Immunochromatographic specific binding assay device

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Unilever NV Munich,

uZ: GM 600 / 29-88 18.07.88

Analytical test device

The invention relates to an analytical test device used at home, in the clinic or in the doctor's office. The test device should provide an analysis result in a short time, whereby the user should be required only a minimum of skill and work. The use of personal test equipment for the purpose of pregnancy assessment and ovulation is currently common practice. A variety of test equipment and kits are commercially available. For all commercially available devices, however, the user must invariably perform a series of steps before the test result is read. These steps necessarily require time and represent possible sources of error.

The invention has for its object to provide a test device that can be easily used by an untrained person and preferably only requires that a part of the device with a sample (for example, a urine stream in the case of a pregnancy or ovulation test) associated and then no further steps have to be taken by the user until the analytical result becomes apparent. Ideally, the result should be readable within minutes of the sample application, for example, in 10 minutes or less.

The use of reagent-impregnated test strips in specific binding assays, such as immunoassays, has recently been suggested. In this procedure, a sample is placed on a portion of the test strip.

so that it can penetrate the strip material, usually with an "iv" c'Mpr ^ en l..ösnr> p, smittel, such as water. At this temperature, the sample moves into or through an area in the test strip where a specific binding reagent is immobilized for a analyte (analyte) expected in the sample. The analyte present in the sample can be bound within the specific range. The extent to which the substance to be detected is bound in this region can be determined by means of labeled reagents which are incorporated into the test strip or are subsequently applied to it.

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Examples of prior proposals according to which these principles have been applied can be found in Thyroid Diagnostics Inc. GB 1589234, Boots Celltech Diagnostics Limited EP 0 225 054, Syntex (USA) Inc. EP 0 183 442 and Behringwerke AG EP 0 186 799th

The present invention is concerned with the adaptation and

Improvements to the known techniques described in the above-mentioned publications in order to provide a diagnostic test device which is particularly suitable for private use and is quick and convenient to use. The user should as little as

must be able to carry out work steps.

According to the invention there is provided a device for use in an assay for a detection substance, which device comprises a porous solid phase material carrying in a first region a labeled reagent held in the first region while the porous material is in the dry state which, however, can freely migrate through the porous material when the porous material is wetted, for example, by applying an aqueous, liquid

QQ sample possibly containing the analyte. The porous one

Material carries, in a second region spatially separated from the first region, an unlabelled specific binding reagent specific for the analyte and capable of reacting with the labeled reagent either in a "sandwich reaction" or:

nc to react in a competitive reaction, the unlabeled specific

Binding reagent is firmly immobilized on the porous material, so that it can not move freely when the porous material is in the

- 3-wet state is located.

The invention also provides an analytical method in which an apparatus as described in the preceding paragraph is contacted with an aqueous liquid solution suspected of containing the analyte, the sample being wicked through the porous solid phase material enters the second area via the first area and moves the labeled reagent together with this from the first area into the second area, wherein the presence of the analyte in the sample is determined by monitoring the extent to which the labeled reagent is optionally bound in the second region.

In one embodiment of the invention, the labeled reagent is a specific binding partner for the analyte. The labeled reagent, the analyte (if present), and the immobilized unlabelled specific binding reagent interact in a "sandwich reaction". This results in the labeled reagent being bound in the second region if analyte is present in the sample. The two binding reagents must have specificities for different epitopes of the analyte.

In another embodiment of the invention, the labeled reagent is either itself the analyte that is labeled with a

ok marking substance has been provided, or there is the marked

Reagent of an analogous substance, ie a chemical entity, which has the identical specific binding characteristics for the analyte and which has been similarly provided with a labeling substance. In the latter case, it is preferable that

"Q the properties of the analyte analogous substance that the

Solubility and dispersibility in an aqueous liquid solution and its ability to migrate through the wet solid phase material should be identical or at least very similar to the properties of the analyte itself. At this

"_r second embodiment migrates the labeled analyte or

their analogue through the porous solid phase material in the second area and binds there with the immobilized reagent. In the

Sample existing analyte occurs in this binding reaction in competition with the labeled reagent. This competition results in a reduction in the amount of labeled reagent that binds in the second region and, consequently, in a decrease in the intensity of the signal observed in the second region as compared to the signal that occurs in the absence of the analyte in the sample.

According to a particularly advantageous preferred embodiment of the invention, nitrocellulose is selected as the carrier material. This has considerable advantages over conventional strip materials, such as paper, because nitrocellulose has a natural ability to bind proteins without the need for prior sensitization. Specific binding reagents, such as immunoglobulins, can be directly transferred to and immobilized on nitrocellulose. · No chemical treatment is required that could interfere with the substantial specific binding activity of the reagent. Unused binding sites of the nitrocellulose can then be blocked by using simple materials such as polyvinyl alcohol. In addition, nitrocellulose is available with a range of pore sizes, so that a carrier material can be selected which can also meet certain requirements, such as requirements for the flow rate of the sample.

According to a further particularly preferred embodiment of the invention, so-called "direct labels" are used with which one of the specific binding reagents is used. Direct marking substances, such as e-twa gold brine and color brine,

QQ are known per se. They can be used to an instant

produce analytical result without the need to add other reagents to develop the detection signal. They are rugged and stable and therefore can easily be used in an analyzer |

used, which is stored in the dry state. Your free

In case of contact with an aqueous sample, it is possible to modulate

for example, by using a soluble glaze.

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An essential aspect of the invention lies in the selection of the technical features which enable the use of a directly labeled specific binding reagent in a carrier analyzer, for example in a strip-type analyzer, for a quick and clear result. Ideally, the assay result should be detectable by eye and to facilitate this, the direct label must be concentrated in the detection area. For this purpose, the directly labeled reagent should be readily and rapidly transportable through the developing fluid. It is further preferable that the total amount of the coil sample liquid is passed through a comparatively small detection area so as to increase the likelihood of obtaining an observable result.

Another important aspect of the invention is the use of a directly labeled binding reagent on a nitrocellulose-containing carrier material. Preferably, the nitrocellulose has a pore size of at least 1 Mkron. Preferably, the pore size of the nitrocellulose is no greater than about 20 microns. According to a particularly preferred embodiment, the direct marking substance consists of a colored latex particle of spherical or almost spherical shape whose maximum diameter is not greater than about 0.5 microro. The ideal size range for such particles is between about 0_sAbout 5 to about 0.5 microns.

