular weight between 100 and 1,500.
Representative polypeptide ligands are angio-tensin I and II, C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, bradykinin, and glucagon.
Representative protein ligands include the classes of protamines, mucoproteins, glycoproteins, globulins, albumins, scleroproteins, phosphoproteins, histones, lipoproteins, chromoproteins, and nucleo-proteins. Examples of specific proteins are pre-albumin, ~l-lipoprotein, human serum albumin, ~1-glycoprotein, transcortin, thyroxine binding globulin, haptoglobin, hemoglobin, myoglobin, ceruloplasmin, -lipoprotein, ~2-macroglobulin, ~-lipoprotein, erythropoietin, transferrin, homopexin, fibrinogen, the immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, and their fragments, e.g., Fc and Fab, comple-ment factors, prolactin, blood clotting factors such as fibrionogen, thrombin and so forth, insulin, melanotropin, somatotropin, thyrotropin, follicle stimulating hormone, leutinizing hormone, gonado-tropin, thyroid stimulating hormone, placental lactogen, intrinsic factor, transcobalamin, serum enzymes such as alkaline phosphatase, lactic dehydro-genase, amylase, lipase, phosphatases, cholinester-ase, glutamic oxaloacetic transaminase, glutamicpyruvic transaminase, and uropepsin, endorphins, enkephalins, protamine, tissue antigens, bacterial antigens, and viral antigens such as heptatitis associated antigens (e.g., HBsAg, HBCAg and HBeAg).

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Representative hapten ligands include the gen-eral classes of drugs, metabolites, hormones, vita-mins, and the like organic compounds. Haptenic hor-mones include thyroxine and triiodothyronine. Vita-mins include vitamins A, B, e.g., B12, C, D, E and K, folic acid and thiamine. Drugs include anti-biotics such as aminoglycosides, e.g., gentamicin, tobramycin, amikacin, sisomicin, kanamycin, and netilimicin, penicillin, tetracycline, terramycin, chloromycetin, and actinomycetin; nucleosides and nucleotides such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD) and its phosphate derivative (NADP), thymidine, guanosine and adenosine; prostaglandins; steroids such as the estrogens, e.g., estriol and estradiol, sterogens, androgens, digoxin, digitoxin, and adrenocortical steroids; and others such as phenobarbital, pheny-toin, primidone, ethosuximide, carbamazepine, val-proate, theophylline, caffeine, propranolol, pro-cainamide, quinidine, amitryptiline, cortisol, desi-pramine, disopyramide, doxepine, doxorubicin, nor-tryptiline, methotrexate, imipramine, lidocaine, procainamide, N-acetylprocainamide, the amphetamines, the catecholamines, and the antihistamines.
The liquid medium to be assayed can be a natur-ally occurring or artifically formed liquid suspected to contain the ligand, and usually is a biological fluid or a dilution thereof. Biological fluids that can be assayed include serum, plasma, urine, saliva, and amniotic and cerebrospinal fluids.

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3 . CARRI ER MEMBER

The carrier member of the present invention can take on a multitude of forms, and is therefore in-tended as being broad in context. It can be mono- or multi-phasic, comprising one or more appropriate materials or mediums of similar or different absorp-tive or other physical characteristics. It can be hydrophobic or hydrophilic, bibulous or nonporous.
In its most efficient embodiment the carrier member can be carefully tailored to suit the characteristics of the particular homogeneous specific binding assay system to be employed.
Thus, as used herein, the term "carrier member"
can comprise any substance, matrix, or surface capable of being incorporated with specific binding assay reagents. It can take on many known forms such as those utilized for chemical and enzymatic reagent strips for solution analysis. For example, U.S.
Patent No. 3,846,247 teaches the use of felt, porous ceramic strips, and woven or matted glass fibers. As substitutes for paper, U.S. Patent No. 3,552,928 teaches the use of wood sticks, cloth, sponge mat-erial, and argillaceous substances. The use of synthetic resin fleeces and glass fiber felts in place of papers is suggested in British Patent No.
1,369,139. Another British Patent No. 1,349,623, suggests the use of a light-permeable meshwork of thin filaments as a cover for an underlying paper carrier element. This reference also suggests im-pregnating the paper with part of a reagent systemand impregnating the meshwork with other potentially incompatible chemical or enzymatic reagents. French Patent No. 2,170,397 teaches the use of carrier members having greater than 50~ polyamide fibers , ,, , ~ . . .. . . _ therein. U.S. Patent No. 4,046,514 dicloses the interweaving or knitting of filaments bearing rea-gents in a reactant system. All such carrier member concepts can be employed in the present invention, as can others. Preferably the carrier member comprises a bibulous material, such as filter paper, whereby a solution or suspension of the reagents of the speci-fic binding assay system is used to impregnate the carrier member. It can comprise a system wherein the ingredients are homogeneously combined with the carrier member in a fluid or semi-fluid state, which later hardens or sets, thereby entrapping the ingre-dients.
Whichever material is chosen for the carrier member, whether it be porous to permit incorporation of ingredients such as through saturation with a solution containing them, whether it be nonporous such as to support or create a continuous coating, whether it be woven or knitted, whatever its com-position or configuration, its selection will in anyevent be dictated by anticipated use and by the reagent system.

. PREPARATION OF THE TEST DEVICE

A method of preparing the test device is provided by the present invention. This is a method for pre-paring a homogeneous specific binding assay element for determining a ligand in or the ligand binding capacity of a liquid sample by incorporating a carrier with a composition which includes a label conjugate, comprising a label component coupled to a ligand moiety or a specific binding analog thereof, and a reagent reactive with the label conjugate, which method comprises (a) incorporating a carrier with ~1~79~Z6i~

the reagent reactive with the label conjugate in a first liquid and drying said carrier; and (b) incor-porating the carrier of (a) with the label conjugate in a liquid effective to prevent its reaction with the reagent reactive with the label conjugate and drying the carrier. The reactive reagent can com-prise, for example, a specific binding partner for the ligand or a specific binding partner for the ligand and a component which is reactive with the label conjugate to cleave the label component from the ligand moiety or specific binding analog thereof.
In a preferred embodiment, a layer of carrier material is impregnated with a first solution or suspension of reagents in a first solvent and dried.
Thereafter, the same carrier material is impregnated with a second solution or suspension of the remaining reagents in a second solvent which prevents inter-action with reagents impregnated by the first solvent and dried. In this way, the reagents in the respec-tive solutions are prevented from substantial inter-action during preparation of the test device and thus do not react prematurely. In a particularly pre-ferred embodiment certain first reagents are impreg-nated into a layer of carrier material using an aqueous solution of these first reagents. For the remaining reagents, a suitable organic solvent is used such as toluene, acetone, chloroform, methylene chloride, n-propanol and ethylene dichloride to prepare the second impregnation solution. This second impregnation solution is adminstered to the carrier which is then set by allowing the organic solvent to evaporate.

11~9~6~

An example of this preferred embodiment is a method for preparing a homogeneous specific binding assay device for determining a ligand in a liquid sample by incorporating a carrier with a composition which includes a ~-galactosyl-umbelliferone-ligand or ligand analog conjugate~ ~-galactosidase, and anti-sera to the ligand which method comprises (a) impreg-nating a carrier with an aqueous solution of ~-galactosidase and antisera to the ligand and drying the carrier; and (b) impregnating the carrier of (a) with an acetone solution of ~-galactosyl-umbelliferone-ligand or ligand analog conjugate and drying the carrier.
Where the carrier comprises additional layers, e.g., paper or other fibrous material, such layers may be maintained in laminar relationship by ad-hesives which permit fluid passage between layers. In preparing integral analytical elements using film formers, the additional layer(s) can be preformed separately and laminated to form the overall element.
The material o the film layer(s) can be a composi-tion comprising a plasticizer and a polymer suitable to impart dimensional stability. Layers prepared in such a manner are typically coated from solution or dispersion onto a surface from which the dried layer can be physically stripped. However, a convenient method which can avoid problems of multiple stripping and lamination steps is to coat an initial layer on a stripping surface or a support, as desired, and thereafter to coat successive layers directly on those coated previously. Such coating can be accom-plished by hand, using a blade coating device, or by machine, using techniques such as dip or bead coating.
If machine coating techniques are used, it is often possible to coat adjacent layers simultaneously using l~.t7~26~

hopper coating techniques well known in the pre-paration of light sensitive photographic films and papers.
Blush polymer layers can be used as the film layer material. The film is formed on a substrate by dissolving a polymer in a mixture of two liquids, one of which is of a lower boiling point and is a good solvent for the polymer and the other of which is of a higher boiling point and is a nonsolvent or at least a poor solvent for the polymer. Such a polymer solution is then coated on the substrate, and dried under controlled conditions. The lower boiling solvent evaporates more readily and the coating becomes enriched in the liquid which is a poor sol-vent or nonsolvent. As evaporation proceeds, underproper conditions, the polymer forms as a porous layer. Many different polymers can be used, singly or in combination, for preparing porous blush polymer layers. Typical examples include polycarbonates, polyamides, polyurethanes and cellulose esters such as cellulose acetate. For layers such as those containing a label conjugate or other reagent, a coating solution or dispersion including the matrix and incorporated active materials can be prepared, coated as discussed herein and dried to form a dimen-sionally stable layer.
The thickness of any layer and its degree of permeability are widely variable and depend on actual usage. Dry thickness of from about 5 microns to 100 microns have been oonvenient, although more widely varying thickness may be preferable in certain circumstances. For example, if comparatively large amounts of interactive material, e.g., polymeric materials like enzymes, are required, it may be desirable to prepare slightly thicker layers.

