EP0621803A1

EP0621803A1 - Assay apparatus and method

Info

Publication number: EP0621803A1
Application number: EP93902397A
Authority: EP
Inventors: Adrian Parton
Original assignee: Genera Technologies Ltd
Current assignee: Genera Technologies Ltd
Priority date: 1992-01-18
Filing date: 1993-01-15
Publication date: 1994-11-02
Also published as: WO1993013856A1; GB9201089D0; CA2127980A1; AU3359193A

Abstract

Dispositif de dosage comprenant une première zone de dosage constituée par une pastille en mousse (26) située sur un premier élément de support solide (10) constitué par une carte en plastique possédant un canal (14) sur une de ses faces bordée par des parois comportant une gorge (12), de façon à recevoir une glissière en plastique (18) en tant que deuxième élément de support solide possédant une fenêtre (24) en tant que deuxième zone de dosage. Les éléments de support s'adaptent l'un à l'autre, afin d'effectuer un déplacement coulissant relatif, de façon que lesdites zones coulissent l'une par rapport à l'autre depuis une première position dans laquelle elles sont séparées l'une de l'autre vers une position dans laquelle elles viennent en contact réciproque au moyen d'un déplacement coulissant. Un anticorps peut être fixé à la pastille en mousse. Un conjugué constitué par un anticorps et par un enzyme peut se trouver non fixé sur ladite pastille. Un réactif de dosage se trouve sous la fenêtre (24), de façon à servir dans une réaction observable avec le conjugué ou avec une espèce à analyser.Dosing device comprising a first dosing zone consisting of a foam pad (26) located on a first solid support element (10) consisting of a plastic card having a channel (14) on one of its faces bordered by walls comprising a groove (12), so as to receive a plastic slider (18) as a second solid support element having a window (24) as a second metering zone. The support elements adapt to each other, in order to effect a relative sliding movement, so that said zones slide with respect to each other from a first position in which they are separated the one another to a position in which they come into reciprocal contact by means of a sliding movement. An antibody can be attached to the foam pellet. A conjugate constituted by an antibody and by an enzyme can be found not attached to said patch. An assay reagent is located below the window (24) for use in an observable reaction with the conjugate or with an analyte species.

Description

ASSAY APPARATUS AND METHOD

The present invention relates to assay apparatus and a method of assay and has particular but not exclusive relevance to immuno-diagnostic assays.

Diagnostic tests based on the specificity of antibodies (e.g. monoclonal antibodies) and DNA technology are continually being developed and provide means for the identification of undesirable infections (bacterial, fungal or viral), or chemical entities, thus facilitating appropriate treatments.

In addition to improvements in the chemistry of diagnostic tests, there has been considerable development in the way in which the tests are formatted. Initially, most diagnostic tests.were conducted in the laboratory due to the constraints imposed by the requirement for equipment, materials and trained operators. There have however been significant developments in format design which allow tests to be carried out in relatively short timescales, e.g. ten minutes or less, whilst requiring minimal input in terms of manual manipulations and operator skill.

Current immuno-diagnostic tests rely on multi-step processes requiring several manipulations and reasonably accurate timing of the steps. This is a major disadvantage in tests for use outside the laboratory such as in doctors surgeries, in the field or in the home. In these circumstances, test procedures may not be followed very accurately by the end user. Although some test kits currently available do not require many steps, they tend to be expensive as the technology needed to achieve these requirements is inherently expensive. There is therefore a need for a simpler device reducing manufacturing costs whilst maintaining simplicity of operation. The present invention provides assay apparatus comprising a first assay zone on a first solid support component for capturing a species to be assayed thereon and a second assay zone on a second solid support component having an assay reagent thereon, means mounting said first and second solid support components together for relative slidingmovement such that said zones are slidable with respect to one another from a first position in which said zones are separated from one another to a position in which said zones are brought into mutual contact by sliding motion.

The first and second support components may be different parts of a single member or may be separate members mounted together. For instance, a single strip may be folded over to bring to end portions together and means may be provided to hold the end portions face to face whilst allowing sliding movement thereof with respect to one another. Preferably however, the first and second assay zones are provided on respective ones of a pair of plastics plate members constituting said first and second solid support components.

Preferably, one said plate member or one end region of such a strip has a pair of oppositely directed channels thereon in which edge regions of the other said plate member are slidingly received to mount said plate members or said strip regions in face to face sliding relationship.

The species to be assayed may itself be such as to produce or catalyse an observable reaction with the assay- reagent in the second assay zone. Otherwise, an assay reagent may be present unbound in said first reagent support zone which is a species capable of binding to the species to be assayed having conjugated thereto a species capable of mediating an observable reaction in the presence of said assay reagent of the second assay zone.

The observable reaction is preferably a colour forming reaction or a reaction giving rise to a species which is observable by fluorescence under UV light. Accordingly, one or both of the support components is preferably transparent at least in its respective assay zone. The unbound assay reagent present in said first assay zone may be an antibody conjugate capable of binding to a species to be assayed. The term "antibody" in this context herein includes antibody fragments having selective affinity for antibody binding sites. It may be a conjugate between a nucleic acid or nucleic acid analogue for binding to a nucleic acid as the species to be assayed and a linker moiety such as biotin capable of binding to a conjugate between an enzyme for mediating such a reaction and a further linking moiety, e.g. avidin or between said nucleic acid component and said enzyme component directly.

