Assay using porosity-reduction to inhibit migration

Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals

Reexamination Certificate

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C422S051000, C422S051000, C422S051000, C422S051000, C422S067000, C422S067000, C422S105000, C435S007100, C435S287100, C435S287200, C435S287800, C435S287900, C435S805000, C436S528000, C436S538000, C436S541000, C436S808000, C436S810000

Reexamination Certificate

active

06472226

ABSTRACT:

TECHNICAL FIELD
This invention relates to assay devices for measuring analytes. In particular, it relates to devices which capture analytes mechanically within a porous material, rather than using conventional immuno-capture techniques.
BACKGROUND ART
The format of the standard rapid test lateral flow device has remained unchanged for around ten years. Typically, the device will comprise a nitrocellulose strip. Sample is applied to an application zone, from which it flows by capillary action through a zone containing a visibly-labelled antibody specific for the analyte in question. Free and bound label continue to migrate to a capture zone, where immobilised antibody specific for the analyte binds the analyte-label complex. Free label (unbound antibody) continues to migrate, leaving an analyte-specific signal in the capture zone. These types of lateral flow device are disclosed in, for example, EP-A-0284232. Numerous variations to the basic assay have been described, including those in WO92/12428, EP-A-0613005, WO97/06439, and U.S. Pat. No. 5,741,662.
In all cases, however, capture of the analyte-label complex is mediated by an immobilised reagent, which is typically an antibody that is specific for the analyte. This is unsatisfactory in many respects.
Firstly, manufacturing quality control is difficult. The solid phase capture membrane is typically made from nitrocellulose, and antibodies are applied to the membrane directly. Nitrocellulose manufacture is not, however, homogeneous. Quality control of the solid phase antibody is therefore limited to testing a statistical sample of devices from the same, but heterogeneous, batch, and assuming that the whole batch will perform within specific tolerances. It is well known, however, that membranes vary considerably, even within a single batch or lot number.
Secondly, they are relatively cumbersome to manufacture. The application of immobilised antibody to the strip requires a separate step from the application of the mobile labelled antibody. The capture antibody can be sprayed directly onto the nitrocellulose strip, but the label antibody has to be soaked into material which is subsequently attached to the nitrocellulose strip, with an overlap to ensure capillary flow.
Thirdly, antibody is immobilised by spraying a solution onto a membrane. Some of the antibody does not bind to the membrane strongly, however, and some remains loosely associated with immobilised antibody. This semi-bound or unbound antibody can become mobile when the solvent front passes over it, resulting in lower binding of label at the detection zone. If the device includes a control line, this will capture the additional label which should have been captured at the detection zone. Tests that rely on a comparison of colour intensity between control and detection lines, such as ovulation prediction kits, may therefore give false results. Furthermore, application by spraying inevitably leads to diffusion into the membrane, leading to a more diffuse and less focused detection signal.
Fourthly, the sensitivity of the devices is limited by their format. Analyte and labelled-antibody react as they migrate through the membrane, and flow rates are therefore adjusted to enable the labelled-antibody to flow at the solvent front in order to maximise the amount of time in which the analyte-label complex can form. The complex passes over the capture antibody for a short time, however, thus imposing constraints on the design of the test and its performance characteristics. The short reaction time decreases sensitivity, and also means that high affinity capture antibodies are required.
Finally, the shelf-life of these test devices is often limited by the collapse of the immobilised capture antibody onto the membrane over time.
These shortcomings in the prior art devices are addressed by the present invention, which does not use immobilised antibody to capture an analyte-label complex.
DISCLOSURE THE INVENTION
The invention provides a device for assaying an analyte, comprising a labelling zone, where a label can bind to the analyte, in communication with a capture zone, wherein the pore size of the capture zone is such that label which is not bound to the analyte can migrate therethrough, whereas label which is bound to the analyte cannot.
During migration from the labelling zone to the capture zone, unbound label can pass into and through the capture zone, whereas bound label will be captured at the junction of the labelling zone and the capture zone. A comparison of the amount of label captured at the entrance to the capture zone with the amount migrating through the capture zone allows the level of analyte to be assessed—as analyte concentration increases, the amount of label retained at the junction of the labelling zone and the capture zone also increases.
It will be apparent that the invention relies upon the label being smaller than the analyte, such that free label is not retarded by the capture zone.
The device is particularly suitable for assaying analytes such as biological cells, which are large in comparison with a label such as a labelled antibody. Preferred cells for assay are spermatozoa and micro-organisms, such as bacteria.
The labelling zone is where label comes into contact with the analyte. It is preferably formed from fibrous material, such as a pad of HDPE material, bonded polyester fibre, glass fibre, or the like. The pore size should be large enough to allow the analyte to move relatively freely, in contrast to the pore size of the capture zone.
The label is typically an antibody which can bind to the analyte of interest, and which has been suitably labelled. The label is preferably visible to the naked eye eg. a fluorescent label, or a particulate label such as colloidal gold (which is visible as a pink colour), or a stain such as eosin. It will be appreciated that the term ‘antibody’ may include polyclonal and monoclonal antibodies, as well as antibody fragments (eg. F(ab)
2
, Fc etc.), provided that the necessary biological specificity is retained.
The capture zone can be made from any suitable porous material through which unbound label can migrate, whilst analyte-bound label cannot. This requirement is reflected in the pore size of the capture zone. In one embodiment, the capture zone will be made from HDPR with a nominal pore size of around 1-75 &mgr;m, preferably 10-50 &mgr;m, and more preferably 20-35 &mgr;m. In second embodiment, the capture zone will be made from nitrocellulose, with a nominal pore size of around 1-15 &mgr;m, preferably 3-10 &mgr;m, and more preferably 5-8 &mgr;m.
In some embodiments, the labelling zone and capture zone may be formed from a single piece of porous material, which contains a region of reduced pore size. By crushing or compressing a region of a porous material, for instance, the pore size can be reduced such that an analyte-bound label cannot enter the compressed region ie. to form a capture zone. As an alternative, the pores of the material could be partially blocked, to achieve the same effect.
As is well known to those in the art, the nominal pore size of a porous material can be determined by hard particle challenge testing ie. by determining the maximum diameter of spherical particles which can pass through the material. Alternatively, the pore size of a material may be determined by measuring its ‘bubble point’. The bubble point is the pressure required to force air through a (water) wet membrane, and correlates with the pore size as measured by particle retention (although at extremes of pressure and pore size, the correlation may be weaker). The bubble point is generally easier to measure than particle retention and is thus the preferred test when assessing pore size.
When the device of the present invention is to be used for detecting and measuring a motile analyte in particular (such as motile spermatozoa or motile bacteria), the appropriate pore size may be determined empirically by routine testing.
In preferred embodiments, the capture zone includes a region which retains label which is not bound to

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