WO2007110702A2

WO2007110702A2 - Assays

Info

Publication number: WO2007110702A2
Application number: PCT/IB2006/004158
Authority: WO
Inventors: Balbir Raj
Original assignee: Inverness Medical Switzerland Gmbh
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2007-10-04
Also published as: WO2007110702A3

Abstract

Provided are assays and devices to detect a target compound in urine.

Description

ASSAYS Related Applications

This application claims the benefit of the filing date of United States Provisional Patent Application serial number 60/739,464, filed November 23, 2005.

Field of the Invention

The invention relates to assays. Background

Assay devices can be used to assay urine for the presence of a target compound. Summaiy

The invention relates to assays. hi one aspect, a device is configured to detect a target compound (e.g., a compound associated with pregnancy (e.g., hCG)) in a sample (e.g., a liquid sample such as urine, saliva, or a blood related sample (e.g., plasma)).

hi some embodiments, the device includes a response zone configured to produce a first discernible response (e.g., a response discernible by visual, optical, or electronic inspection) indicative of the presence (e.g., the presence above some threshold) of the target compound in the sample and a second discernible response (e.g., a response discernible by visual, optical, or electronic inspection) indicative of the absence (e.g., the presence below some threshold) of the target compound in the sample.

The first and second discernible responses are typically different (e.g., the responses can be distinguished by visual, optical, or electronic inspection). For example, the presence of the target compound can be indicated by the appearance of a first color (e.g., a colored line, such as a blue line) within the response zone and the absence of the target compound can be indicated by the appearance of a second, different color (e.g., a colored line, such as a white line) within the response zone. hi some embodiments, the response indicative of at least one of the absence of and/or the presence of the target compound results from an immunochemical event. hi some embodiments, the device includes a sample receiving zone configured to receive a sample (e.g., a liquid sample) that is suspected of having a target compound. The response zone may be disposed within (e.g., on) a support (e.g., a nitrocellulose membrane) configured to support movement (e.g., lateral flow) of sample liquid along the support (e.g., from the sample receiving zone to the response zone).

In some embodiments an assay improves indication of a negative hCG result as compared to known consumer pregnancy tests. A single line is used to relate both negative and positive hCG results. A negative test result is indicated by the development of a first colored line (e.g., white) on a nitrocellulose membrane, which line is formed during the running of the test. In the instance where the sample contains hCG at a level that signifies pregnancy this line no longer appears the first color, instead it is seen to be a second color (e.g., blue). The assay can be compatible with the materials and procedures used in Unipath Rapid Assay Tests.

In some embodiments, a device produces a first physical change indicative of pregnancy and a second, different physical change in the absence of pregnancy. Such a physical change can signal a negative result by the development of a colored line within the device. Furthermore, this indication typically results from an immunochemical event, manifesting itself during the running of the test in a negative sample. In the instance where hCG is present at a level expected in pregnancy, the same line would take on a different color, to indicate a positive result.

The assays described herein can be compatible with the materials and procedures currently used in the generation of Rapid Assay Tests at Unipath. White latex particles can be used to create a white line and a blue line can be formed using blue latex particles.

In some embodiments, devices described herein may include a second zone configured to indicate (e.g., in a manner suitable for determination by visual, optical, or electronic inspection) whether or not the test has (or is) operated properly. The first and second zones may overlap at least partially (e.g., the test and control zones may be contiguous).

Brief Description of the Drawings

FIG. 1 illustrates an assay device.

FIG. 2 illustrates an assay device.

FIG. 3 illustrates generation of a discernible response.

FIG. 4 illustrates response of an assay device. Detailed Description

Referring to FIG. 1, an assay device 10 includes a lateral flow support 12 (e.g., a nitrocellulose membrane) having a sample application zone 14, a test zone 16, and a control zone 18 spaced apart from the test zone 16. Device 10 can be used to assay a liquid sample (e.g., urine) for the presence of a target compound (e.g., a hormone such as hCG indicative of pregnancy). Li use, sample is received by the application zone 14 and travels along support 12 to the test and control zones 16, 18. Test zone 16 produces a discernible response (e.g., the formation of a color) in the presence of the target compound but gives no visually discernable response in the absence of the target compound. Test zone 16 typically operates by an immunochemical reaction (e.g., a sandwich or competitive assay). Control zone 18 gives a discernible response (e.g., the formation of a color) whether or not the target compound is present. Control zone 18 also typically operates by a immunochemical reaction.