The assay reagent in said second assay zone may be a substrate for a colour forming or other observable reaction mediated by said conjugate. The species to be assayed is to be captured on to the first assay zone so that its attachment is resistant to washing procedures during the assay. In some cases this may be achieved by direct physical capture or absorption. For instance, some bacteria or proteins will bind directly onto a roughened plastics strip or mesh. Alternatively and more generally the first assay zone may have bound thereon an assay reagent which is capable of binding the species to be assayed.

This bound reagent may be an antibody, which in this context includes antibody molecules and also fragments thereof having selective binding affinity for antibody binding sites, a nucleic acid or a nucleic acid analogue, depending upon the nature of the species to be assayed.

Where the bound an /or the unbound assay reagent in the first assay zone is an antibody or antibody conjugate the antibody or antibody fragment is preferably a monoclonal antibody or fragment thereof.

Preferably, all of the reagents are present in dry form, the solvent (usually water) needed for the assay, reaction being derived from the addition of the sample. There may be an absorbent pad present in the first assay zone for holding a quantity of liquid during the assay. This pad may for instance be of open cell foam or of absorbent paper such as filter paper or similar materials.

The arrangement may be such that the pad is compressed when the second solid support component is slid into position over the first assay zone.

The invention includes a method of assay comprising capturing a species to be assayed on a first assay zone provided on a solid support component, and (a) sliding thereover a second support component bearing a substrate for an observable reaction mediated by said species to be assayed so as to contact said substrate with said bound species to produce said observable reaction or (b) binding thereto a conjugate between an enzyme and a species capable of binding to the species to be assayed, washing to remove unbound conjugate and sliding thereover a second solid support component bearing a substrate for an observable reaction mediated by said conjugate so as to contact said substrate with said bound conjugate to produce said observable reaction.

The invention will be further described and illustrated by the following description of preferred embodiments with reference to the accompanying drawings in which:-

Figure 1 shows in perspective view an example of apparatus according to the invention in use in three stages of an assay procedure, and Figure 2 shows stages, including sample taking, involved in a specific form of assay procedure described in Example 1 below using the same apparatus according to the invention.

As shown in Figure 1, apparatus according to the invention comprises a first solid support component 10 in the form of a plastics plate of about the size of a credit card having a pair of upstanding walls 12 bordering a central strip portion of lesser thickness 14. • The faces of the walls 12 bordering the strip 14 are undercut as shown at 16. A second solid support component 18 comprises a plastics strip dimensioned to fit snugly between the walls 12 and having chamfered edges 20 which locate in the undercut shown at 16 to mount the support component 18 for sliding movement in the channel formed between the walls 12 on the support component 10. A first assay zone 22 is provided on the first solid support component 10 and a second assay zone 24 is provided on the second solid support component 18. At least the region of the said assay zone 24 on the second support component 18 is made transparent. A foam pad 26 is present in the first assay zone 22 securely adhered in place on the first support component 10. A substrate for an enzyme mediated colour forming reaction is present in dry form in the second assay zone 24.

Whilst the above described features are sufficient to define an apparatus according to the invention in its broadest form, the apparatus illustrated in Figure 1 further comprises unbound on the foam pad 26 an enzyme-antibody conjugate, the enzyme being suitable to catalyse a colour forming reaction with the substrate present on the second assay zone 24.

Also present in the foam pad 26 but bound thereon is a further antibody. The bound antibody and the antibody component of the unbound antibody conjugate may be the same or may be different. Both are preferably monoclonal anti¬ bodies although polyclonal reagents are still suitable.

In use, a sample such as a liquid containing micro¬ organism cells is applied to the foam pad. The cells are bound by the bound antibody and a "sandwich" is formed by binding to the bound cells of the antibody conjugate. The apparatus may then be washed under running water, which may be tap water, to remove excess antibody conjugate from the sample area and the second support component 18 is then slid over the first support component 10 to bring the first and second assay zones into position one over the other. The foam filter pad is thereby compressed against the underside of the second support component. Excess liquid is squeezed out from the foam pad by the leading edge of the second support component passing over it and a good contact is ensured between the foam pad 26 and the second assay zone 24 which contains in dried form a substrate for the antibody conjugate. This is resuspended by the residual liquid on the foam pad and the colour forming reaction takes place and is observable through the transparency of the second support component. The following examples illustrate the use of the apparatus shown in Figure 1.