9~6~

It can also be desirable to include within a carrier one or more reflective layers, optionally absorptive to detecting radiation, such as to faci-litate signal detection by reflection radiometry, e.g., reflection photometry or a similar technique.
Such reflector can be provided by one of the above-described layers or it can be provided by an addi-tional layer that may not have an additional function within the element. Reflective pigments, such as titanium dioxide and barium sulfate, can be used to advantage in a reflecting layer. Blush polymers can also constitute a suitable reflecting material. In one preferred aspect, blush polymer layers can also incorporate a pigment to enhance reflectivity or other functions. The amount of pigment that can be included in a layer together with a blush polymer is highly variable, and amounts of from about 1 to about 10 parts by weight of pigment per part by weight of blush polymer are preferred, with from about 3 to about 6 parts pigment per part of blush polymer being most preferred.
It can be advantageous to incorporate one or more surfactant materials, such as anionic and non-ionic surfactant materials, in the layers of the carrier. They can, for example, enhance coatability of layers formulations and enhance the extent and range of wetting in layers that are not easily wetted by liquid samples in the absence of an aid such as a surfactant.
As mentioned previously herein, the integral analytical elements can be self-supporting or coated on a support. The support can be opaque or trans-parent to light or other energy. A support of choice for any particular carrier will be compatible with the intended mode of signal detection. Preferred supports include transparent support materials capable of transmitting electromagnetic radiation of a wave-length within the region between about 200 nm and about 900 nm. The support need not, of course, transmit over the entire 200-900 nm region, although for fluorometric detection of analytical results through the support it is desirable for the support to transmit over a wider band or, alternatively, to transmit at the absorption and emission spectra of the fluorescent materials used for detection. It may also be desirable to have a support that transmits one or more narrow wavelength bands and is opaque to adjacent wavelength bands. This could be accom-plished, for example, by impregnating or coating the support with one or more colorants having suitable absorption characteristics.

5. DETECTABLE RESPONSE

As previously noted, many of the recently de-vised homogeneous specific binding assay systems provide, or can be readily adapted to provide, a detectable response such as a color change, chemi-luminescence, or fluorescence related to the presence or amount of the ligand under assay in the liquid sample.
The term "detectable species", and similar terms as used herein, refer to atoms, chemical groups (i.e., a portion of a molecule) or chemical compounds that are themselves directly or indirectly detectable and the term "detectable response", and similar terms as used herein, refer to the detectable manifestation of the presence of such species. Examples are electro-magnetic radiation signals such as fluorescence, phosphorescence, chemiluminescence, a change in light . .

~179Z62 absorption, or reflectance in the visible spectrum thereby producing a visible color change, a change in light absorption or reflectance outside the visible range, such as in the ultraviolet or infrared range.
As will be apparent to one skilled in the art of specific binding assays, the phrase "detectable response'~, as used herein, is intended in its broad-est sense. In addition to electromagnetic radiations signals the term "detectable response" is also meant to include any observable change in a system para-meter, such as a change in or appearance of a reac-tant, observable precipitation of any component in the test sample or a change in any other parameter, whether it be in the assay system or the test sample.
Such other detectable responses include electro-chemical responses and calorimetric responses. More-over, the detectable response is one which can be observed through the senses directly or by use of ancillary detection means, such as a spectrophoto-meter, ultraviolet light-sensing equipment, fluoro-meter, spectrofluorometer, pH meter or other sensing means.
After the analytical result is obtained as a detectable change, it is measured, usually by passing the test element through a zone in which suitable apparatus for reflection, transmission or fluores-cence photometry is provided. Such apparatus serves to direct a beam of energy, such as light, at the element. Th~e light is then reflected from the element back to a detecting means or passes through the element to a detector in the case of transmission detection. In a preferred mode, the analytical result is detected in a region of the element totally within the region in which such result is produced.
Use of reflection spectrophotometry can be advan-tageous in some situations as it effectively avoids .. . . . . .. . . . . _ . ... _ 11~7~62 optical interference from any residues, such as blood cells or urine sediment, which have been left on or in the layers of the element or from atypical urine colors. Conventional techniques of fluorescence spectrophotometry can also be employed if desired.
Furthermore, transmission techniques can be used to detect and quantify the indicating reaction products by reacting a flow of radiant energy, for example, ultraviolet, visible or infrared radiation at one surface of the element and measuring the output of tha~ energy from the opposing surface of the element.
Generally, electromagnetic radiation in the range of from about 200 to about 900 nm has been found useful for such measurements, although any radiation to which the element is permeable and which is capable of quantifying the product produced in the element can be used. Various calibration techniques can be used to provide a control for the analysis. As one example, a sample of a standard solution of the ligand under assay can be applied adjacent to the area where the drop of sample is placed in order to permit the use of differential measurements in the analysis.

~79Z62 ~XAMPLES

The following examples describe experiments which were performed in developing the present invention. While they illustrate preferred embodi-ments, they are in no way to be interpreted aslimiting the scope of the invention.

E~amp~e I - Substrate-LabeZed F~orescent Imm~noassay E~ement for Centamicin Gentamicin is a water-soluble, broad spectrum aminoglycoside antibiotic derived from Micromo~ospora purpurea, an actinomycete. Peak serum concentration, in micrograms/milliliter (~g/ml) is usually up to four times the single intramuscular dose, which is usually 1-3 milligram~s)/kilogram of body weight (mg/kg), administered three times daily. It is potentially nephrotoxic.

Conjugate Preparation The reaction sequence for the preparation of the glycone-dye-drug conjugate is given in Figure 2 in the drawings. 3-carboethoxy-7-hydroxycoumarin (2) was prepared by a Knoevenagel condensation of 2,4-dihydroxybenzaldehyde (Aldrich Chemical Co., Mil-waukee, Wisconsin, USA) with diethylmalonate in acetic acid, benzene, and piperidine as described in J. Am. Chem. Soc. 63:3452 (1971). The potassium salt of 7-~-galactosylcoumarin-3-carboxylic acid (3) was prepared by the reaction of 3-carboethoxy-7-hydroxy-coumarin (2) and 2,3,4,6-tetraacetyl-~-D-galactosyl bromide (I, Sigma Chemical Co., St. Louis, Missouri, USA) as described by Leaback for the preparation of methyl-umbelliferyl-~-D-galactoside in C~i~. Chim.
Acta 12:647 (1965). The potassium salt of this , - . . . . . , ~

9~6Z

compound was purified by chromatography on silica gel-~0 (E. Merck, St. Louis, Missouri, USA) with a gradient of n-butanol/methanol/water (4/2/1 volume) and methanol/water (1/6). After recrystallization s from acetone-water, the correct melting point of the product was 258-263C (decomp.).
Analysis: Calculated for C16H1501oK: C, 47,28:
H, 3.73; K, 9.62.
~ound: C, 47.30; H, 3.74; X, 9.34.
[~]D = ~77 4 (c 1.0, H20), NMR Spectrum (D20): ~ 8.2 (s, lH), 7.6 (m, lH), 7.0 (m, 2H), 5.0 (s, lH), and 4.0 (m, 6H).
Infrared Spectrum (KBr~: 1705 cm 1 (carbonyl), 1620 cm 1 (C=C).
~ -galactosyl-umbelliferone-sisomicin (g) was prepared by mixing 50 milligrams (mg) (117 ~mol) of the potassium salt of 7-~-galactosylcoumarin-3-carboxylic acid (3) with 171 mg of sisomicin sul-fate (223 ~mol of sisomicin free base, ScheringCorp., Bloomfield, New Jersey, USA) in 2 ml of water.
The pH was adjusted to 3.8 by dropwise addition of 1 molar hydrochloric acid. The sol~tion was cooled in an ice bath and 30 mg (150 ~mol) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (Pierce Chemical Co., Rockford, Illinois, USA) was added. After 2 hours the mixture was chromatographed at 25C on a 2.5 x 50 centimeter (cm) column of CM-Sephadex C-25 (Pharmacia Laboratories, Inc., Piscataway, New Jersey, USA) 5.8 ml fractions were collected, and their absorbance was conitored at 345 nanometers (nm). The column was washed with 200 ml of 50 mmol/liter ammonium formate to elute unreacted 7-~-galactosylcoumarin-3-carboxylic acid (3). A
linear gradient formed with 400 ml of 50 mmol/liter * Trade Mark ~17~62 and 400 ml of 1.8 mol/liter ammonium formate, was applied to the column. A peak of material absorbing at 345 nm eluted at approximately 1.4 mol/liter ammonium formate. After the gradient, the column was washed with 600 ml of 1.8 mol/liter ammonium formate.
Three 345 nm absorbing peaks were eluted in this wash. Eluted unreacted sisomicin was well separated from the last 345 nm absorbing peak.
The carbodiimide-activated reaction leads to the formation of amide bonds between the carboxylic acid of ~-[7-(3-carboxy-coumarinoxy)]-galactoside and the primary amino groups of sisomicin. The major peak of ~-galactosyl-umbelliferone-sisomicin (the last 345 nm absorbing peak) was used in the present studies.
Ammonium formate was removed by lyophilization.
Because the absorptivity of isolated label conjugate is currently unknown, the relative concentration is presented in terms of A345 units. One A345 unit is the quantity of material contained in 1 ml of a solution that has an absorbance of 1.0 at 345 nm when measured with a 1 cm light path.