Referring to FIG. 2, an assay device 20 includes a support 22 (e.g., a lateral flow support such as a nitrocellulose membrane) having a sample application zone 24 and a response zone 26. Device 20 can be used to assay a liquid sample (e.g., urine) for the presence of a target compound (e.g., a hormone such as hCG indicative of pregnancy).

In use, sample is received by the application zone 24 and travels along support 22 to response zone 26. Response zone 26 produces a first discernible response in the presence of the target compound (e.g., in the presence of the target compound above some threshold (e.g., in the presence of hCG at a level that indicates pregnancy)) and produces a second, different discernible response in the absence of the target compound (e.g., in the presence of the target compound below some threshold).

The first discernible response includes capture of first particles in the response zone. The second discernible response includes capture of second particles in the response zone. The first and second particles have different colors. The first discernible response includes the formation of a first color in the response zone and the second discernible response includes the formation of a second, different color in the response zone.

Typically, both the first and second discernible responses can be distinguished from the response (or lack of response) that may result from improper functioning or utilization of device 20 (e.g., from failure of sample to reach response zone 26, such as can be caused by the application of too little sample). Thus, response zone 26 can typically indicate the presence or absence of the target compound and whether the device has operated or been utilized properly.

Referring to FIG. 3, an assay forming produces a white line (from white latex particles) in the absence of hCG and a blue line (from blue latex particles) in the presence of hCG. In negative hCG samples, the white latex particles would bind at the test line and reveal a white line in the test window. Samples containing levels of hCG at a level associated with pregnancy would produce a blue line (e.g., visible through a test window). The white particles may still be present. The appearance of the white particles may be masked by ihe increased number of blue particles being specifically bound via hCG. As the level of hCG increased, one may expect the binding of the white particles at the test line to be influenced, (reduced) since the blue particles may take priority for 'space' at the test line.

The Examples discussed below demonstrate that a single line can be used to indicate negative, as well as positive hCG results. Furthermore, the negative result can be instigated by specific binding events which occur whilst the test is running. Here, a single line has been seen to appear as a white line in negative hCG samples, yet the same line appears blue when hCG is present at a level indicative of pregnancy.

In the instance where the test line appears blue, it is likely that white latex particles are still present at the line. The appearance of the white particles becomes masked by the intense color of the blue anti-hCG coated beads. However, the binding of white latex particles at the line may be hindered when hCG is present at a high level. Here, one could postulate the blue beads take precedence over the white, for locating on the available space at the line on the nitro-cellulose, (the blue beads can easily bind at the line due to the high levels of hCG in the sample). The ease with which the white latex particles were visualised at the line, on grey nitro-cellulose was quite unexpected. However, the contrast of this white line may be improved by subtle modifications in the background of the nitrocellulose membrane. Use of grey tinted Mylar covers for example, may enhance the definition of the white line.

Colors other than white for the indication of a negative hCG result can be used. For example, yellow latex could be used to form a yellow line in hCG negative samples. A dry assay can be used (e.g., a true one-step pregnancy test). The Examples discussed below demonstrate use of an assay having a single line that produces a first discernible response in the presence of hCG in a buffer and a second discernible response in the absence of hCG in the buffer. An alternative second assay can be used for analysis in urine. Such a second assay can be directed at a protein or other substance, which is unlikely to be found in human urine. One example is the use of anti- fluorescein isothiocyanate, (FITC), in combination with BSA-FITC conjugate. The development of a system that incorporates two assays, which function independently, without non-specific effects in patient samples, may. be useful. A second assay for the generation of a negative hCG result may be eliminated using other assay formats. For example, coating white latex particles with a controlled amount of hCG would help provide binding of these particles to the line in negative hCG samples. Once hCG was present in the sample at a level indicative of pregnancy, the blue latex particles would begin to localise at the line to relate a positive result.

The devices described herein may omit a separate control line, hi the assay formats described in this report the user should perceive a line that is either white or blue. If no line appears this would indicate a false result and thus instigate a re-test. Furthermore, both negative and positive results emerge as a consequence of particles migrating from one portion of the test strip to another. In addition, the binding of these particles at the test line occurs as a consequence of immuno-chemical interactions that occur between reagents at the line and those on the surface of the particles. The presence of a line that is white or blue is proof that the test has been performed correctly, and also that the reagents within the device are functional. Generating a negative hCG result by virtue of white latex particles coated in hCG provides some form of control, since it indicates the reagents within the test stick are reactive towards the analyte of interest.

