GB2232486A - Immunoassay - Google Patents

Abstract

A method of estimating in an aqueous sample the quantity of a first component, which has specific binding affinity for a second component, comprises adding a known quantity of the sample to a device formed of a microporous material having the second component uniformly immobilised thereon, the device being shaped so that the sample can diffuse by capillary attraction in substantially only one or two dimensions, and detecting the extent of diffusion of the first component. A kit comprising the device and a supply of the first component labelled with a detectable marker is also provided. The second component is conveniently a monoclonal antibody.

Description

OUANTITATIVF. CAPILLARY IMMUNOASSAY This invention relates to an assay technique and concerns a method and kit for estimating in a sample the quantity of a first component, e.g. an antigen or antibody, which has specific binding affinity for a second component e.g. the corresponding antibody or antigen.

A variety of such assay techniques are known, but all have various draw-backs. In particular, assays of this type tend to be merely qualitative - serving to detect the presence of a particular substance, for example an antibody, by its reaction with a substance having affinity. Another problem is that tests of this type generally involve immunodiffusion, for example in an agarose film or slab. Typical is the so-called Mancini technique in which bi-functional precipitating antibodies are used. In this technique, the sample is allowed to diffuse slowly through the solid agarose until it reaches the equilibrium point at which a band of precipitin is deposited. This can take a substantial time, e.g. 24 hours, and it is an object of this invention to provide a technique which is quicker and more quantitative.

It has now been found that a simple saturation assay based on capillary diffusion through a microporous substrate can provide an effective estimate of the quantity of the test material in a sample, in a matter of seconds or minutes. The technique relies not on precipitating antibodies, but on saturation of immobilised monoclonal antibodies or the like.Thus, according to one aspect of the present invention there is provided a method of estimating in an aqueous sample the quantity of a first component which has specific binding affinity for a second component, comprising adding a known quantity of the sample to a device formed of a microporous material having the second component uniformly immobilised thereon, the device being shaped so that the sample can diffuse by capillary attraction in substantially only one or two dimensions, and detecting the extent of diffusion of the first component.

As mentioned, the test works by a saturation principle. Thus, if the microporous material had no second component immobilised thereon, the sample and the first component contained in it would diffuse by capillary diffusion throughout the whole extent of the device. However, when the second component is immobilised on the microporous material, the first component is bound by the immobilised second component as it diffuses through the device until the second component is progressively saturated allowing the remaining unbound first component to continue diffusing until all the first component has been bound.As the diffusion is only in one or two dimensions, detection of the extent of travel of the first component provides a simple estimate as to the quantity of first component present in the sample, in that the higher the content of first component there is present. the further it will diffuse before being completely taken up by the second component. It should be noted thaL this test does not rely on precipitating antibodies, but instead on detection of the extent of travel of the first component.

Where the diffusion is in two dimensions, for example in a thin sheet of microporous material, determination of the size of the circle of diffusion (i.e. the area) gives the required quantitative estimate. Where the diffusion is essentially in one dimension, for example in a microporous rod or thin strip, measurement of the length of travel provides the quantitative estimate.

The length or area of diffusion is preferably determined by visual means, for example by partially or completely labelling the first component with a substance capable of providing a visible reaction, e.g.

dyestuffs, chromogenic couples, colloidal gold etc. In some cases, of course, the first component will itself be visible, either directly in the visible spectrum, or by fluorescence under Uv. As an alternative to labelling, it may under certain circumstances be possible to develop the test device by exposure to a reagent capable of colouring the first component in situ rather as one develops a thin layer chromatography plate.

Non-visual detection can be achieved by various means including, for example, radio-labelling of a tracer amount of the first component.

It is preferred that the label, visible or otherwise, is associated with the first component. One preferred technique involves adding to the sample to be tested a known quantity of suitably labelled first component. The labelled material then acts a trace for the distribution of the total first component and the concentration of the first component in the sample can be estimated by a comparison of the extent of diffusion of the trace alone and of the sample containing the trace.