Antiserum Preparation Antiserum to gentamicin was prepared as des-cribed in NatuYe New BioZ. 239: 214 (1972).

Element Preparation A~ueous Solution To 5.00 milliliters (ml) of antiserum was added

6.4 ml of 1 molar N, N-bis-(2 hydroxyethyl)-glycine (Bicine) buffer (Nutritional Biochemicals Corp.) containing 0.1 molar magnesium chloride, and 2.84 ml of a 73.9 units/ml ~-galactosidase solution. The pH
was adjusted to 8.3 by adding 1.1 ml of a 0.16 il79~6i~

molar sodium hydroxide solution. A sheet of Whatman 31 ET paper (h'hatman~ Inc. Clifton, New Jersey) was impre~nated to saturation with the above solution and dried at 50C for 15 minutes.
Organic Solution The paper was then impregnated a second time with a conjugate solution prepared by adding 0.5 ml of 32.5 micromolar (~m) B-galactosyl-umbelliferone sisomicin conjugate solution in 0.05 molar sodium formate (pH 3.5) to 20 ml of acetone.
The resulting immunoreagent paper was laminated onto silver Mylar and one side of double-faced adhe-sive tape. After cutting into 1 centimeter (cm) ribbons the material was laminated onto one surface of 8.3 x 12.7 cm polystyrene supports. The laminated supports were cut into 8.3 cm x 1 cm configuration each having 1 cm x 0.5 cm assay element.
Final reagent contents per gentamicin immuno-assay element were as follows:

20 Component Content Antiserum 7.7 ~1 Conjugate ~-gal-umb-sisomicin (quantity) 65 picomoles Buffer (bicine~ 10.8 micromoles 25 Magnesium chloride 1.0 micromole ~-galactosidase 0.33 units Sodium formate 0.1 micromole Analytical Procedure 35 microliter (~1) aliquots of drug solution were pipetted onto the exposed surface of the ana-lytical elements prepared as described above.

O ,~
.,~ .
* Trade Mark The fluorescence generated at room temperature at the end of 15 minutes was measured in a fluoro-meter equipped with a mechanical holder suitable for horizontally positioning the analytical element. The fluorometer had been adjusted to provide an excita-tion light source at 405 nm, which struck the surf~ce at 90 and to detect light emitted at a wavelength of 450 nm. A front face measurement of fluorescence was made at a 90 angle from the pad.
The concentratlon ranges assayed were as fol-lows:
RANGF GENTAMICIN
Therapeutic Range 5-10 ~g/ml Dose Response Range Checked O-S.O ~g/ml The dose response range checked covers the thera-peutic range since solutions containing up to 10 ~g/ml were checked after 1:2 dilution in distilled water.

Results -The data obtained by the above-described proce-dure are graphically illustrated by Figure 3. The ordinate units are expressed in terms of millivolts (mv~. A millivolt is one thousanth of a volt.

Conclusion The resultant data show that integral analytical elements, prepared according to the invention provide quantitatively detectable signals which are respon-sive to the concentration ranges of the gentamicin present. Increasing concentrations of gentamicin ~S-1182 results in a drug dependent increase in fluorescence of the respective analytical elements.

, ` 117~i~6~

E~amp~e II - Substrate-~abe~ed ~Zuorescent Immunoassay EZement for Tobramy~i~

Tobramycin is another water-soluble, broad spectrum aminoglycoside antibiotic derived from Streptomyees tenebrari~s, an actinomycete. Peak serum concentration (~g/ml) is usually up to four times the single intramuscular dose, which is usually 1-3 mg/kg administered three times daily. Like other aminoglycoside antibiotics, it is potentially nephro-toxic.

Con~ugate Preparation The reaction sequence and methodology for thepreparation of the tobramycin conjugate were basic-ally those of Example I.
With 55 mg (135 ~mol) of the potassium salt of

7-~-galactosylcoumarin-3-carboxylic acid was mixed 150 mg (220 ~mol) of tobramycin (Eli Lilly ~ Co.
Indianapolis, Indiana, USA) in 1.5 ml of distilled water. The pH was adjusted to 3.65 by the dropwise addition of lN hydrochloric acid and the resulting solution cooled in an ice bath. To initiate the coupling reaction, 30 mg (160 ~mol) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were added. After overnight incubation of 4C, two drops of lN sodium hydroxide were added to give a pH of 6.1.
The product was purified by chromatography on carboxymethyl Sephadex gel (Pharmacia Laboratories, Inc.) with ammonium formate as eluant. After an initial wash with 0.05M ammonium formate to remove unreacted galactoside, 1.5M ammonium formate was used il75~Z

to elute conjugated products. Five peaks of material absorbing at 345 nm were eluted, with the third peak being selected for use in this study.

Antiserum Preparation Antiserum to tobramycin was prepared as des-cribed for gentamicin in ~ature ~ew Bio ~ . 239: 214 (1~72).

Element Preparation Preparation of the analytical element was as described in Example 1, with the exceptions that the conjugate and antiserum used were those prepared as described in this Example.
Final reagent contents per tobramycin immuno-assay element were as follows:

Component Content Antiserum 7.7 ~1 Conjugate ~-gal-umb-tobramycin (quantity) 59 picomoles Buffer (bicine) 10.8 micromoles Magnesium chloride 1.0 micromole ~-galactosidase 0.33 units Sodium formate 0.5 micromoles Analytical Procedure The procedure followed in performing the assays reported by this Example were identical with those described in Example 1.
The concentration ranges assayed were as fol-lows:

.. . . . . . ..

6;:

RANGE TOBRAMYCIN
Therapeutic Range 5-10 ~g/ml Dose Response Range Checked 0-12 ~g/ml The dose response range checked includes the thera-peutic range after a 1:2 dilution.

Results The data obtained by the above-described pro-cedure are graphically illustrated by Figure 4. The ordinate units are expressed in terms of electrical output.

Conclusion .

The resultant data show that integral analytical elements, prepared according to the invention, pro-vide quantitatively detectable signals which areresponsive to the concentration ranges of the tobra-mycin present. Increasing concentrations of tobra-mycin results in a drug dependent increase in fluor-escence of the respective analytical elements.

6~

~amp~e III - Substrate-labe~ed FZuorescent Immu~oass~y ~ement for Amika~in Amikacin sulfate is a water-soluble, broad spec-trum, semisynthetic aminoglycoside antibiotic derived from kanamycin, which is obtained from Streptomyces kanamyceticus. In normal adults, average peak serum concentrations of about 12, 16 and 21 ~g/ml are obtained about 1 hour after intramuscular administra-tion of 3.7 mg/kg, 5 mg/kg, and 7.5 mg/kg single doses, respectively. Normal dosage is 15 mg/kg daily, divided into 2-3 equal doses.

Conjugate Preparation The reaction sequence and methodology for the preparation of the amikacin conjugate were basically those of Figure 2.
290 mg (540 ~mol) of amikacin (Bristol Labora-tories, Syracuse, New York, USA) were mixed 110 mg (270 ~mol) of the potassium salt of 7-~-galactosyl-coumarin-3-carboxylic acid in 3 ml of distilled water. The pH was adjusted to 4.1 by addition of lN
hydrochloric acid. After the solution had been cooled in an ice bath, 55 mg (292 ~mol) of l-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride were added to initiate the reaction. After overnight incubation at 4C, the reaction mixture was chroma-tographed on carboxymethyl Sephadex gel. After washing the column with 0.05M ammonium formate to remove unreacted galactoside, 1.5M ammonium formate was used to elute the desired conjugate. Three peaks of material absorbing at 345 nm were obtained, with the last peak being used for this study.

Antiserum Preparation Antiserum to amikacin was prepared as described for gentamicin in Natu~e New Bio~. 239:214 (1972).

Element Preparation Preparation of the analytical element was as described in Example 1, with the exceptions that the conjugate and antiserum used were those prepared as described in this Example and that the conjugate was dissolved in dimethylsulfoxide (DMSO).
Final reagent contents per amikacin immunoassay element were as follows:

Component Content Antiserum 7.7 ~l Conju~ate 3-gal-umb-amikacin (quantity) 77.8 picomoles Buffer (bicine) 10.8 micromoles Magnesium chloride 1.0 micromole ~-galactosidase 0.33 units DMSO 0.17 ~1 Analytical Procedure The procedure followed in performing the assays reported by this Example were identical with those described in Example l.
The concentration ranges assayed were as fol-lows:

RANGE AMIKACIN
Therapeutic Range 15-25 ~g/ml Dose Response 0-10 ~g/ml Range Checked -1~79Z6~

The dose response range checked includes the thera-peutic range after a 1:2 dilution.

~esults The data obtained by the above-described pro-cedure are graphically illustrated by Figure 5. The ordinate units are expressed in terms of electrical output.

Conclusion __ The resultant data show that integral analytical elements, prepared according to the invention, pro-vide quantitatively detectable signals which are responsive to the concentration ranges of the amika-cin present. Increasing concentrations of amikacin result in a drug dependent increase in fluorescence of the respective analytical elements.

