This invention relates to medical diagnostic test systems and a liquid carrier matrix for use with such a system.

5

Background

Many countries have limited financial resources for healthcare and few highly trained healthcare workers. Low cost diagnostic tests which are easy to interpret are therefore very attractive to such country's healthcare organisations. Medical diagnostics, in

10 particular, is often expensive, and the necessary equipment often requires training and skill to operate, as well as often needing a clean environment to operate. The development of cheap biomedical devices that can be easily operated would be greatly appreciated not only within resource-limited nations, but also in emergency situations where time is of the essence and specialized equipment is not immediately available. Paper-based biomedical

15 devices have been developed to address this need. However, they have a number of limitations. One limitation is that the chemicals commonly used to prepare these devices are toxic. Another limitation is that conventional filter paper cannot be used to immobilize proteins in a way that does not damage the proteins, and is therefore unsuitable for many standard biomedical assays such as the western blot. (Analytical Chemistry, 82 (1 ), 329-

20 335 DOI)

To partially address the above needs, a large number of rapid diagnostic test systems are currently based on the combination of mostly cellulosic membranes with papers and/or glass fiber pads. The most dominant test system available on the marketplace is the so- 25 called "lateral flow test" (see e.g Posthuma-Trumpie et al. Anal. Bioanal Chem (2009), 393, pp569 - 582, and references cited therein).

The system allows for the rapid diagnosis of very diverse physiological conditions for example: fertility; infectious diseases; drugs of abuse; marker molecules for cardiac issues 30 and the like from bodily fluids. The diagnostic test is in most cases be performed at a point of care, but it some cases even a layperson may run the diagnostic test at home (e.g. pregnancy and fertility tests).

The membrane materials currently available on the marketplace range in thickness between about 100 and about 150 μηη. This membrane layer needs to be filled completely with capture reagents although the signal that is usually being generated by colored particles linked to detector reagents is visible only for a part of the membrane thickness (Rapid Lateral Flow Test Strips, Millipore Corporation (2008)), thus resulting in an unnecessary waste of precious capture reagents, and as a consequence, manufacturing costs higher than necessary.

Therefore, there is a need to provide a liquid carrying membrane matrix that consistently reduces the reagent without compromising the analytical signal, combined with a superior consistency and reproducibility of the diagnostic test result.

In rapid diagnostic tests using conventional membranes with visual indications, including colorimetry, chemiluminiscence, fluorescence and the like, the signal that is visible to the test user is generated within the first few micrometers of the membrane surface. Any signal that is generated deep within the matrix does not contribute to the visible signal. Nevertheless, the whole membrane volume underneath the surface needs to be filled with reagents, and the whole membrane volume needs to be filled with sample liquid when the test is being used. This is an unnecessary waste of reagents which can be very expensive depending on the test system under consideration. Also the amount of sample liquid consumed is high, which is problematic where the sample volume is low.

Summary of the invention

In one aspect, a membrane matrix material is provided that has an effective thickness of far less than 100 μηη, with a consistency in terms of thickness and capillary flow, for example a membrane having a thickness variance of 5% or less and a capillary flow with a variance of 10% or less . The membrane of the invention can be manufactured in a dry cast process by forming a liquid layer of a casting mix consisting of nitrocellulose, organic solvents, water and surfactants onto a moving support, and then forming a thin membrane on the support by evaporating the solvents in the formed layer, under controlled conditions using a counter- current air or gas flow. The porosity of the membrane can be controlled by adjusting the water content of the casting mix to required percentages varying between 5 and 15 % (v/v).

Preferably, for ease of handling, the membrane is left on the support on which it has been formed. Other options are the use of a fleece material in the casting mix to stabilize the forming membrane, or to laminate the membrane onto self-adhesive supports. The thickness of the casting mix layer on top of the support and the consistency of this thickness needs to be controlled very carefully. Prior to the evaporation, for the new thin membrane materials, the liquid layer of casting mix is adjusted to values below 100 μηη whereas for conventional membranes, the thickness of the layer is usually a couple of hundred micrometers, more specifically around 800 μηη which requires less accurate control.

This thin casting mix layer also demands for a very exact control of heat, humidity, speed of the moving support and of the countercurrent gas stream.