In another embodiment of the invention, the porous solid state phase material is bonded to a porous receiving member to which the liquid sample can be applied and from which the sample can migrate into the porous solid phase material. Preferably that is

QQ porous solid phase material in a moisture-proof housing

and the porous receiving member to which the porous solid phase material is bonded extends out of the housing and acts as a means for introducing a liquid sample into the housing and entering the porous solid phase material

leave oc. The housing should be provided with means, such as suitably placed apertures, which allow the second region of the porous solid phase material (which is the immobilized) to be immobilized

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f_t 1 unlabelled specific binding reagent) from the outside of the

L housing is observable, so that the result of the assay is detected

'· can be. If desired, the housing can with other means

be provided, which allow the further region of the porous solid state phase material from the outside of the housing observable

^, the other area containing control reagents containing a

t Allow indication of whether the assay procedure has ended.

■ Preferably, the housing is with a removable cap or a

Enclosing the protruding porous receiving part during ^{of the} Protects storage before use. If desired, the cap or sheath may be re-set on the protruding porous anode after application of the sample while the assay procedure is being performed.

An important embodiment of the invention is a pregnancy test device having an elongate hollow housing containing a dry porous nitrocellulose carrier which communicates indirectly with the outside of the housing via an absorbent urine receiving member which extends beyond the housing and serves as a reservoir which ^^r*ⁿ *ⁿ ^^s P^or° sen carrier is released. The carrier contains, in a first region, a highly specific anti-hCG antibody carrying a colored "Direkf tag, wherein the labeled antibody is freely mobile within the porous carrier when in the wet state. In a second region, which is spatially distant from the first region, there is a highly specific unlabeled anti-hCG antibody that is permanently immobilized on the support material and therefore is not mobile in the wet state. The labeled and unlabelled antibodies have specificities for various hCG epitopes. The two areas are like this

QQ arranged that a urine sample applied to the porous support

can penetrate the first area in the second area. The housing is made of opaque or translucent material with at least one opening through which the analysis result can be observed. The housing also includes a removable and re-attachable

₃G ble cover for the above absorbent urine receptacle part. One

Ovulation test equipment, essentially as described above, except that the analyte is LH, is an important one

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The porous sample receiving member may be made of any absorbent, porous or fibrous material capable of rapidly absorbing liquid. The porosity of the material can be unidirectional (ie

the pores of the emitters are wholly or preferably parallel to an axis of the part) or multidirectional (so that the part has an amorphous, sponge-like structure). Porous plastic materials, such as polypropylene, polyethylene (preferably with very high mobility)

^ q kulargewicht), polyvinylidene fluoride, ethylene vinyl acetate copolymer,

Polyacrylonitrile and polytetrafluoroethylene can be used. It may be advantageous to pre-treat the part with a surfactant during manufacture, as this reduces any inherent hydrophobicity of the receiving part, thereby rapidly and effectively increasing its ability to pick up and continue a wet sample. Porous sample receiving members may also be made of paper or other cellulosic materials, such as nitrocellulose. Materials currently used as nibs of pens are particularly suitable. These materials may be molded or extruded in a variety of lengths and cross-sectional shapes that may be used in conjunction with the invention. Preferably, the material of the porous receiving part should be chosen so that the porous part can be saturated with aqueous liquid in a few seconds. The material should also remain stable in the wet state, which is why paper and similar materials are less suitable in those embodiments in which the porous receiving part protrudes from the housing. The liquid must then migrate freely from the porous sample receiving member into the porous solid phase material.

If present, the control panel may be constructed to be a

Provides a signal to the user indicating that the device has been working. For example, the control region may be loaded with an antibody that binds to the labeled antibody from the first region, e.g., an "anti-mouse" antibody, if the labeled body

one that has been derived using a murine hybridoma. to confirm that the sample passes through the test strip

Has. Alternatively, the control region may contain an anhydrous reagent which, when moistened, causes a color change or color formation, such as anhydrous copper sulfate, which turns blue when in contact with an aqueous liquid. In another alternative, the control region may contain immobilized analyte that reacts with excess labeled reagent from the first region. Since the purpose of the control area is to indicate that the testing has been completed, the control area must be located downstream of the second area, in which the partial result desired is recorded. A positive

Indicator thus informs the user that the sample has traveled the required distance through the tester.

The labeling substance may be any composition whose presence can be easily detected. Preferably, the

Labeling substance is a direct marking substance, ie a composition that can be readily observed in its natural state either with the naked eye or by means of an optical filter and / or excitation, such as by UV light to produce 2Q fluorescence. For example, tiny ones

colored particles, such as color sols, metal sols (for example, gold), colored latex (polymer) particles, very suitable. The concentration of the label on a small area or volume should produce a readily detectable signal, such as a highly colored area. This can be detected by eye or, if desired, with instruments.

Indirect labels such as enzymes, for example, alkaline phosphatase and horseradish peroxidase can be used, but

2Q these usually require the addition of one or more |

Development reagents, such as substrates, before a visible signal can be detected. For these reasons, these are less preferred. Such additional reagents may be incorporated in the porous solid phase material or in the sample receiving member, if present

G₅ be inserted so that they in the aqueous liquid sample ■

dissolve or disperse. Alternatively, the development reagents may be added to the sample prior to contact with the porous material.

or the porous material may be exposed to the development reagents after the binding reaction has taken place.

In all embodiments of the invention, it is essential that the labeled reagent in the first area migrate from the first area to the second area along with the liquid sample. Preferably, the flow of the sample is beyond the second range and a sufficient amount of the sample is applied to the porous material.

JO so that it can take place and thus jcgliCncf «JucrSChüß you mdtkiel tem

Reagent from the first region, which does not participate in a binding reaction in the second region, is flushed out of the second region by this continuous flow. If desired, an absorbent sink may be provided at the outboard end of the substrate. For example, this absorption sink may consist of Whatman 3MM chromatography paper and should have sufficient absorption capacity so that any unbound conjugate is washed out of the second region. Alternatively to such a dip, it may also be sufficient to provide a length of porous solid phase material extending beyond the second region.

The presence or intensity of the signal of the label bound in the second region can provide a qualitative or quantitative measurement of the analyte in the sample. A variety of detection areas on the porous solid phase material through which the aqueous liquid sample can progressively pass can also be used to allow quantitative measurement of the analyte, or the detection areas

gQ individually loaded with different specific binding agents

to create a multiple analyzer.

The immobilized specific binding reagent in the second region is preferably a highly specific antibody and in particular a monoclonal antibody as mentioned above. In the embodiment of the invention with the

Sandwich reaction, the labeled reagent is also preferably a highly specific antibody, especially a monoclonal antibody.

• ·

Preferably, the carrier material is in the form of a strip or sheet on which the reagents are applied in spaced regions, and the liquid sample traverses the sheet or strip from one side to the other.