1~7~6Z

E~ampZe IV - Su~strate-LabeZed ~Zuorescent Immunoassay EZement ~or TheophyZZine Theophylline [1,3-dimethylxanthine, cf. The Merek Inde~, 9th ed., p. 1196 (1976)] is a drug useful in the management of asthma. In most pat-ients, the therapeutic range of serum concentration lies between lO and 20 ~g/ml whereas toxicity almost invariably appears at blood levels over 35 ~g/ml.

Conjugate Preparation ~-galactosyl-umbelliferone-labeled theophylline conjugates are prepared according to the reaction scheme shown in Figure 6. This synthetic route is exemplified by the following method of preparing 8-~3-(7-~-galactosylcoumarin-3-carboxamido)propyl]
theophylline ~8), n = 3.

8-(3-Aminopropyl)theophylline (6) A mixture of 2.66 g (0.01 mol) of 8-(3-carboxy-propyl)theophylline (5) [Cook et aZ, Res. Commun.
Chem. Path. PharmaeoZ. 13(3):497-505 (1976)], 20 ml of chloroform, and 3 ml of concentrated sulfuric acid was stirred at 50C under an argon atmosphere.
To this was added 1.3 g of solid sodium azide pro-tionwise over a 90 minute period ~cf. Organie Reac-tions 47: 28 (1967)]. The reaction was cooled and the solvent removed under reduced pressure. The residue was combined with enough sodium bicarbonate solution to bring the pH to 7.5. Ten grams of celite (Fisher Scientific Co., Pittsburgh, Pennsylvania) was added and the water evaporated. The impregnated celite was placed atop a column of 200 g of silica gel (E. Merck Co., Darmstadt, West Germany) made up in 9:1 (v:v) .. .. .... . . . . . .. .. .... .. ..... _ _ 6~, ethanol - 1 molar aqueous triethylammonium bicar-bonate. The column was eluted with this solvent and 15 ml fractions were collected. Fractions 171 to 225 were combined and evaporated to give 500 mg of a white powder. This substance was rechromatographed on a column of CM-Sephadex, ammonium form (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA), eluting with 0.5 molar ammonium bicarbonate. The bed volume was 3 cm by 50 cm; and 10 ml fractions were collected.
Fractions 65 to 110 were combined and evaporated to give 250 mg of a white solid. It was taken up in dilute hydrochloric acid, then reevaporated.
The residue was recrystallized from methanol to give 90 mg (3% yield) of the hydrochloric acid salt of (6) as pale tan needles tha~ did not melt below 300C.
Analysis: Calculated for CloH16N5C102: C, 43.88;
H, 5.89; N, 25.59.
Found: C, 43.77; H, 5.88; N, 25.46.
Infrared Spectrum (KCl): 1695 cm 1 and 1655 cm 1 (amide carbonyls).

8-[3-(7-~-galactosylcoumarin-3-carboxamido)propyl]
theophylline (8).

A reaction mixture was prepared containing 24 g of potassium hydroxide, 80 ml of water, 240 ml of methanol and 20 g (0.035 mmol) of ethyl 7-3-galactosyl-coumarin-3-carboxylate ~Burd et a~, C~in. Chem. 23:1402 (1977)]. The reaction was stirred at 50C for 15 hours. When cool, the methanol was removed under reduced pressure. The concentrated aqueous solution was acidified to pH 2.0 with concentrated hydrochloric acid. The white precipitate was collected, washed with cold water, and recrystallized from hot water.

~7~6Z

The crystals were collected, washed with acetone, and dried at 80C for 1 hour. This gave 12 g of 7-~-galactosylcoumarin-3-carboxylic acid as whi~e crystals, mp 250-255C.
A mixture of 1.45 g (0.004 mol) of 7-~-galactosyl-coumarin-3-carboxylic acid, 404 mg (0.004 mol) of triethylamine, and 40 ml of dry dimethyl formamide (DMF) was cooled to -10C while stirring under argon.
To this was added 546 mg (0.004 mol) of isobutyl chloroformate (Aldrich Chemical Co., Milwaukee, Wisconsin) to form the mixed anhydride (7). Ten minutes later, an additional 404 mg of triethylamine and 949 mg (0.004 mol) of 8-(3-aminopropyl)theophyl-line (6) was added to the flask. After stirring for 30 minutes at -10C, the reaction was allowed to warm to room temperature. It was combined with 10 g of silica gel and the DMF removed under high vacuum. The impregnated silica gel was placed atop a column of 170 g of silica gel and the column eluted with anhydrous ethanol and collecting 15 ml fractions. Fractions 41 to 475 were combined and evaporated to give 545 mg of a yellow solid. It was dissolved in water, filtered, and concentrated to a 20 ml volume. A small amount of precipitate formed and was discarded. The filtrate was chromatographed on a 2.5 cm by 57 cm column of Sephadex LH-20 ge]
(Pharmacia Fine Chemicals, Piscataway, New Jersey), eluting with water and collecting 15 ml fractions.
Fractions 18 to 23 were combined, evaporated, and residue recrystallized from water to give 55 mg (2% yield) of the label conjugate (8) as a light yellow solid, mp 190-192C.
Analysis: Calculated for C26H29N5Oll: C, 53.15;
H, 4.98; N, 11.92.
Found: C, 52.65; H, 5.01; N, 11.80.

~179~6~

The above-described synthesis of the ~-galactosyl-coumarin-theophylline conjugate (8), n = 3, can be modified to yield label conjugates wherein n = 2 through 6 by replacing the starting material 8-(3-carboxypropyl)theophylline (5), n = 3, wi.th theappropriate 8-(~-carboxyalkyl)theophylline as fol-lows:

_ Alkylene 2 ethylene 4 butylene pentylene 6 hexylene Antiserum Preparation Antiserum was collected from rabbits immunized with a theophylline immunogen conjugate prepared as described by Cook et aZ, Res. Comm. Chem. Path.
Pharma~o~. 13: 497-505 ~1976).

Element Preparation Preparation of the analytical element was as described in Example 1, with the exceptions that the conjugate and antiserum used were those prepared as described in this Example and that the conjugate was dissolved in dimethylsulfoxide ~DMSO) rather than formate.
Final reagent contents per theophylline immuno-assay element were as follows:

.

117~1~6~

Component Content _ _ __ Antiserum 7.7 ~1 Conjugate X-gal-umb-theophylline ~quan~ity) 76 picomoles Buffer ~bicine) 10.8 micromoles Magnesium chloride 1.0 micromole ~-galactosidase 0.33 units DMSO 0.17 ~1 Analytical Procedure .

The procedure followed in performing the assays reported by this Example were identical with those described in Example 1.
The concentration ranges assayed were as fol-lows:

RANGE THEOPHYLLINE
Therapeutic Range 10-20 ~g/ml Dose Response 1-10 ~g/ml Range Checked The dose response range checked includes the thera-peutic range after a 1:2 dilution.

Results The data obtained by the above-described pro- .
cedure are graphically illustrated by Figure 7. The ordinate units are expressed in terms of electrical output.

. .

Conclusion The resultant data show that integral. analytical elements, prepared according to the invention, provide quantitatively detectable signals which are respon-sive to the concentration ranges of the theophyllinepresent. Increasing concentrations of theophylline results in a drug dependent increase in fluorescence of the respective analytical elements.

.

1 ~7~6~

E~amp~e V - S~bstrete-Labe~ed FZuores~ent Immunoassay E~ement for Carbam~zepine Carbamazepine [5H-dibenz[b,f]azepine-5-car-boximide, cf. The Merck Inde~, 9th ed., p. 226 (1976)]
sold under various trademarks including Tegretol, is an anti-convulsant drug useful in the management of epilepsy. The therapeutic range of serum concentra-tion in most patients lies between 8 and 12 ~g/ml whereas toxic signs may appear at blood levels over 12 ug/ml.

Conjugate Preparation 3-galactosyl-umbelliferone-labeled carbamazepine conjugates are prepared according to the reaction scheme shown Figure 8 in the drawings. This synthetic route is exemplified by the following method of preparing N-[4-[7-~-galactosylcoumarin-3-carbox-amido)-butyl]aminocarbonyl-5H-dibenz[b,f]azepine (12), n = 4.

N-(4 -Aminobutyl)aminocarbony1-5H-dibenz[b,f]azepine (10) Phosgene gas was bubbled into a room temperature suspension of 14.1 g (0.073) of 5H-dibenz[b,f]azepine (Aldrich Chemical Co., Milwaukee, Wisconsin) in 180 ml o dry toluene until 15 g was absorbed. The warm mixture was stirred for 2 hours, heated at reflux for 2 hours, then stirred at room temperature overnight.
The yellow solution, now containing N-chloroca:rbonyl-5H-dibenz[b,f]azepine (9), was concentrated by boiling to about 100 ml volume. It was added dropwise over 1 hour to a solution at room temperature of 26 g (0.29 ~17~

mol) of l,4-diaminobutane in 250 ml of toluene. A
white crystalline solid began to precipitate imme-diately. After the addition was ccmplete, the re-sulting slurry was stirred at reflux for 3 hours.
It was then cooled, filtered, and the precipitate washed with toluene. The filtrate was evaporated and excess butane diamine was removed by heating to 100C
at 0.2 mm. The residual oil was ta~en up in dilute hydrochloric acid and some insoluble material fil-tered off. The solution was made basic to pH 9.5 with sodium carbonate and extracted with chloroform.
Evaporation of this extract gave a glass that soli-dified when triturated with ether. This gave 15.8 g (70% yield) of the amine (10), as a solid, mp 114-116C.
Analysis: Calculated for ClgH2lN3O; C, 74.24;
H, 6.89; N, 13.67.
Found: C, 73.92; H, 6.71; N, 13.64.
Infrared Spectrum (KCl); 1655 cm 1 (amide carbonyl).