In a further aspect, the present invention provides a kit for use in estimating in a sample the quantity of a first component which has specific binding affinity for a second component comprising a device formed of a microporous material having the second component uniformly immobilised thereon, the device being shaped so that the sample can diffuse by capillary attraction in substantially only one or two dimensions; and a supply of the first component labelled with a visible or otherwise detectable marker.

The invention finds application in a wide range of assays, but particularly in immunoassays in which the components are antigens and their corresponding antibodies. By use of monoclonal antibodies, highly specific assays for a wide range of substances can be performed, e.g. on biological fluids such as blood, serum or plasma, urine etc.

Although the invention is particularly of use in estimating the quantity of first component present in a sample, at its crudest level this test can comprise a simple investigation as to the presence or not of the first component, i.e. a qualitative test.

The microporous material forming the device should be sufficiently thin so that the diffusion is substantially in two dimensions, e.g. in the plane of a membrane, or in one dimension, e.g. along the length of a thin strip or rod, with no significant diffusion in the other one or two dimensions. Typically, the membrane will have a thickness of 0.5 mm or less and a strip or rod should have a width of about 3 to 6mm.

The microporous material for use in membranes and strips conveniently comprises a cellulose ester, e.g.

the acetate, butyrate, propionate etc or, especially, cellulose nitrate. Microporous material of this type is available in thin sheet form having high, uniform permeability, permitting rapid transport of the first component in the aqueous sample, by capillary attraction.

The microporous material for use in rods and the like conveniently comprises microporous plastics, as made by the Porex Technologies Company of Georgia, U.S.A. or Sintair Ltd of Norfolk, U.K.

The sensitivity of the test may be controlled by varying the amount of immobilised second component.

The device may be of any suitable size for convenient handling, depending on the nature of the test sample, typically a small card, e.g. credit card size, may bear an array of test wells, e.g. in a 1 cm grid.

The spacing of the wells may be varied as appropriate depending on the sensitivity of the test material and the expected amount of diffusion.

In one preferred embodiment, the membrane is provided with a backing of rigid, or semi-rigid hydrophobic plastics material apertured to define an array of test holes. The plastics backing provides the membrane with rigidity. Furthermore, by choosing the diameter of the apertures, the size of the loading zone can be varied as required. In practice, the wells will be very small, e.g. pin pricks containing no more than 10 microlitres of sample. In use, liquid sample is applied in known volumes to the apertures in the backing, so that a measured quantity is applied to the membrane via each aperture. The size of the diffusion regions, if detected by their visibility, may be seen either directly through the plastics backing (if transparent) or alternatively by inspection of the reversed face of the membrane.The membrane is conveniently covered on both sides for storage before use, for example in a sealed foil jacket.

An alternative preferred embodiment comprises a microporous rod coupled at one end to reservoir means capable of holding a small known volume of sample, for example a very small plastic sponge. In use, the sponge would be applied to a source of the sample and would absorb a pre-calibrated amount. The sample would then diffuse along the rod to provide a detectable length of invasion by the first component. In a particular example of this technique, the rod and sponge could be coupled with a needle point for punturing the skin to obtain a blood sample which is then absorbed by the sponge. In particularly preferred version, the sponge itself is treated with material capable of lysing the cell walls of erythrocytes to release haemoglobin, for example a surfactant.The sample, containing released haemoglobin can then diffuse along the rod in which are immobilised anti-haemoglobin antibodies. In this particular test, the visual detection of the extent of diffusion depends simply on the colour of the haemoglobin itself.

In general, the invention is found to give reliable results very rapidly, generally in less than five minutes, resulting in a substantial time saving as compared with immunodiffusion techniques of the type discussed above. Furthermore, the method of the invention is easy to use and can be carried out reliably by unqualified personnel in any part of the world. The test does not require the facilities of a laboratory.

Depending on the nature of the immobilised second component, the membrane or rod may be dried or may require storage in a moist condition. It is particularly desirable to stabilise any immobilised material by the presence of trehalose as disclosed and claimed in International Patent Application number The following Examples illustrate the invention.