If desired, a device according to the invention may comprise two or more separate bodies of porous solid phase material, such as separate strips or sheets, each of which

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may be arranged in parallel, for example so that a single application of the liquid sample to the device simultaneously triggers the flow of sample in the two separate bodies. The separate analytical results that can be obtained in this way can be used as control results or, if different reagents on different

Carriers are used - to be used for the simultaneous determination of a variety of detection substances in a single sample. Alternatively, multiple samples may be individually applied to a number of carriers and analyzed simultaneously.

The material with the porous solid phase is preferably nitrocellulose. This has the advantage that the antibody can be firmly immobilized in the second-tone region without prior chemical treatment. For example, when the porous solid state phase material contains paper, the immobilization of the antibody in the second region must be carried out by chemical coupling, for example using CNBr, carbonyldiimidazole or tresyl chloride.

After applying the antibody to the detection area, the QQ residue of the porous solid phase material should be treated to

to block remaining binding sites. The blocking can be achieved, for example, by treatment with protein (for example bovine serum albumin or milk protein) or with polyvinyl alcohol or ethanolamine or with any combination of these agents. The labeled reagent for the first region can then be applied to the

dry carrier and is moved in the carrier when it enters the wet state. Between each of these

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various process steps (sensitization, application of unlabelled reagent, blocking and application of labeled reagent), the porous solid phase material should be dried.

To aid free mobility of the labeled reagent when the porous support is wetted with the sample, preferably the labeled reagent should be coated on the support rather than impregnated throughout its thickness. As a result, an interaction between the carrier material and the labeled reagent can be minimized. According to a preferred embodiment of the invention, the support is pretreated with a glaze material in the area in which the cas reagent is to be applied. The glazing can be achieved, for example, by applying an aqueous sugar or cellulose solution, for example sucrose or lactose, to the carrier at the important sites and by drying. The labeled reagent can then be applied to the giarierte part. The rest of the carrier material should not be glazed.

Preferably, the porous solid state phase material is a nitrocellulose sheet having a pore size of at least 1 micron, with a pore size greater than 5 microns being preferred and a size of about 8 to 12 microns being more desirable. A very suitable nitrocellulose sheet having a nominal pore size of up to about 12 microns is commercially available from Schleicher and Schuell GmbH.

Preferably, the nitrocellulose sheet is reinforced or supported, for example, by plastic sheets, so that its handling strength is increased. This can easily be achieved by making a thin layer of nitrocellulose on a sheet base material. The actual pore size of the cellulosic material supported in this manner is typically smaller than that of the unsupported material.

Alternatively, the preformed sheet of nitrocellulose may be firmly entrapped between two solid support plates, such as plastic sheets.

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For an aqueous sample, the flow rate through the porous solid phase material should be such that for the untreated material, the aqueous liquid migrates at a rate of 1 cm in no more than 2 minutes. However, it is also possible, if desired, to work with smaller flow velocities.

The spatial distance between the regions and the flow characteristics of the porous support material can be selected to allow sufficient reaction times during which the necessary specific binding reactions can take place and the labeled reagent in the first region to dissolve or disperse in the liquid sample and through the Carrier can wander. Further influencing these parameters can be achieved by incorporating a viscosity modifier (e.g., sugar and modified cellulose) into the sample to slow down reagent migration.

Preferably, the immobilized reagent in the second region is impregnated throughout the thickness of the backing (e.g., throughout the thickness of the sheet or strip if the backing is in this form). Such impregnation can enhance the extent to which the immobilized reagent can capture any analyte from the migrating sample.

The reagents can be applied to the support material in various ways. Various printing techniques have heretofore been proposed for applying the liquid reagents to the supports, such as micro-syringes, metered-coil springs, direct pressure and jet-jet printing. Each of these techniques may be used in the present context

are used. To simplify the manufacture of the carrier

(For example, a sheet) are treated with the reagents and then divided into smaller sections (for example, small narrow strips, each having the areas containing the necessary reagents) to a plurality of identical carrier units

to produce gc.

For example, only preferred examples of

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- ΠΙ embodiments of the invention described in detail with reference to the accompanying drawings.

Embodiment 1

Figures 1 and 2 show a typical strip of porous solid phase material for use in an assay assay according to the invention and illustrate the basic principles upon which the invention operates.

FIG. 1 shows the assay test strip 10 as a rectangular strip whose (for the purpose of this description) longitudinal axis runs in the vertical direction. In the region of the lower end 11 of the strip 10 is a narrow band or a region 12 which extends over the entire width of the strip. A small area 13 of the strip 10 lies vertically below the area 12. Above the area 12 is a second area \ k, which lies a certain distance further up on the strip JO and extends similarly over the entire width of the strip. The region 15 of the strip 10 between the regions 12 and K can have any height, as long as only the two said regions are separated. Another region 16 of the strip extends above the region 1 (f. At the top of the strip, there is a porous pad 18 fixedly connected to the strip 10 so that the pad 18 acts as a "sink" for sample liquid which rises through the strip 10 by capillary action.

Region 12 is loaded with a first antibody that has a visible (direct) label (for example, colored latex particles, a

QQ color sol or a gold sol). This reagent can be used in presence

to move a liquid sample freely through the strip. In the area Ik, the strip is impregnated with a second antibody which has a specificity for different epitopes of the same analyte as the first antibody. The second antibody is firmly immobilized on the strip.

FIG. 2 shows what happens when the assay strip is analyzed in an anaerobic fashion.

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lytic method is used. The lower end 11 of the dry strip is contacted with a liquid sample (not shown) which may contain the analyte to be determined. By capillary action, the liquid in the strip rises and eventually reaches the pad 18. The sample passes through the region 12 and the labeled antibody will dissolve or disperse in the sample and migrate with it through the strip. When migrating in the direction of region 14, the labeled antibody can be bound to the analyte present in the sample: ι. Upon reaching the region 14, each molecule of the analyte should be bound to the second antibody so as to immobilize the "sandwich" thus prepared. If there is a significant concentration of the analyte to be detected in the liquid sample, then, for a short time, a distinct accumulation of visible label in the region 14 should occur.

As an analytical example for which the embodiment can be used, the analyte may be hCG, the reagents in regions 12 and 14 may be monoclonal antibodies to hCG participating in a sandwich reaction with hCG, and the label may be a particular dye, a gold sol, or colored latex particles.

Although the above embodiment has been described in terms of a "sandwich" reaction, it will be understood by those skilled in the art that the embodiment may also be modified for a "competitive" reaction (competitive reaction) wherein the labeled reagent is in the range 12 is the analyte or an analogous substance to the analyte.