N-[4-(7-~-~alactosylcoumarin-3-carboxamido)butyl]-aminocarbonyl-5H-dibenz[b,f]azepine (12).

A mixture of 24 g of potassium hydroxide, 80 ml of water, 240 ml of methanol, and 20 g (0.035 mol) of ethyl 7-3-galactosylcoumarin-3-carboxylate [Supra, Burd et aZ, CZin. Chem. ] was prepared. The methanol was removed under reduced pressure. The concentrated aqueous solution was acidified to pH 2.6 with concen-trated hydrochloric acid. The white precipitate was 3d collected, washed with cold water, and recrystallized from hot water. The crystals were collected, washed with acetone, and dried at 80C for 1 hour. This gave 12 g (54% yield) of 7-~-galactosylcoumarin-3-carboxylic acid as white crystals, mp 250-255C.

Z6i~

A mixture of 1.02 g (5 mmol~ of dicyclohexyl-carbodiimide, 575 mg (5 mmol) o~ N-hydroxysuccini-mide, and 50 ml of dry dimethylformamide (DMF) was stirred at room temperature under argon for 30 min-utes. The clear, colorless solution was cooled to -5 and 1.835 g (5 mmol) of 7-~-galactosylcourmarin-3-carboxylic acid was added. The reaction was al-lowed to warm to room temperature and stirred for 2 hours. The mixture was then cooled in an ice bath and the precipitate of dicyclohexyl urea rçmoved by filtration under argon. The filtrate, now containing the N-hydroxysuccinimide ester (11), was combined with 1.54 g (5 mmol) of N-(4-aminobutyl)aminocarbonyl-SH-dibenz]b,f]azepine ~10~ dissolved in 5 ml of DMF.
The reaction was stirred overnight at room tempera-ture. The solvent was removed at 50C/12 mm on the rotary evaporator and the residue trîturated with dilute aqueous sodium bicarbonate solution. The insoluble material was chromatographed on 100 g of silica gel (E. Merck Co., Darmstadt, West Germany) eluting with a gradient of 2 L of ethyl acetate to 2 L of ethanol and 20 ml fractions were collected.
Fractions 190 to 250 were combined, evaporated, and the residue recrystallized twice from ethanol. This 25 gave 1.0 g (30% yield) of the label conjugate (12) as a white powder, mp 150-160C (decomposed).
Analysis: Calculated for C35H35N3Olo:
H, 5.35; N, 6.39.
Found: C, 63.55; H, 5.77; N, 6.14.
Mass Spectrum (Filed desorption): m/e 658, [P + 1].
Optical Rotation: [~]D = -46.84 (c 1.0, MeOH).

, . . . . . . . . . . .

1~7~3~Z6Z

The above-described synthesis of the 3-galactosyl-coumarin-carbamazepine conjugate (12), n = 4, can be modified to yield label conjugates wherein n = 2 through 6 by replacing the starting material 1,4-diaminobutane with the appropriate ~,~-diaminoalkane as follows:

n ,~-diaminoalkane 2 ethylenediamine 3 1,3-diaminopropane 1,5-diaminopentane 6 1,6-diaminohexane Antiserum Preparation Antiserum was obtained by immunization of rabbits with carbamazepine-bovine serum albumin immunogen con-jugate.

Element Preparation Preparation of the analytical element was asdescribed in Example 1 with the exception that the conjugate and antiserum used were those prepared as described in this Example and that the conjugate was dissolved in DMSO.
Final reagent contents per carbamazepine immuno-assay element were as follows:

Component Content Antiserum 7.7 ~1 Conjugate ~-gal-umb-carbamazepine (quantity) 65 picomoles Buffer (bicine) 10.8 micromoles Magnesium chloride 1.0 micromole 3-galactosidase 0.33 units DMSO 0.12 ~1 .. . . .

6~

Analytical Procedure The procedure followed in performing the assays reported by this Example were identical with those described in Example 1.
The concentration ranges assayed were as follows:

RANGE CARBAMAZEPINE
Therapeutic Range 8-12 ~g/ml Dose Response 0-1.25 ~g/ml 10 Range Checked The dose response range checked includes the thera-peutic range after a 1:10 dilution.

Results The data obtained by the above-described pro-cedure are graphically illustrated by Figure 9. Theordinate units are expressed in terms of electrical output.

Conclusion The resultant data show that integral analytical elements, prepared according to the invention, pro-vide quantitatively detectable signals which are responsive to the concentration ranges of the car-bamazepine present. Increasing concentrations of carbamazepine result in a drug dependent increase in fluorescence of the respective analytical elements.

l~t7~6~ ~

E~ampZe VI - S~bstY~e-LabeZed FZuore~cen~ Immunoassay E~ement For Primidone Primidone [5-ethyl-5-phenylhexahydrophyrimidine-4,6-dione, cf. The Merc~ Index, 9th ed., p. 1003 (1976)], sold under various trademarks including Mysoline, is an anti-convulsant drug useful in the management of epilepsy. The therapeutic range of serum concentration in almost all patients lies between 5 and 10 ~g/ml whereas toxicity almost invariably appears at blood levels over 15 ~g/ml.

Conjugate Preparation The ~-galactosyl-umbelliferone labeled primidone conjugates are prepared according to the reaction scheme shown in Figure 10 in the drawings. This synthetic route is exemplified by the following method of preparing 5- [4- (7-~ galactosylcoumarin-3-carboxamido)-butyl]-5-phenyl-2-desoxybarbituric acid (18), n = 4.

Diethyl (3-Cyanopropyl)phenylmalonate (13) A 50% mineral oil dispersion containing 16.8 g (0.7 mol) of sodium hydride was placed in a 3-liter, three-necked, round-bottom flask and washed free of oil with 500 ml of dry hexane. To this was added 1.2 liters of dry dimethylformamide (DMF) and 165.4 g (0.7 mol) of diethyl phenylmalonate (Aldrich Chemical Co., Milwaukee, Wisconsin). After hydrogen ceased to be evolved (3.5 hours?, 104.2 g (0.7 mol) of 4-bromobutyronitrile (Aldrich Chemical Co.) was added, and the reaction heated at 65C overnight. The solvent was removed under reduced pressure, and the residue suspended in 1 liter of ethyl acetate. It ~1~79~iZ

was filtered, the filtrate reevaporated, and the residue evaporatively distilled at 170C/0.1 mm to give 168 g (79% yield) of the diester (15) as a yellow liquid.
Analysis: Infrared Spectrum (C~ ~3):
2245 cm 1 ~CN); 1730 cm 1 (ester carbonyl).
NMR Spectrum (CDC13):
~ 1.3 (6H, t, J = 8Hz);
~ 7.3 (5H, s).

5-(3-Cyanopropyl)-5-phenyl-2-thiobarbituric acid (14) A solution of 11.5 g (0.5 g-atom) of sodium and 47.6 g (0.625 mol) of thiourea in 320 ml of absolute ethanol was stirred at reflux under an argon atmo-sphere. Over the next 30 minutes, 75.9 g (0.25 mol)of diethyl (3-cyanopropyl)phenylmalonate (13) was added dropwise. Heating was continued for 18 hours.
When cool, the solvent was removed under reduced pressure, and the residue partitioned between 500 ml of water and 500 ml of ethyl acetate. The aqueous phase was separated, washed with ether, and acidified to pH 1 with concentrated hydrochloric acid. This aqueous mixture was allowed to evaporate to dryness to give a semicrystalline mass. It was digested with 350 ml of boiling chloroform, filtered, and cooled to give 20 g of light tan solid, mp 130-145C.
Recrystallication from ethanol gave 8 g (11%
yield) of the cyano-thiobarbituric acid (14), as white crystals, mp 199C.
Analysis: Calculated for C14H13N3SO2: C, 58-52;
H, 4.56; N, 14.62.
Found: C, 58.59; H, 4.39; N, 14.27.
Mass Spectrum (70 e.v.): m/e 287 [P ];
m/e 240 [PH minus CHzCH2CH2CN].
~IS-1182 i ~7~ ~ Z

5-(3-cyna~ropyl~-S-phenyl-2-desoxybarbituric acid (15) and 5-(4-aminobutyl)-5-phenyl-2-desoxybarbituric acid (18).