This work has provided a valuable insight into a concept of improving the visualisation of a negative hCG result in a Clearblue One-step Home pregnancy test, hi addition, this approach has the added advantage of being compatible with the materials and procedures currently used in the production of rapid assays at Unipath. With this in mind, there is great potential for realising improvements in the Clearblue One-step product in the near future. Examples

A pregnancy test produced a test line that appeared white in negative samples and blue in positive hCG samples.

The model system described here utilised two separate assays, which manifest themselves on a single line. Solid phase reagents for both these assays were mixed in solution and deposited as a single line onto nitro-cellulose membrane. This line was used to generate the two assays for indicating both negative and positive hCG results. The conventional RAT assay for hCG was the first assay, utilising blue latex particles coated in anti-α hCG as a label. A sandwich assay for hCG was produced when these blue latex particles were paired with nitro-cellulose membrane comprising a zone of anti-β hCG.

The second assay utilised white latex particles, which generated a white line on nitro-cellulose membrane in samples that were negative to hCG. The E3G assay was used for this purpose as a model assay, with white latex particles coated in anti-E3G antibody. Solutions of anti-β hCG and ovalbumin-E3G conjugate were mixed and deposited as a single reagent onto nitro-cellulose membrane. This mixture formed a test line, which was capable of performing assays for both hCG and E3G. In the absence of hCG, a white test line would be visualised as white latex particles coated in anti-E3G bound to the ovalbumin-E3G conjugate on the nitro-cellulose membrane. When hCG was present at a level to signify pregnancy, the blue latex particles begin to bind at the test line by virtue of the hCG in the sample in a classical sandwich assay. As the blue latex particles build up on the test line, the test line is no longer perceived to be white, but appears blue to signify a positive hCG result.

The experiments described in this report were performed in buffer standards. Urine samples might contain various amounts of E3G, which would interfere with the second assay. Buffer standards containing various amounts of hCG were thus used, however, without any E3G. These preliminary experiments were designed to prove the principle of this concept. In light of this, all testing was performed in the wet state, with a suspension of latex particles being mixed with buffer standards and this mixture applied to test strips arranged on a vertical support, (wet assay).

Referring to FIG. 3, in hCG negative samples, a white line was perceived on grey nitro-cellulose. As the level of hCG increased, the test line no longer appeared white, instead it was seen to be blue in coloration. The format was seen to function as expected, however, the white test line indicating the negative hCG result was only visible whilst the nitro-cellulose membrane was wet. The photograph in Figure 4, shows test strips run with 0 and 50mIU/ml hCG buffer standards, (taken whilst the strips were still moist). On drying, the nitro-cellulose membrane turned from grey to white. As a result the distinct white test line generated in hCG negative samples was not visible on the dry white nitro-cellulose membrane.

Claims

ClaimsWhat is claimed is:

1. A device, comprising: a support comprising a sample receiving zone and a response zone, the support being configured to support movement of liquid along the support from the sample receiving zone to the response zone, the response zone being configured to produce a first discernible response in the presence of the target compound in the liquid and a second, different discernible response in the absence of the target compound in the liquid.

2. The device of claim 1, wherein the target compound is a compound associated with a pregnant state of a female.

3. The device of claim 1, wherein the support comprises a lateral flow strip.

4. The device of claim 1, wherein the first discernible response results from the presence of first colored particles within the response zone and the second discernible response results from the presence of second, differently colored particles within the response zone.

5. The device of claim 1, wherein the device comprises first and second different particles and the response zone is configured so that first particles are captured in the response zone in the presence of the target compound in the liquid and second particles are captured in the response zone in the absence of the target compound in the liquid.

6. The device of claim 5, wherein the first and second particles have different colors.

7. The device of claim 5, wherein the response zone is configured so that second particles are captured in the presence of the target compound in the liquid.

8. An assay device for performing an immunochemical determination of the presence or absence of a target compound in a liquid sample, the device comprising: a lateral flow support comprising a response zone, the response zone being configured to produce a first color response in the presence of the target compound in liquid of the sample and a second, different color response in the absence of the target compound in liquid of the sample.