An assay based on the above principles can be used to determine a variety of analytes by choosing suitable specific binding reagents. The analytes may include, for example, proteins, haptens, immunoglobulins,

gg hormones, polynucleotides, steroids, drugs, pathogens for

Infectious diseases (of bacterial or viral origin), such as Streptococcus, Neisseria and Chlamydia s ~ in. Sandwich assays

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For example, for detection agents such as hCG, LH, and infectious agents, competitive assays may be performed on, for example, detection reagents such as E-3-G and P-3-G.

The determination of more than one analyte which may be present in the sample may have considerable clinical value. For example, the ratio; the level of apolipoproteins A and B is an indication of the susceptibility of coronary heart disease. In

Similarly, the ratio of the levels of glycated hemoglobin may be similar

(HbH) to unglycated (HbAo) or to total hemoglobin (Hb) in the treatment of diabetes. In addition, it is possible to work out tests to determine two steroids simultaneously, for example E-3-G and P-3-G. For example, a duplicate test, ie a test for the detection of two detection substrates? ' n, for the apolipoproteins

A. and B prepared by wet that in two spatially separated areas antibody specific for apolipoprotein A.₍ in a first region and an antibody specific for apolipoprotein B is applied throughout an entire second region of a porous support matrix.

After applying the two antibodies in the respective regions by a suitable application method (for example, inkjet printer metering pump and airbrush), the remainder of the porous material should be treated with a reagent such as bovine serum albumin, polyvinyl alcohol or ethanolamine to provide any remaining binding sites To block. A third and fourth reagent carrying a tagging agent may then be applied to the dry support in one or more areas near one end of the strip, the strip being dried in the same area between the applications of the two reagents

OQ will. Reagents 3 and 4 can be conjugates of anti-Apolipo

contain protein A j antibodies and anti-apolipoprotein B antibodies. These two conjugates become mobile in and on the carrier when they reach the wet state. The reagents 3 and 3 can migrate with the solvent stream when an aqueous sample is applied to the

"5- first end of the carrier strip is applied. During the hike

towards the two regions along the strip, the reagent 3 may comprise apolipoprotein A present in the sample and the reagent

k bind apolipoprotein B present in the sample. Upon reaching the first region with the second antibody (anti-apolipoprotein A antibody region), anti-apolipoprotein A molecules should be bound to the second antibody, thereby immobilizing the labeled sandwich produced in this manner. No labeled apolipoprotein S molecules are bound to this first region. Upon reaching the second region with the second antibody (anti-apolipoprotein B antibody region), the apolipoprotein B molecules should be bound to the second antibody (solid phase antibody)

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No labeled apolipoprotein A. molecule will bind to the second region. Accumulation of each of the direct labels may take place at either or both of the regions to a weaker or stronger level, yielding a visible signal at one or both of the solid state antibody regions.

Excess unbound conjugate (from both reagent 3 and reagent k) can pass freely over the two antibody regions and is washed into the outboard end of the strip.

on The development of a quantifiable color in both of the areas for

the second antibody can be detected by suitable instruments which provide a ratio of color density between the two sites.

ok Determining the presence of more than two analytes

(multiple system) in a sample can have considerable clinical value. For example, the detection of the presence of various serotypes of a bacterium or the detection of the presence of soluble serological markers in humans can

ο «be helpful. For example, a multiple test for detection

the presence of different serotypes of Streptococcus for groups A, B, C and D. A mixture of monoclonal antibodies, each of which is specific for several pathologically important group serotypes, or a polyclonal antiserum raised against a particular streptococcal group, can be applied to a porous carrier strip in line across the width of the strip of about 1 mm become. Multiple lines should

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in spatially separated areas, each area containing one or more immunochemically reactive components suitable for binding the analyte of interest. After applying the multiple areas according to a suitable application method (for example, ink jet printing, metering pump and nib, airbrush), the remainder of the porous material should be mixed with a reagent (eg Bovine serum albumin, polyvinyl alcohol, ethanolamine) to block any remaining binding sites. Labeling conjugates, for example a color sol,

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Bacterial group can then be applied either to a single area at the bottom of the strip, near the sample application area, or as a series of separate areas.

Figures 3, k and 5 of the drawings show a complete apparatus operating with a porous strip as just described. Figure 3 shows the complete apparatus seen from the front, Figure k shows the same apparatus with the front wall partly broken away to show the details of the inside strip and Figure 5 shows the apparatus from below.

The device according to Figure 3 has a flat rectangular body 30, the front side 31 is provided with a hole or window 32 which releases the view of the porous test strip 10 within the body. The region of the test strip 10 to be seen through the window 32 includes a narrow horizontal region 1 * f.

As can be seen in Figure 4, the apparatus has a dry rectangular test strip made of a porous material and

QQ extends from the lower end 33 of the body 30 within the body

Between the front plate 31 and the rear plate 34 of the body, near the lower end 11 of the strip 10, there is a horizontal region 12 carrying a labeled specific binding reagent for a detection substance, which binding reagent in the

ok test strip is mobile in the wet state. Next up in the

Test strip is a narrow horizontal area Ik, which is visible through the window 32. At the top 17 of the test strip

10 is a porous "sink" 18 that can absorb sample fluid that has migrated up the strip.

As can be seen in Figure 5, the lower edge 35 of the body 30 has a lateral opening in which the lower end 11 of the strip lies.

In use, the lower end 32 of the body 30 is immersed in a liquid sample (eg urine) so that the liquid sample is absorbed by the lower end 11 of the test strip 10 and rises by capillary action to the upper end 17 of the test strip and the sink 18. In this case, the liquid sample moves over region 12 to region 14. Specific binding reactions take place as described above and the test result is visible to the user through window 32.

Embodiment 2

Figures 6 and 7 show another test device according to the invention.

FIG. 6 shows the complete device seen from the front, and FIG. 7 shows FIG. 20

the same device with partially broken parts to visualize details of the test strip contained in the device body.

As shown in FIG. 6, the apparatus has an elongate body 200 which abuts

its lower end 201 into a small integral receptacle 202 25

passes, which can take a predetermined amount of a liquid sample, such as urine. The front panel has two small square windows 204 and 205 arranged one above the other.