A mixture of 8 g (0.029 mol) of 5-(3-cyanopropyl)-5-phenyl-2-thiobarbituric acid (16), 50 ml of an isopropanol slurry of freshly prepared W-5 raney nickel (R. L. Augustine, CateZyti~ Hydroge~at~o~, Marcel Dekker, Inc., New York, 1965, page 27) and 300 ml of ethanol was stirred at reflux under a hydrogen atmo-sphere for 4 hours. It was filtered while hot andthe filtrate cooled in an ice bath. The catalyst was washed with 200 ml of hot ethanol and then combined with the filtrate. When concentrated to a 50 ml volume, a yellow precipitate formed that amounted to 4.1 g when dry. This was chromatographed on 200 g of silica gel 60 (E. Merck Co., Darmstadt, West Germany).
The column was eluted with 9:1 (v:v) toluene:methanol and 20 ml fractions were collected. Fractions 70 to 200 were combined, evaporated, and the residue twice recrystallized from ethanol to give 1.8 g (25% yield) of the cyano-desoxybarbituric acid (17) as fine white crystals, mp 253-254C.
Analysis: Calculated for C14H15N3O2: C, 65.35;
H, 5.88; N, 16.33.
Found: C, 65.09; H, 5.56; N, 15.71.
NMR Spectrum (d6-DMSO): ~ 4.0 (m, 2H~;
~ 7.4 (s, 5H)-The ethanol filtrate from the original crystal-lization was evaporated to give a glassy solid that was chromatographed on 250 g of silica gel using a solvent prepared by equilibrating equal volumes of chloroform, methanol, and concentrated ammonium 1 1~7 hydroxide. The lower phase of this mixture was used to elute the column, and 15 ml fractions were col-lected. Fractions 66 to 100 were combined, evaporated, and the crystalline residue slurried in 2-propanol, filtered, and dried. This gave 180 mg (2% yield) of white crystals of the amino-desoxybarbituric acid (16), mp 242-244C.
Analysis: NMR Spectrum (d4-CH30H): ~ 3.0 (2H, t, J = 8 Hz); ~ 4.3 (2H, m);
~ 7.3 (s, 5H) Mass Spectrum (70 e.v.): m/e 261 [P ];
218 [P minus CH2 = CHNH2].

5-[4-(7-3-~alactosylcoumarin-3-carboxamido)butyl]-5-phenyl-2-desoxybarbituric acid (18).

A mixture of 24 g of potassium hydroxide, 80 ml of water, 240 ml of methanol, and 20 g (0.035 mol) of ethyl 7-~-galactosylcoumarin-3-carboxylate [Burd et aZ, CZin. Chem. 23:1402 (1977)] was stirred at 50C
for 15 hours. When cool, the methanol was removed under reduced pressure. The concentrated aqueous solution was acidified to pH 2 with concentrated hydrochloric acid. The precipitate was collected, washed with cold water, and recrystallized from hot water. The crystals were collected, washed with acetone, and dried at 80C for 1 hour. This gave 12 g (54% yield) of 7-~-galactosylcoumarin-3-carboxylic acid as white crystals, mp 250-255C.
A mixture of 210 mg (0~57 mmol) of 7-~-galactosyl-coumarin-3-carboxylic acid and 5.7 ml of 0.1 M solu-tion of triethylamine in dry DMF was cooled to 10Cwhile stirring under argon. To this was added 78 mg (0.57 mmol~ of isobutyl chloroformate. After 15 minutes at -10C, the reaction was allowed to warm to 0C for an additional 15 minutes. To this solution, , . . .. . . .. . .

7~6~

now containing the mixed anhydride (17), was added 150 mg (0.57 mmol~ of 5-(4-aminobutyl)-5-phenyl-2-desoxy-barbituric acid (16) and another 5.7 ml o-f 0.1 M triethylamine-DMF solution. The reaction was stirred at 0C for 15 minutes, then allowed to come to room temperature overnight.
Two gram of silica gel 60 was added to the reaction mixture and the solvent evaporated under high vacuum. The impregnated silica gel was placed atop a column of 50 g of silica gel made up in ethyl acetate. The column was eluted with a gradient of 1 liter of ethyl acetate to 1 liter of ethanol, and 10 ml fractions were collected. Fractions 120 to 160 were combined and evaporated to give a white solid.
Recrystallization from methanol gave 180 mg (51%
yield) of the label conjugate (18) as a white powder, mp 181-183C.
Analysis: Calculated for C30H33N3Oll: C, 58.91;
H, 5.44; N, 6.89.
Found: C, 55.86; H, 5.28; N, 6.37.
Mass Spectrum (Field Desorption):
m/e 612 [pH ].
Optical Rotation: [~]D = -48.38 (c 1.0, CH30H)-The above described synthesis of the ~-galactosyl-coumarin-primidone conjugate (20), n = 4, can be modified to yield label conjugates wherein n = 2 through 6 by replacing the starting material 4-bromo-butyronitrile with the appropriate ~-bromoalkyl nitrile as follows:

, . . .. . . . . . . . . . . ... .. . .

~7~6Z

~-bromoalkyl nitrile 2 2-bromoacetonitrile 3 3-bromopropionitrile 5-bromovaleronitrile 6 6-bromocapronitrile Antiserum Preparation Antiserum was obtained by immunization of rabbits with a primidone-bovine serum albumin immuno-gen conjugate.

Element Preparation Preparation of the analytical element was as described in Example 1 with the exception that the conjugate and antiserum used were those prepared as described in this Example and that the conjugate was dissolved in DMSO.
Final reagent contents per primidone immunoassay element were as follows:

Component Content Antiserum 7.7 ~l Conjugate ~-gal-umb-primidone (quantity) 67 picomoles Buffer (bicine) 10.8 micromoles Magnesium chloride 1 micromole ~-galactosidase 0.33 units DMSO .09 microliter 11~7~

Analytical Procedure The procedure followed in performing the assays reported by this Example were identical with those described in Example 1.
The concentration ranges assayed were as follows:

RANGE PRIMIDONE
Therapeutic Range 5-12 ~g/ml Dose Response 0-9 ~g/ml Range Checked The dose response range checked includes the thera-peutic range after a 1:3 dilution.

Results The data obtained by the above-described proce-dure are graphically illustrated by Figure 11. The ordinate units are expressed in terms of electrical output.

Conclusion f The resultant data show that integral analytical elements, prepared according to the invention, pro-vide quantitatively detectable signals which areresponsive to the concentration ranges of the primi-done present. Increasing concentrations of primidone result in drug dependent increase in fluorescence of the respective analytical elements.

6~

E~ampZe VII - FZuorescence Quenehing Immunoassay EZement foY TheophyZZine In the experiment described by this example, a single layer element was prepared and tested for its ability to perform a direct quenching fluoroimmuno-assay which quantitatively determined, as read by front face fluorometry, the presence of theophylline in a liquid sample.

Antiserum Preparation Antiserum to theophylline was prepared by the method described in Example IV.

Conjugate Preparation Umbelliferone-theophylline (conjugate) was pre-pared by hydrolysis of 0.16 mM galactosyl-umbelliferone-theophylline (GUT) by 0.1 U/ml ~-galactosidase. GUT
was prepared by the method described in Example IV.

Element Preparation The solutions used in preparing the theophylline specific element contained the following components:

Aqueous solution Component Content Antiserum 50 ~1 Double distilled water 30 ~1 Bicine, 0.5 M (pH 8.5) 20 ~1

9;~6~

Organic solution Component Content Toluene 1.00 ml Umbelliferone-theophyl-line (0.05 M, Bicine pH
8.5) 4.00 ~1 A 1 cm x 1 cm layer of Whatman 31 ET paper was laminated onto silver Mylar. The silver Mylar layer was then mounted, by double-faced adhesive tape, on a 8.3 cm x 1 cm polystyrene support. Then, 20 ~1 of the above prepared aqueous solution was pipetted onto the layer of Whatman 31 ET paper. The paper was dried in a convection oven at 40C for 20 minutes.
The organic solution (20 ~1) was then pipetted onto the paper containing the dried residue of the aqueous solution and dried in a convection oven at 40C for 15 minutes.

Test Solution Theophylline was added to aliquots of 0.05 M
Bicine (pH 8.5) to give final theophylline concen-trations of 0.125, 0.25, 0.50, 1.00, and 2.00 ~g/ml, respectively.

Analytical Procedure The analytical elements which had been prepared and fixed to supports as described above were inserted into a mechanical holder suitable for horizontally positioning the device in a fluorometer. Just prior to inserting the element and holder into the fluoro-meter, a 70 ~1 aliquot of one of the theophylline solutions, prepared as described above, was pipetted onto the exposed surface of the element.

.. . . . . . . . . ...

~7~6~

The fluorometer had been adjusted to provide an excitation light source at 405 nm, which struck the surface of the element at a 90 angle, and to detect light emitted at a wavelength of 450 nm. A front face measurement of fluorescence was made at a 90 angle from the pad.
The fluorescence response of each was measured after 2 minutes.

Results The data obtained in the above-described pro-cedure are graphically illustrated by Fig. 12. The ordinate units are expressed as percent quenching.

Conclusion The resultant data show that the analytical element provides a quantitatively detectable response to the theophylline concentration of each of the aliquots tested.
Under these conditions, fluorescence is quenched in the presence of antiserum and this quenching can be progressively overcome by increasing the concen-tration of theophylline.