F,xample 1 Nitrocellulose papers were impregnated with purified IgG from sheep anti-rat IgG serum. The impregnated paper was washed and dried and then stored at room temperature until required for use. The paper was backed with plastics film containing four lmm diameter holes at a 12.5 mm spacing. Test material was loaded into the holes as follows: Hole 1: 10 microlitres of a mixture of 5 microlitres of rat IgG coupled to colloidal gold (2 mg IgG per 50 ml gold suspension) and 5 microlitres water (referred to below as RCG); Hole 2: 10 microlitres of a mixture of equal volumes of RCG and rat IgG at 4.35 mg/ml; Hole 3: 10 microlitres of a mixture of equal volumes of RCG and rat IgG at 1.01 mg/ml; Hole 4: 10 microlitres of a mixture of equal volumes of RCG and rat IgG at 0.25 mg/ml.

The four samples diffused by capillary attraction to give different sizes of circular diffusion patterns.

The diameters of the diffusion patterns were measured and then checked by scanning densitometry and the difference in area between the samples of holes 2, 3 and 4, and the RCG of hole 1 was calculated: Sample diam. area area minus Content (mm) area due to RCG 1 17.5 240 RCG 2 23.5 434 194 RCG+4.35mg/ml net IgG 3 20 314 74 RCG+l.Olmg/ml 4 18 255 15 RCG+o.25mg/ml When the difference in area - Bkg was plotted versus concentration of unlabelled rat IgG this gave a straight line of best fit which extrapolated through zero. This confirms the quantitative precision of the assay.

As a control, various concentrations of RCG and a sample of Streptavidin coupled to colloidal gold were loaded onto paper which had no antibody coupled to it.

These gave similar sized reacion patterns which did not vary with the concentration of the label thus showing that the reaction was specific for rat IgG and sheep anti-rat IgG antibody.

ExamPle 2 Nitrocellulose paper was soaked for 15 minutes in a pre-diluted sample of sheep anti-equine IgG serum. The paper was then washed in 0.9% saline and dried at room temperature.

The prepared nitrocellulose paper was spotted with 1 microlitre of each of the following: Position A - Purified equine IgG coupled to colloidal gold (ECG).

Position B - a mixture of equal volumes of ECG and purified equine IgG containing 4g/l.

Position C - A mixture of equal volumes of ECG and equine serum containing 5.2g/l IgG.

Position D - A mixture of equal volumes of ECG and equine serum containing 8.0gel IgG.

Position E. - A mixture of equal volumes of ECG and equine serum containing 10.Og/l IgG.

The diameter of each circular area formed was measured upon completion of diffusion (about 2 minutes) and the area calculated. This area minus the area due to ECG was plotted against the concentration of unlabelled IgG in the sample.

Sample Diameter Area Area minus area Concn. of IgG (mm) due to ECG in sample (unlabelled) A 3.1 7.54 B 3.9 11.94 4.40 4g/l C 4.2 13;85 6.31 5.2g/l D 4.5 15.89 8.35 8.0g/l E 4.6 16.61 9.07 10.0g/l

Claims (5)

1. CLAIMS: 1. A method of estimating in an aqueous sample the quantity of a first component which has specific binding affinity for a second component, comprising adding a known quantity of the sample to a device formed of a microporous material having the second component uniformly immobilised thereon, the device being shaped so that the sample can diffuse by capillary attraction in substantially only one or two dimensions, and detecting the extent of diffusion of the first component.
2. 2. A method according to claim 1 in which the detection of the extent of diffusion is by visual means.
3. 3. A method according to claim 1 or claim 2 in which the first and/or second component is an antibody.
4. 4. A kit for use in estimating in an aqueous sample the quantity of a first component which has specific binding affinity for a second component comprising a device formed of a microporous material having a second component uniformly immobilised thereon, the device being shaped so that the sample can diffuse by capillary attraction in substantially only one or two dimensions; and a supply of the first component labelled with a detectable marker.
5. 5. A kit according to claim 4 in which the device comprises a membrane, strip or rod of microporous material.