As shown in Fig. 7, the elongated part of the body 200 is hollow 30

and contains a test strip 206 that extends almost the entire length of the body. This test strip has a similar structure to that described in connection with embodiment 1 and contains a horizontal region near its lower end 207.ⁿ*. el ·

carries a labeled specific binding reagent, which in the 5

Strips can migrate freely when wet. In the area of ​​the windows 204 and visible through these are two circular areas

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^ 209 and 210. The strip ends at its upper end 211 in one

porous sink 212. At the lower end of the test device, the receptacle 202 communicates with the cavity of the body via a lateral opening 213 in connection. '

In operation, a liquid sample is brought to the bottom of the apparatus and a predetermined volume of the sample fills the receiver 202. From the receiver 202, the liquid rises by capillary action through the test strip 206 and conveys the labeled reagent from the region 2OS to the two circular regions 209 and 210. A series of specific binding reactions as described with reference to Embodiment 1 take place. In this embodiment, the second circular region 210 may serve as a control region (for example, producing a color signal whether or not the sample contains the analyte to be determined) while the determination of the analyte is in the first circular region 209. The user can determine if the analyte is present in the sample by comparing the signals generated in the two regions.

For example, if the test is intended to determine the presence of

hCG in the urine as part of a pregnancy test, the circular control region 210 may contain immobilized hCG which binds a labeled antibody coming from the region 208 with the _K wandering fluid sample is carried upward. The same

labeled antibody may participate in a sandwich reaction with hCG in the sample and is bound in the first circular region 209 by another specific anti-hCG antibody immobilized there. If desired, the control panel can also

alternatively, only a non-specific signal to the user 30th

which shows that the device has worked. For example, the second circular region may be loaded with an antibody that binds to the labeled antibody from region 208, for example, an "anti-mouse" antibody if the labeled antibody is one

which has been derived using a murine hybridoma, 35

to confirm that the sample has passed the test strip.

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- 20-, Embodiment 3

Fig. 8 of the drawings shows an isometric view of an assay device according to the invention and Fig. 9 shows the device shown in Fig. 8 cut parallel to a narrow side.

As seen in Fig. 8, the apparatus comprises a housing 500 of oblong rectangular shape having at a end 501 a portion 502 of reduced cross-sectional area. A cap 503 may be placed on the portion 502 and abuts the shoulder 504 at the end 501 of the housing. The cap 503 is shown separated from the housing 500. Extending beyond the end 505 of section 502 is a porous member 506. When the cap 503 is placed on the portion 502 of the housing, it covers the porous part 506. The top wall

507 of the housing 500 includes two openings 508 and 509. 15

As can be seen in Fig. 9, the housing 500 is hollow. The porous member 506 extends into the housing 500 and communicates with a strip of porous substrate 510. The porous part 506 and the

Strips 510 overlap each other to ensure that a 20

there is sufficient contact area between these materials and that the liquid sample applied to the part 506 can pass through this part 506 and enter the strip 510. The strip 510 extends further into the housing 500. The strip 510 is through

a support strip 511 reinforced, made of transparent, moisture-25

impermeable plastic material is made. The strip 510 extends beyond the openings 508 and 509. By means of webs 512 and 513 in the housing 500, the strip 510 is held firmly in place. The internal structural details of the housing do not constitute a significant aspect of the invention, it is important that the strip is firmly f

ii

held in place within the housing and the porous part 506 f

is also held in the housing, and that a sufficient |

Fluid passage contact between the part 506 and the strip 510 is ensured. The transparent reinforcing strip 511 is located

between the 5treifen 510 and the openings 508 and 509 and can as 35

Seal and prevent the ingress of moisture from the outside into the housing through these openings. If desired, the

·· 111

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- 21-

^ Residual space 51 ^ moisture-absorbing within the housing

Material such as silica gel to keep the strip 510 dry during storage. The reagent-containing areas of the strip 510 are not shown in FIG. 8, but the first area

g with the labeled reagent, which is mobile when the strip

is within the range between the porous portion 506 and the opening 508. The second area containing the immobilized unlabelled reagent is in the area visible through the opening 508 so that in use of the apparatus in an assay Result through the opening 508 can be observed. The opening 509 is a means by which a control area containing other reagents that allow the sample to pass appropriately through the strip can be observed.

In use, the protective cap 503 is removed from the holder and the portion 506 is exposed to a liquid sample, for example held in a urine stream in the event of a pregnancy test. After the portion 506 has been exposed to the liquid sample for a time sufficient to saturate the portion 506 with the sample, the cap 503 may be re-exposed and the instrument may be re-used by the user for an appropriate time (e.g., 2 or 3 minutes) Page as the sample passes through the test strip 510 to provide the analytical result. After the appropriate time, the user can observe the test strip through openings 508 and 509 and through

Observing the control area through the opening 509 determine whether 25

the assay is complete. It can read the result of the assay by observing the second area through the opening 508.

In manufacture, the device can be easily assembled from, for example, plastic material, with the housing 500 being cast in two parts (e.g., the upper and lower halves 515). These halves can be securely fastened together (for example, by ultrasonic welding) after the porous part and the test strip are inserted into one of the halves and then enclosed by the two halves. In this sandwich construction, the porous part and the test strip can be pressed against each other to ensure sufficient contact between the two parts

• • »it ·· · ItIt

- 22-

sure. The cap 503 can be cast as a separate complete component. If desired, apertures 508 and 509 may be provided with transparent inserts which provide greater security against the ingress of moisture from outside the housing. Due to a tight fit between the end 505 of the 5th

Housing 500 and the above porous member 506 ensures that a sample applied to the protruding part does not directly enter the device and the part 506 bypasses. The part 506 thus provides the only access route for the sample to the strip within the

Housing and deliver the sample to the strip in a controlled 10

Wise. The device as a whole thus combines the function of sampling and analyzing.

When using the test strip materials and reagents as in

As described below, according to FIGS. 8 and 9, a device 15

Especially suitable as a pregnancy test kit or ovulation cycle kit for home or hospital use. The user need only apply a urine sample to the exposed porous part and then (preferably after re-starting

the cap), the test result through the opening 508 within a few 20

Watch for minutes.

Although the device is described with reference to pregnancy and ovulation cycle tests, it may become a liability

a variety of detection substances are used when 25

suitable reagents are introduced into the test strip. The opening 509 is. redundant and can be omitted if the test strip contains no control agents. The general shape of the housing and the cap both in terms of their length and in terms of their

Cross-section and other physical features can be considerably 30

be varied without the mind de^r Deviated from the invention.

Similarly, the labeled reagent may be omitted from the test strip when this reagent is added to the sample prior to application of the sample to the test device. Alternatively, the marked

Reagent may be contained in the above porous member 506.

Fig. 10 shows on an enlarged scale the porous receiving part and the test strip in the apparatus shown in Figs. 8 and 9.