1~ 6~

E~ampZe VIII - Pros-~heti~ ~ro~p l,abeZ CoZorimetri~
mmunoassay EZement for Theophy~Zine In order to study various parameters of incor-porating the prosthetic group label homogeneous immunoassay reagent system described in British Pat.
No. 2,023,607 into a dry test device format, a theo-phylline specific system was experimentally devised.
Reagents comprising the immunochemical components of the system included antibody to theophylline, a con-jugate of theophylline and flavin adenine dinucleo-tide (FAD), and apoglucose oxidase.
The system was designed to respond to theophyl-line by exhibiting color due to the activation of apoglucose oxidase (formation of the holoenzyme) by the theophylline-FAD conjugate. Theophylline-F~ Iconjugate which does not become bound by antibody is directly proportional to theophylline concentration. It is detectable by its ability to combine with the apo-glucose oxidase to produce active glucose oxidase enzyme. Thus, the response system included, in addition to apoenzyme, antibody and conjugate, a glucose oxidase detection system comprising glucose, 3, 3', 5, 5'-tetramethylbenzidine (TMB), and hor-seradish peroxidase. Upon activation of the apo-. 25 enzyme to glucose oxidase, a blue color formed due to the oxidation of glucose to hydrogen peroxide and subsequent conversion of TMB to its blue oxidized state in the presence of peroxidase.

Preparation of Apoenzyme Apoenzyme was prepared from a sample of highly purified glucose oxidase obtained from Miles Labora-tories, Inc. (Catalog No. 31-619). 10.5 milliliters (ml) of this enzyme solution (1000 units/ml) was llt7~6~:

mixed in a glass beaker with 4.5 ml of glycerol, and the mixture was cooled to a temperature of 0-4C.
The pH of the mixture was lowered, using a 10%
aqueous solution of sulfuric acid, until a pH of 1.4 was reached. This procedure was carried out with constant stirring with the beaker immersed in an ice bath, and the stirring was continued for 2 hours.
After that time, the solution was poured over a 1.5 cm by 43 cm column of Sephadex G-50 (medium) cross-linked gel filtration media. The Sephadex had beenequilibrated previous to the introduction of the enzyme solution with a 30% by volume aqueous glycerol solution having a pH of 1.4. Following the intro-duction of the enzyme solution onto the Sephadex column, more of the 30% glycerol solution was used to elute apoenzyme. The effluent was separated into fractions and observed using UV absorbance at 280 nanometers (nm). Those fractions having absorbance at this wavelength were combined with a buffer solu-tion containing 50 milligrams (mg) of activatedcharcoal and 25 mg of dextran (Pharmacia Company No.
T-70). The buffer comprised an aqueous solution which was 1 molar (M) tris-(hydroxymethyl)-amino-methane to which glutamic acid was added until a pH
of 7.0 was reached. The pH of the resultant effluent solution was then readjusted to 7 using a saturated solution of tris(hydroxymethyl)aminomethane. This final solution was allowed to stir in an ice bath for 1 hr. The apoenzyme solution was then centrifuged and the supernatant was filtered through 0.5 micrometer (~m) and 0.22 ~m filters obtained from Millipore Corporation.

Conjugate Synthesis The conjugate molecule whereby FAD is bound covalently to theophylline was prepared as follows.
1,3-Dimethyl-1,6,7,8-tetrahydropyrido[1,2e]-purine-2,4,9-[3H]-trione (0.9 mg/3.62 ~mmol), prepared according to the method of Cook et al. was added to 0.2 ml dimethylsulfoxide containing 2.4 ~mol N6-(aminohexyl) FA~. See Cook, C. E., Twine, M.E., Meyers, M., Amerson, E., Kepler, J.A. ~ Taylor, G.F.
Res. Commun. Chem. Path. PharmacoZ. 13, 497-505 (1976). After 4 hours a further 1.8 mg (7.3 ~mol) of the trione was added. The solution was stirred overnight, the solvent evaporated under vacuum (0.1 mm Hg), and the residue chromatographed on a column (2.5 x 90 cm) of Sephadex LH-20 equilibrated with 0.3M-triethyl-ammonium bicarbonate (pH 7.8).
The crude product, eluting between 216 and 246 ml of effluent, was collected, applied to a 20 cm x 20 cm x 100 ~m silica gel plate and chromatographed using ethanol/lM-trie~hylammonium bicarbonate, pH 7.8 (8:2 by volume). The band containing the desired product (~ 0.77) was scraped from the plate, extracted with lM-triethylammonium bicarbonate buffer, pH 7.8, filtered and concentrated. Final purification by chromatography on Sephadex LH-20 equilibrated with 0.3 M buffer gave 1.26 ~mol of theophylline-F~ las determined by the absorbance at 450 nm, which re-presented a yield of 53~.

Antiserum Preparation ~0 The antiserum to theophylline was collected from rabbits immunized with a theophylline immunogen conjugate as described by Cook, et aI., Res. Com~.
Chem. Path. PharmacoZ. 13: 497-505 ~1976).

6~

Element Preparation Using this theophylline-FAD conjugate, reagent strips for the assay of theophylline were prepared.
Pieces of Buckeye S-22 paper measuring 4 cm square were impregnated with a first dip solution comprising 5 mM TMB in acetone containing 0.1 gm per 100 ml of an emulsifier known as ON-870, marketed by General Aniline and Film Corporation. After dipping into the first dip solution, the impregnated papers were dried i0 at 50C for 1 minute in a forced air oven.
Following drying, the papers were then impregnated with a second dip solution. The second dip was an aqueous solution which was adjusted to give a pH of 6.4, 0.1 M in glucose, 19 units/ml of horseradish peroxidaseJ glucose oxidase apoenzyme (1.0 nmoles FAD
binding sites per ml), partially purified antibody to theophylline (0.14 ml antibody per ml dip solution), 0.5 mg/ml bovine serum albumin and 0.5 gm of poly-vinyl alcohol per 100 ml. After brief immersion of the dried papers in the second dip solution, a second drying was effected at 50C for 12 minutes in a forced air oven.
The doubly impregnated papers were then further impregnated with a third solution containing FAD-theophylline conjugate at a concentration of 0.5 ~Min acetone. These papers were then dried at 50C for 1 minute in the forced air oven.
~ lements were prepared having 0.5 cm squares of the triply impregnated papers mounted on stTips of polystyrene measuring 0.5 x 8.3 cm utilizing double-faced adhesive known as Double-Stick (3M Company).

; * Trade Mark -~:1 7~iZ

Test S~olution To test the performance of the test devices prepared above, solutions of theophylline in water were prepared having concentrations of theophylline in the range of 0-8 ~M.

Analytical Procedure The performance of the reagent device prepared and incubated as above-described was analyzed instru-mentally using a device known as the "Rapid Scanner".
This device is a scanning reflectance spectrophoto-meter interfaced with a PDP-12 computer obtained from the Digital Equipment Corporation. The instrument is used for the rapid measurement of reflectance spectra in the visua] range. The computer allows for the storage of spectral data and computations.
The Rapid Scanner instrument was constructed by the Ames Company Division of Miles Laboratories, Inc., Elkhart, Indiana U.S.A., from whom complete information with respect to structural and perfor-mance characteristics are obtainable.
The elements were dipped in the test solutionsand, after a period of 2 minutes, were analyzed using the Rapid Scanner. The reflectance data was obtained at 660 nm, which is the maximum absorption wavelength in the blue color range of oxidized TMB.

Results The dose-response curve is shown in Fig. 13 wherein K/S is plotted against theophylline con-centration.

.. . . . . . . . ... .. . .. . . . . .

il7~3~6~

K/S is defined as follows:

K = (l-R) in which K is a constant, S is the scattering co-efficient of the particular reflecting medium, and R
is the fraction of reflectance from the test strip.
This relationship is a simplified form of the well known Kubelka-Munk equation (Gustav KortUm, "Reflec-tance Spectroscopy", pp. 106-111, Springer-Verlaz, New York (1969~. K/S is a function of the co'orant concentration. It increases with increasing ligand lQ concentration, thereby indicating the efficacy of the device.

Conclusion .. .. ..
The plotted data shows that there is an ob-servable color intensity change for varying amounts of theophylline in solution, and that the change is indicative of the particular theophylline concentra-tion.

.

E~amp~e IX - Prosthetie Group Labe~ CoZor~metri~
Immunoassay EZement ~or Phenytoin In the experiment described by this example, the same type of assay described in Example VIII was adapted to the quantitative determination of a different ligand, phenytoin.

Conjugate Preparation To 14.7 mg (40.0 ~mol) of 5-(2'-carboxybutyloxy-phenyl-5-phenylhydantoin in 1.8 ml of molecular sieve-dried dimethylformamide (DMF), under argon, was added 0.10 ml (40.0 ~mol) of a 400 ~M solution of isobutyl chloroformate in DMF. The reaction was stirred one hour at room temperature. To the reac-tion mixture was added a solution of 10.0 ~mol of N6 (aminohexyl) FAD in 2.0 ml of molecular sieve-dried dimethylsulfoxide (DMSO), followed by 0.05 ml of a 400 ~M solution of thioethylamine in DMF. The mix-ture was stirred 19 hours at room temperature, then was diluted to 450 ml with water and was applied to a 1.5 cm x 30 cm column of Whatman DE-52 cellulose anion exchange resin (bicarbonate form) with the aid of a peristaltic pump. The column was then eluted with a gradient of 1.5 liters of water to 1.5 liters of 0.3 M triethylammonium bicarbonate aqueous solu-tion. Fractions of approximately 16 ml were collected,with fractions 70-88 determined as containing the product on the basis of activity with apoglucose oxidase. These fractions were combined and the solution adjusted to pH 7. The yield was determined as 4.78 ~mol (47.8% yield? on the basis of the absor-gance of the solution at 450 nm using the millimol`ar extinction coefficient of FArl(E450 = 11.3).