In another aspect, a diagnostic test is provided that uses the thin membrane mentioned above. Thereby said test has the same sensitivity/specificity/runtime properties as a diagnostic test using conventional membrane materials with a thickness of 100-150 μηη or more, but capture reagent volume needs of 30 - 50 % or less compared to conventional membrane reagent volume needs

Brief Description of the Drawings

Figure 1 shows a scanning electron microscope (SEM) image of the surface of the thinner membrane of the invention, at 500x magnification. The image shows a nitrocellulose membrane consisting of a meshwork of nitrocellulose fibers without surface impurities; and Figure 2 shows a picture and analysis of a pregnancy test at the limit of detection with the thinner membrane after use in a test strip, as obtained by reflectance spectroscopy . All pads were removed prior to analysis by reflectance spectroscopy. Diagnostic test example

A generally conventional pregnancy diagnostic test was prepared using conventional paper sample pads and wicks, and glass fibre conjugate pads for all test strips investigated, while the thinner membranes mentioned above were used in this setup. The detector reagent used was a mouse anti-beta-hCG antibody conjugated to 40 nm colloidal gold. The capture antibody is a mouse anti-alpha-hCG antibody. hCG hormone was diluted into the sample liquid in order to obtain the required final concentration. The membrane test strip width was 5 mm. Test line results were obtained using a reflectance reader.

Thin membrane example

Thin membranes with a capillary flow of about 125 seconds/4 cm (test liquid: water) were obtained by using a casting mix comprising:

Ethanol 39,9%

Methylacetate 38,4%

Nitrocellulose 1 1 ,7%20

Water 9,9%

Surfactants 0,1 %

These membranes were manufactured by moving a support surface under the casting mix and depositing a layer of the mix onto the support. In this case, the casting mix was poured onto a 100 μηη PET foil support at a casting speed of 30 cm/min and a maximum casting mix thickness of 90 μηη and the membrane was formed by drying the mix in a conventional manner, to form a membrane having a final mean thickness of 43 μηη plus the thickness of the foil. The pore size was approximately 8 μηη. Membranes with a smaller or larger pore size (0.1 μηη to 20 μηη) were obtainable by decreasing or increasing the water content of the casting mix. Additional membranes were cast resulting in a dried membrane thickness of 25 μηη, (without foil thickness) . For all membranes produced, the thickness variability was less than 5%-coefficient of variance(CV), and capillary flow time variability was less than 10 % (CV). It was determined that the minimum thickness for a membrane using the manufacturing process mentioned above is around 10 μηη.

The membranes were used in the pregnancy test described above, and the amount of test line antibody required to detect hCG at a concentration of 25 mlU/ml was determined for the new membranes as well as conventional PET-backed NC membranes with similar capillary flow time properties as shown in table 1 .

Table 1 : Capture line reagent requirements on different membranes

The examples shown above illustrate that thinner membranes can be manufactured using generally conventional so-called dry casting technologies, and that they need less (up to 80% less) reagent as compared to conventional membranes when used in lateral flow diagnostic test. It follows that the same is true for flow-through diagnostic test systems, line assays and Western Blots were liquids flow through the volume of the membrane laterally (in a planar manner) or vertically- (surface to surface) in a generally capillary or forced manner. Such membranes enable the manufacturing of rapid tests at lower costs, and allow for miniaturisation of test systems. It has been shown that there is no reduction in test performance. Whilst one example of a membrane has been described above, and one example of a diagnostic test employing the membrane has been described, it will be appreciated that other membranes could be manufactured, and those membranes could be employed in other tests, all within the scope of the claims.

For example, a PET foil membrane support is described. Other supports include other foils such as metal foils or other plastics foils such as PVC or polystyrene; integrated fleeces or webs, such as non-woven polyester webs; fibrous materials such a glass fibre materials, or papers, for example high quality chromatography papers.

Whilst a range of pore sizes between 0.1 and 20 μηη has been described above, specific sub-ranges within the 0.1 and 20 μηη range will be useful, for example 2 to 20 μηη for use with lateral flow diagnostic tests where capillary fluid flow is important and where the 8 μηη pore size example given above is about right. In other examples the sub range is 2 to 6 μηη for use in a lateral flow diagnostic test where the cost of reagents is high and so as little fluid, and reagent, as possible is used for an assay. All other sub ranges or specific values within the 0.1 to 20 μηη are possible.