The porous receiving portion 506 is coated with the porous test ¹J10, which is reinforced by a transparent plastic sheet 511, connected, so that liquid in the direction of the arrows drawn through the porous A'jfnahmeteil can flow into the porous strip. The test area 517 contains the immobilized specific binding reagent and the control area 518 contains a reagent which indicates that the sample is a,,_N has passed through a sufficient piece along the test strip. A part

the surface of the test strip opposite to the reinforcing strip 511 and near the porous receiving part 506 carries a glaze 519 on which a layer 520 of a labeled specific binding reagent is attached. The thickness of these two layers, as shown in FIG. 10

shown is greatly exaggerated for purposes of illustration. In the 15th

Practice, the glaze is not a surface layer, but that

Glaze material will penetrate the thickness of the strip to some extent. Likewise, the labeled reagent then applied will also penetrate the strip. Nonetheless, the key objective of reducing interactions between the ZO

labeled reagent and the carrier material forming the strip reaches. An aqueous sample, which is placed on the receiving part 506, flows therefrom in the longitudinal direction of the strip 510, dissolving the glaze 519, mobilizing the labeled reagent and carrying it

labeled reagent along the strip through area 517. 25

Embodiment k

11 and 12 show a further embodiment of the invention, which is shown in Fig. 11 in plan view and in Fig. 12 in section, wherein the section along the line A in Fig. 11 is made.

As shown in FIG. 11, the test apparatus has a flat rectangular housing 600 having a centrally disposed rectangular opening 601

near the left end 602 and two more openings 603 and ..

60 ^ near the center of the device, so that the openings 601, 603 and 604 on the central longitudinal axis of the device accordingly

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- 24-

the section line A lie. Although all three openings are shown as rectangular, their actual shape is not critical.

After the longitudinal section of Fig. 12, the device is hollow and contains a

porous sample receiving part near the end 602 of the housing 600, 5

which lies directly below the opening 601. A test strip of similar construction to that described with reference to Embodiment 4 and which includes a porous strip 606 reinforced by a transparent plastic sheet 607 is also provided within the housing 600 and extends

extends from the porous receiving portion 602, with which the porous carrier is in fluid communication contact, to the other end of the housing. The transparent reinforcing sheet 607 is in firm contact with the upper interior surface 608 of the housing 600 and provides a seal against the openings 603 and 604 to prevent ingress of moisture or moisture

Sample liquid into the housing to prevent. Although not shown in the drawings, the test strip 606 contains a labeled specific binding reagent, a test area, and a control area that are in appropriate relation to the openings 603 and

604 lie, as described with reference to the embodiment 3. 20

In use, an aqueous sample is applied through the orifice 601, for example by means of a syringe, to saturate the porous receiving member 605. Thereafter, the aqueous sample can pass through the test strip, and after a suitable time, the test result by

the openings 603 and 604 are observed.

Embodiment 5

A further embodiment of the invention is shown in FIGS. 13 and 14

shown. FIG. 13 shows a device with a rectangular housing 700, which has a rectangular opening 702 in its upper surface 701. An end wall 703 of the device includes an opening 704 through which a porous test element communicates with the outside of the device. An opening 702 is disposed in the upper surface 701 at a point that is relatively far from the end 703 with the opening 704.

»■

- 25-

Fig. 14 shows the apparatus of Fig. 13 with parts partially broken away. The hollow device includes a porous test strip 705 that extends almost the entire length of the housing 700, starting from the opening c 70V. The test strip 705 includes a first region 706

a labeled specific binding reagent and a further region 707, away from the opening 704, which contains an immobilized specific binding reagent. The area 706 is directly under the opening 702 and is therefore from the outside of the housing to_1Q observe. Below the strip 705 and near the area

707 is a frangible element 708 containing one or more substrates or reagents which may be used to generate a detectable signal when released into the region 707 if labeled reagent of 706 is present in the region 707 during use the device has been bonded. The release of the reagents from the part 708 may be achieved by applying pressure to the outside of the housing at this point to break the part and express the reagent therefrom.

In operation, the first test element may be exposed to an aqueous sample, for example, by dipping the end 703 of the housing 700 into a vessel containing the sample. The liquid sample will then pass the length of test strip 705, taking labeled reagent from region 706 and passing through region 707 where the labeled reagent will be bound, for example by a "sandwich" reaction involving the analyte contained in the sample , When the sample has passed the test strip, reagent may be released from the frangible portion 708 and the result of the test may pass through the opening 702

to be watched. 30

For purposes of example only, various test strip materials, reagents and methods for their preparation will be described below.

1. Selection of liquid-conducting material

Representative examples of liquid-conducting materials include paper, nickel, and nitrile membranes. Essential properties of the materials are the ability to bind protein, the rate of liquid conduction; and, if necessary, after pretreatment, the ability to pass labeled antibodies along the strip. If it is a direct label, it may be desirable for the material to control the flow of particles as small as a few microns (usually less as 0.5μ). Examples of flow rates achieved with various materials are given below:

Nitrocellulose (unreinforced) (by Schleicher + Schuell)

<tgroup cols="2"><tbody><row><entry>pore size</entry><entry>Needed time</entry></row><row><entry></entry><entry>for 45mm flow</entry></row><row><entry></entry><entry>away (minutes)</entry></row><row><entry>3μ</entry><entry>3.40</entry></row><row><entry>5μ</entry><entry>3.30</entry></row><row><entry>8μ</entry><entry>3.00</entry></row><row><entry>12μ</entry><entry>2.20</entry></row><row><entry>8μ (nominal)</entry><entry>3, ' "0</entry></row><row><entry>5</entry><entry>19.20</entry></row><row><entry>3</entry><entry>4.00</entry></row><row><entry>5</entry><entry>3.20</entry></row></tbody></tgroup>

polyester reinforced

Whatman nitrocellulose Pail "Immunodyne" (nylon)

The speed of the test procedure is determined by the flow rate of the material used. While any of the above materials may be used, some perform faster than others.

Nitrocellulose had the advantage that no activation was necessary and that it strongly immobilized proteins by absorption. "Immunodyne" is pre-activated and does not require any chemical treatment. Papers, such as Whatman 3MM, require chemical activation, for example. · .- .; ■■ - ?. it is carbonyldiimidazole to successfully immobilize the antibody.

Second markers

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Preparation of the marking substances

A selection of tags that can be used is given below. The list is not exhaustive.

A) Gold sol production

Gold sols may be prepared for use in immunoassays from commercially available colloidal gold and an antibody preparation, such as a preparation of anti-alpha human chorionic gonadotrophin .