6~

Antiserum Preparation The antiserum was raised against o-caproyl-diphenylhydantoin, similarly as in Example VIII.

Element Preparation The test devices were prepared by consecutive immersion of a piece of paper into 3 solutions, each of which contained different components of an immuno-assay system potentially responsive to the presence of phenytoin, with drying between each immersion.
Accordingly, a piece of paper measuring 4 cm square (S-22, Buckeye Cellulose Corp., Memphis, TN) was immersed in a 5 mM solution of TMB in acetone containing 0.1~ (w/v) of an emulsifier known as OH-870 ~General Aniline ~ Film Corp.). It was dried at 50C for 1 minute.
The paper was then immersed in a second, aqueous, solution which was 0.2 M in tris-glutamate buffer, pH
6.4, 0.1 M in glucose, horseradish peroxidase (19 units/ml), apoglucose oxidase (1.0 nmoles FAD binding sites/ml), 0.5 mg/ml bovine serum albumin, 0.5 g/100 ml polyvinyl alcohol (Type 20-30, Monsanto Co~,), and phenytoin antiserum (0.14 ml antiserum per ml).
After drying at 50C for 12 minutes in a forced air oven, the paper was impregnated by immersion in a third solution containing the FAD-phenytoin conjugate (0.5 ~M) in n-propanol with 0~1 g/100 ml-Gafquat 734, a polymer having pendant quaternary amine groups (General Aniline ~ Film Corp.).
Following drying at 50C for 3 to 4 minutes, the impregnated paper was used to make test strips having a 0.5 cm square of the reagent-laden paper mounted at one end of a strip of a polysyrene film measuring 0.5 by 8.3 cm. Mounting was achieved using a double-faced adhesive tape known as Double-Stick (3M Com-pany).

Test Solution . _ The aqueous test solutions used contained the analyte at concentrations ranging from O to 8 Analytical ~rocedure The elements were analyzed in the Rapid Scanner 2 minutes after inoculation as in Example VIII.

Results Fig. 14 shows the change in K/S with respect to phenytoin concentrations of 0, 0.5, 1, 2, 4 and 8 ~M
in water.

Conclusion The data presented in Fig. 14 show that the test device responds well to the presence of phenytoin, enabling facile determination of different concen-trations of the analyte, either instrumentally or visually.

. . . :

Claims (35)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for preparing a homogeneous speci-fic binding assay element for determining a ligand in or the ligand binding capacity of a liquid sample by incorporating a carrier with a composition which includes a label conjugate, comprising a label com-ponent coupled to a ligand moiety or a specific binding analog thereof, and a reagent reactive with the label conjugate, which method comprises (a) incorporating the carrier with the reagent reactive with the label conjugate in a first liquid and drying the carrier; and then (b) incorporating the carrier of (a) with the label conjugate in a liquid effective to prevent reaction with the reagent reactive with the label conjugate prior to contact of the element with the sample and drying the carrier.

2. The method of claim 1 wherein the reactive reagent comprises a specific binding partner for the ligand.

3. The method of claim 1 wherein the reactive reagent comprises a specific binding partner for the ligand and a component which is reactive with the label conjugate to cleave the label component from the ligand moiety or specific binding analog thereof.

4. The method of claim 1 wherein the liquid of (a) is a aqueous.

5. The method of claim 1 wherein the liquid of (c is an aqueous solvent and the liquid of (b) is an organic solvent.

6. The method of claim 5 wherein the organic solvent is selected from toluene, acetone, chloro-form, n-propane, methylene chloride and ethylene dichloride.

7. The method of claim l wherein the conjugate includes a substrate for an enzyme linked to a ligand or binding analog thereof.

8. The method of claim 7 wherein the substrate is a .beta.-galactoside moiety and the enzyme is .beta.-galactosidase.

9. The method of claim 8 wherein the conjugate is .beta.-galactosyl-umbelliferone-ligand or binding analog thereof.

10. The method of claim 1 wherein the conjugate is a fluor bound to the ligand.

11. The method of claim 1 wherein the conjugate is unbelliferone-ligand.

12. The method of claim 1 which, prior to (a) comprises the additional step of incorporating the carrier with an indicator reagent in a liquid ef-fective to prevent reaction of the indicator reagent with the reagent of (a) prior to contact of the element with the sample.

13. The method of claim 12 wherein the liquid of the indicator reagent solution is an organic solvent.

14. The method of claim 13 wherein the organic solvent is the same as that used in (b).

15. The method of claim 13 wherein the organic solvent is different from that used in (b).

16. The method of claim 12 wherein the indica-tor reagent comprises 3,3',5,5'-tetramethylbenzidine.

17. The method of claim 12 wherein the reagent of (a) comprises glucose, glucose oxidase apoenzyme, peroxidase and antibody to the ligand and the reagent of (b) comprises a flavin adenine dinucleotide-ligand conjugate.

18. The method of claim 17 wherein the reagent of (a) further comprises bovine serum albumin and polyvinyl alcohol.

19. A method for preparing a homogeneous speci-fic binding assay element for determining a ligand in a liquid sample which method comprises:
(a) impregnating a carrier with .beta.-galactosidase in an aqueous solvent and antibody to the ligand and drying the carrier; and then (b) impregnating the carrier of (a) with .beta.-galactosyl-umbelliferone-ligand or ligand analog conjugate in acetone and drying the carrier.

20. A method for preparing a homogeneous speci-fic binding assay element for determining an amino-glycoside antibiotic in a liquid sample which method comprises:
(a) impregnating a carrier with .beta.-galactosidase in an aqueous solvent and antibody to the aminogly-coside antibiotic and drying the carrier; and then (b) impregnating the carrier of (a) with .beta.-galactosyl-umbelliferone-aminoglycoside antibiotic conjugate in acetone and drying the carrier.

21. A method for preparing a homogeneous speci-fic binding assay element for determining gentamicin in a liquid sample which method comprises:
(a) impregnating a carrier with .beta.-galactosidase in an aqueous solvent and antibody to gentamicin and drying the carrier; and then (b) impregnating the carrier of (a) with .beta.-galactosyl-umbelliferone-sisomicin conjugate in acetone and drying the carrier.

22. A method for preparing a homogeneous speci-fic binding assay element for determining tobramycin in a liquid sample which method comprises:
(a) impregnating a carrier with .beta.-galactosidase in an aqueous solvent and antibody to tobramycin and drying the carrier; and then (b) impregnating the carrier of (a) with .beta.-galactosyl-umbelliferone-tobramycin conjugate in acetone and drying the carrier.

23. A method for preparing a homogeneous speci-fic binding assay element for determining theophylline in a liquid sample which method comprises:
(a) impregnating a carrier with .beta.-galactosidase in an aqueous solution and antibody to theophylline and drying the carrier; and then (b) impregnating the carrier of (a) with .beta.-galactosyl-umbelliferone-theophylline conjugate in acetone and drying the carrier.

24. A method for preparing a homogeneous speci-fic binding assay element for determining theophyl-line in a liquid sample which method comprises:
(a) impregnating a carrier with antibody to theophylline in an aqueous solution and drying the carrier; and then (b) impregnating the carrier of (a) with umbel-liferone-theophylline conjugate in toluene and drying the carrier.

25. A method for preparing a homogeneous speci-fic binding assay element for determining theophyl-line in a liquid sample which method comprises:
impregnating the carrier with 3,3',5,5'-tetra-methylbenzidine in acetone and dried; then impregnating the carrier with glucose, peroxi-dase, glucose oxidase apoenzyme and antibody to theophylline in an aqueous solvent and drying the carrier; and then impregnating the carrier with flavin adenine dinucleotide-theophylline conjugate in acetone and drying the carrier.

26. A method for preparing a homogeneous speci-fic binding assay element for determining phenytoin in a liquid sample which method comprises:
impregnating the carrier with 3,3',5,5'-tetra-methylbenzidine in acetone and dried; then impregnating a carrier with glucose, peroxidase, glucose oxidase apoenzyme, and antibody to phenytoin in an aqueous solvent and drying the carrier; and then impregnating the carrier of flavin adenine dinucleotide - phenytoin conjugate in acetone and drying the carrier.

27. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 1, 2 or 3.

28. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 4, 5 or 7.

29. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 10, 11 or 12.

30. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 19, 20 or 21.

31. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 22, 23 or 24.

32. An analytical element for determining a ligand in or the ligand binding capacity of a liquid sample, which element is prepared by the method of claim 25 or 26.

33. A method for determining a ligand in or the ligand binding capacity of a liquid sample which com-prises contacting the sample with an analytical ele-ment prepared by the method of claim 1, 2 or 3, and observing any detectable response.

34. A method for determining a ligand in or the ligand binding capacity of a liquid sample which com-prises contacting the sample with an analytical ele-ment prepared by the method of claim 4, 5 or 7, and observing any detectable response.

35. A method for determining a ligand in or the ligand binding capacity of a liquid sample which com-prises contacting the sample with an analytical ele-ment prepared by the method of claim 10, 11 or 12, and observing any detectable response.