For example, colloidal gold G20 (20nm particle size, supplied by Janssen Life Sciences Products) is adjusted to pH 7 with 0.22p-filtered 0.1 molar K-CO, and 20ml is placed in a clean glass container. 200μl of anti-alpha hCG antibody prepared in 2mM Borax buffer pH9 at 1mg / ml and 0.22μ filtered is added to the gold sol and the mixture is stirred continuously for 2 min.

0.1 molar K₂CO, is used to adjust the pH of the antibody-gold-sol mixture to 0.9 and 2 ml of 10% (w / v) BSA (bovine serum albumin) is added.

The antibody gold is digested in a series of three centrifugation steps at 1200g, 30 min, and ^⁰C, whereby only loose parts of the pellet are resuspended for further use. The final pellet is resuspended in 1% (w / v) BSA in 20 mM Tris, 15 mM NaCl pH 8.2.

B) Color Sol Preparation

Coltsols can be prepared from commercially available hydrophobic dyes such as Foron Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer). 50 g of dye is dispersed in distilled water by mixing with a magnetic stirrer for 2 to 3 minutes. The fractionation of the dye dispersion may be carried out by an initial centrifugation step at 1500 g for 10 minutes at room temperature to obtain larger sol particles

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in the form of a solid pellet (ball), the supernatant (suspension A) being retained for further centrifugation.

Suspension A is centrifuged at 3000 g for 10 min at room temperature, the supernatant discarded and the pellet resuspended in 500 ml of distilled water. This procedure is repeated three more times, with the last pellet resuspended in 10 ml of distilled water.

The spectra of the color sols prepared as described above can be measured. Their lambda max is around 657nm for Foron Blue and 690nm for Resolin Blue. The absorbance at Lambda-Max on learning path length is used as an arbitrary measure of the dye concentration.

C) Colored particles

Latex (polymer) particles for use in immunoassays are commercially available. These may be based on a number of synthetic polymers such as polystyrene, polyvinyltoluene, styrene-acrylic acid copolymer and polyacrolein. The monomers used are normally water-insoluble and are emulsified in aqueous surfactants to form monomeric mycelia, which are then allowed to polymerize by adding an initiator to the emulsion. Essentially, spherical polymer particles are produced.

Colored latex particles can be produced, either by adding a suitable dye, such as anthraquinone, to the emulsion prior to polymerization, or by staining the preformed particles. In the latter approach, the dye should be dissolved in a water-immiscible solvent, such as chloroform, which is then added to an aqueous suspension of latex particles. The particles take up the non-aqueous solvent and the dye and can then be dried.

Preferably, such latex particles have a maximum dimension of less than 0.5 microns.

* "* ■■ ·

-29-

Colored latex particles can be sensitized with protein and especially antibodies to provide reagents for use in immunoassays. For example, polystyrene granules about 0.3 micron in diameter (available from Polymer Laboratories) may be sensitized with anti-alpha human chorionic gonadotrophin in the process described below:

0.5 ml (12.5 mg solids) of a suspension are diluted with ImI 0.1 M borate buffer, pH 8.5, in an Eppendorf tube. These particles are washed four times in borate buffer, each wash consisting of a 3 minute centrifugation at 13000 rpm in an MSE microcentrifuge at room temperature. The remainder of the pellet is resuspended in ImI borate buffer, mixed with 300 μg of anti-alpha hCG antibody and the suspension rotated end-over-end for 16 to 20 h at room temperature. Centrifuge the antibody latex suspension at 13000 rpm for 5 min, discard the supernatant and resuspend the pellet in 1.5 ml borate buffer containing 0.5 mg bovine serum albumin. over-end), the suspension is washed three times in 5 mg / ml BSA in phosphate buffered saline pH 7.2, by centrifugation at 13000 rpm for 5 min. The pellet is resuspended in Jmg / ml BSA / 5% (w / v) glycerol in phosphate buffered saline pH 7.2 and incubated at 4⁰C stored until use.

(A) Preparation of anti-hCG color sol

Protein can be coupled to a dye sol in a process that involves passive adsorption. The protein may be, for example, an antibody preparation, such as anti-alpha human chorionic gonadotrophin prepared in phosphate buffered saline pH 7, * t at 2 mg / ml. A reaction mixture is prepared containing ΙΟΟμΙ antibody solution, 2 ml of coltsol, 2 ml of 0.1 M phosphate buffer pH 5.8 and 15.9 ml of distilled water. After gentle mixing of this solution, the preparation is allowed to stand for 15 minutes at room temperature. Excess binding sites can be blocked by addition of, for example, bovine serum albumin: 4 ml of 150 mg / ml BSA in 5 mM NaCl pH 7.4 is added to the reaction mixture.

! • If ·· ···

-30-

After 15 min of incubation at room temperature, the solution is centrifuged at 3000 g for 10 min. The pellet is resuspended in 10 ml of 0.25% (w / v) dextran / 0.5% (w / v) lactose in 0.04M phosphate buffer. The antibody-dye sol-conjugate is best stored in freeze-dried form.

B) Preparation of the LH color sol

Due to the structural homology between the alpha subunits of hCG and LH, alpha hCG antibodies can be used to target LH

to detect in a cross-reactive immunoassay. Thus, a labeled antibody for use in an LH assay can be prepared in the same manner as described in Example 1 using anti-alpha hCG antibody. 15

Third Production of the reaper strip

Partial impregnation of liquid-containing materials

A liquid-conducting material with a limited range of immobilized protein, in particular an antibody, can be prepared, for example, as follows:

A rectangular sheet of 8p-nitrocellulose, reinforced (Schleicher and Schueil) with a length of 25cm and a width of 20cm should get a reaction area by applying material in a line of γγγ ,, width in intervals of 5cm in the longitudinal direction which extends over the entire 20cm width. For example, the material may suitably be selected from an antibody preparation such as anti-beta (human chorionic gonadotrophin) having an affinity Ka of

10, prepared in phosphate buffered saline pH 7.4 at 2 mg / ml, which is suitable for immunoassay for human chorionic gonadotrophin using a second (labeled) anti-hCG antibody in sandwich format. This solution can be applied by means of a microprocessor controlled microsyringe delivering precise volumes of reagent through a nozzle.

voi / Algsweise with 2mm diameter. After the material has been allowed to dry for 1 h at room temperature (excess binding of the nitrocellulose with an inert component such as polyvinyl alcohol (1% w / v in 2 mM Tris pH 7, &lt;*) is blocked for 30 min at room temperature rinsed with distilled water and then 30 min at 30⁰C dried.

In one embodiment, the liquid-conducting material may then be cut into numerous strips 5 cm in length and one lenth in width, each strip bearing a limited area of ​​immobilized antibody which acts as an immunoabsorbent part way (for example, about half way) of its length , In this example, the test strip is used with a liquid mark which is mixed with the sample. In use, this limited area then becomes a test reaction area where the immunoassay reactions take place.

According to another embodiment, the mark may be applied in a limited area before the liquid-conducting material is cut into strips. For example, this reagent may be a colourant or a dye-polymer-conjugated anti-hCG antibody prepared as described above under "Preparation of the colourant". This reagent is retained in the area when the material is in the dry state, but it can freely migrate through the substrate when the material is wetted, for example, by applying the liquid sample containing the analyte to be determined. This area with the mobile reagent is applied, for example, as follows:

A sheet of 8M nitrozellose, reinforced (Schleicher and Schuell), 25cm in length and 20cm in width, and immobilized antibody areas at 5cm longitudinal distances are prepared as described above. Before applying the dye-labeled antibody, an intermediate layer of, for example, 60% w / v. Sucrose in distilled water by air brushing applied to the microprocessor-controlled system at 6cm intervals along the length of the sheet. Then in several passes (for example in three

Runs) of dye-labeled antibody coated in 1% Methacel KAM (trademark for methylcellulose from the Dow Chemical Company) and 0.6% (w / v) polyvinyl alcohol by air brushing or by a microsyringe directly onto the top of the backsheet. The leaves are then dried and cut into strips 5cm in length and learning width and ready for use in the finished device.

Gold sols or colored polystyrene labels can be applied in a similar process.

In addition to the test area, various control areas can be operated as desired. For example, a range of anti-species IgG may be applied behind the test area. 15

'F. Sandwich assays using the strip format

A sandwich-type reaction can be used to detect human chorionic gonadotrophin (hCG) in a liquid sample. The marker used is preferably a direct label that can be easily seen by the naked eye. Colloidal sols, gold sols or colored latex particles can be bound to the anti-hCG antibody as described above.

For direct markers, assays can be performed in which fresh urine samples are transferred directly from the urine stream or from a suitable volume (eg, ΙΟΟμΙ) from a container to the absorbent wick of the Teitg device with the aid of a pipette. Each sample is run for 5 minutes in the portrait and the color produced in the reactive region is read either by eye or using a light reflectometer.

Indirect labels, such as enzymes, such as alkaline phosphatase, may also be used, but require the addition of a substrate to produce a colored end-point.

Using conventional techniques, enzyme assays

in which the anti-hCG antibody is conjugated to alkaline phosphatase, dissolved 1: 100 in 0.01M phosphate buffered saline pH 7 with 3% polyethylene glycol 6000, 1% (w / v) bovine serum albumin and 0 , 02% Triton X305 (trademark - available from Rohm and Haas) before application to the sheet. Fresh urine samples are then applied to the absorbent wick of a test device, either directly from the urine stream or by providing a suitable volume (eg, ΙΟΟμΙ) from a container using a pipette. Each sample is run for 5 minutes before a pad of liquid-swellable material contacted with BCIP substrate (at 1 mg / ml in IM Tris / HCl pH 9.8) is brought into contact with the antibody immobile area. After a further 5 minutes, the pad is removed and the color produced read by eye or using a light reflectometer.

A similar embodiment can be made using luteinizing hormone (LH) instead of hCG.

5th Competitive assays

For example, a competitive type assay may be performed by estrone-3-glucuronide, a urinary metabolite of estrone. Conjugates of estrone-3-glucuronides and bovine serum albumin are prepared as follows:

Preparation of BSA-estrone-3-glucuronide

The conjugation of E-3-G and BSA can be achieved using a mixed anhydride. All glass containers, solvents^unc* Reagents used in the preparation of the activated species must be carefully dried using a furnace, desiccator or molecular sieve for at least 24 hours.

Solutions of E-3-G (2 mM) in dry dimethylformamide (DMF) and tri-n-butylamine (TnB) (10 mM) in dry DMF were separated at.⁰C equilibrates. Using pre-cooled glass containers

Add E-3-G in DMF (1.25 ml) and TnB in DMF (0.25 ml) to the pre-cooled 5 μl of a liquid containing a magnetic stirrer Isobutylchloroformate in dry DMF (10 mM) was prepared and a portion (0.25 ml) was cooled to &lt;* ° C. and added to the reaction vessel. The contents of the reaction vessel were incubated for 20 min⁰C. and a solution of BSA (1 mg / ml) was prepared in bicarbonate buffer (0.5%). When the mixed anhydride incubation was complete, the contents of the reaction vessel were added to the BSA solution (2.5 ml) and stirred with a magnetic stirrer U h at 4 ° C. The conjugate preparation was equilibrated by passing over a Tris buffer, Pharmacia PD-10 S ^ phadex G-25 column, transferred to a brown glass storage container and stored at 4 ° C.

Preparation of the BSA-E-3-G Farbsols

A dispersion of dye (5% w / v) in distilled water was prepared by careful mixing and aliquots were centrifuged at 3850 rpm (1500g) for 10 minutes in a bench top centrifuge. The pellet was discarded and the supernatant was maintained and centrifuged in aliquots at 4850 rpm (3000g) for 10 minutes in a benchtop centrifuge. The supernatant was decanted and the pellet was resuspended in half of the original volume in distilled water. This step was repeated four times to wash the pellet. The pellet was finally resuspended in distilled water and the absorbance at lambda max determined.

Solutions of the color sol in distilled water and E-3-G / BSA conjugate dissolved in phosphate buffer were mixed to a final concentration of 10 μg / ml conjugate (based on BSA content) and to an extrapolated optical density of the color sol of 20 at the absorption maximum. The reaction mixture was incubated for 10 min at room temperature and blocked for 15 min at room temperature with BSA in NaCl solution (5 mM, ph 7, k) to finally give a final BSA concentration of 25 mg / ml. The reaction mixture was centrifuged at 48&gt; C w.in (3000g) for 10 minutes in a bench top centrifuge. The supernatant was drained off and the pellet resuspended in half of the original volume in dextran (0.25% w / v) / lactose (0.5%).

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-35 μg / volume) of phosphate (0.04M pH 5.8) buffer.

Preparation of the E-3-G test strips

Antibodies to E-3-G were applied as described in Example 3.

BSA E-3-G color sol was applied to the strip as described in FIG.

Determination of E-3-G

Using the reagents described above, a standard curve can be generated by exposing the strips to samples containing known concentrations of E-3-G. The color in the immobilized area can be read, for example, using a Minolta chromameter, and the concentration of E-3-G can be calculated by extrapolation from the reflectance value.

The invention described above extends to all such modifications and variations as would be obvious to one skilled in the art, and also extends to all combinations and subcombinations of the features of this specification and the accompanying drawings.