EXAMPLE 1 - Assay for E.coli by Detection of β-Glucuronidase There is a requirement in the water industry for a convenient assay for detecting E. coli in water. β- glucuronidase is an enzyme expressed by a limited number of micro-organisms which is accepted in the water industry as being a 95% positive test for E. coli. To exemplify the use of the apparatus described above, a sample of water is taken up into a syringe 27 and is discharged via a micro-organism trapping 0.2μm filter 28. The micro-organisms initially present in the liquid sample are now held in concentrated form in the liquid in the dead space of the syringe/filter assembly. A defined amount of a suitable micro-organism growth medium is drawn up into the filter. The syringe/filter assembly is now placed with the syringe vertical and the lower face of the housing of the filter on a heating block by which the filter is warmed to an incubating temperature. After a predetermined period of incubation to multiply the bacteria, the temperature of the heating block may be raised to a temperature suitable to produce lysis of the bacteria for a period of about one minute. Apparatus according to Figure 1 is employed having a polyclonal antibody to β-glucuronidase bound to the foam pad 26. In this case no antibody conjugate is present on the foam pad as β-glucuronidase itself catalyses a colour forming reaction. The liquid content of the syringe/filter assembly may then be discharged on to the first assay zone 22 of apparatus as shown in Figure 1 and may be left at room temperature for a period sufficient to enable binding of the β-glucuronidase from the micro-organism lysate by the bound antibody on the first assay zone, e.g. for abou-t ten minutes. The assay apparatus is optionally then washed and the second support component 18 is slid into position as shown in step 3 in Figure 1. The colour forming reaction is allowed to take place and is observed. Optionally, a series of syringe and filter assemblies are prepared at the outset of the procedure and these are incubated for differing periods of time, the length of time necessary for incubation depending upon the level of contamination present. Optionally, to enhance sensitivity, the growth medium may contain a substrate suitable to induce expression of the β-glucuronidase by the micro-organisms to produce elevated levels of enzyme in the cultured cells, e.g. an R-β-D-glucuronide. Also to enhance sensitivity the colour forming reaction may be replaced by a reaction giving rise to a UV-fluorescent compound by appropriate selection of the enzyme substrate on the second support component 18.

EXAMPLE 2 - Sandwich Assay for β-Glucuronidase

To enhance sensitivity the apparatus employed in Example 1 is modified by the inclusion of a conjugate between an anti β-glucuronidase antibody and a colour forming reaction catalysing enzyme in dried form unbound in said foam pad 26. The procedure followed is as in Example 1 up to the end of the lysis step. When the liquid content of the syringe/filter assembly is discharged on to the foam pad a sandwich is formed in which the conjugate and the bound antibody both bind to the β-glucuronidase. The foam pad is washed to remove unbound conjugate and the second support component is slid into position. The assay reagent thereon is selected to take part in a colour forming reaction in the presence of the bound conjugate.

EXAMPLE 3 - Assay for β-Glucuronidase Trapped Directly

The procedure of Example is modified in that the bound antibody on the foam pad 26 is omitted and the foam material is selected such that it has a non-specific affinity for proteins including β-glucuronidase. The lysate as applied to the foam pad and proteins therein are bound to the foam. Bound β-glucuronidase is further bound by the free antibody conjugate. The presence of bound conjugate is then detected by washing and developing as in Example 2. Alternatively, the antibody conjugate is also omitted and the trapped β- glucuronidase acts directly to catalyse the colour forming reaction as in Example 1.

Many modifications and variations of the methods and apparatus described above are within the scope of the invention.

Claims

1. Assay apparatus comprising a first assay zone on a first solid support component for capturing a species to be assayed

5 thereon and a second assay zone on a second solid support component having an assay reagent thereon, means mounting said first and second solid support components together for relative sliding movement such that said zones are slidable with respect to one another from a first position in which 0 said zones are separated from one another to a position in which said zones are brought into mutual contact by sliding motion.

2. Apparatus as claimed in Claim 1, wherein an assay reagent is present unbound in said first reagent support zone which 5 is a species capable of binding to the species to be assayed having conjugated thereto a species capable of mediating an observable reaction in the presence of said assay reagent of the second assay zone.

3. Apparatus as claimed in Claim 2, wherein said unbound 0 assay reagent present in said first assay zone is an antibody conjugate, which antibody conjugate is capable of binding to a species to be assayed and wherein said assay reagent in said second assay zone is a substrate for an observable reaction

• • mediated by said antibody conjugate. 5 4. Apparatus as claimed in Claim 3, wherein said antibody conjugate is a monoclonal antibody conjugate.

5. Apparatus as claimed in Claim 1 or Claim 2, wherein said unbound assay reagent is a conjugate between an enzyme and a nucleic acid or nucleic acid analogue probe. 0

6. Apparatus as claimed in any preceding claim, wherein said first assay zone has bound thereon an assay reagent which is capable of binding a species to be assayed.

7. Apparatus as claimed in Claim 6, wherein said assay reagent bound in said first assay zone an antibody or antibody 5 fragment, or a nucleic acid or nucleic acid analogue capture probe.

8. Apparatus as claimed in Claim 6, wherein said bound assay reagent is a monoclonal antibody.

9. Apparatus as claimed in any preceding claim, wherein said first and second assay zones are provided on respective ones of a pair of plastics plate members constituting said first and second solid support components.

10. Apparatus as claimed in Claim 9, wherein one said plate member has a pair of oppositely directed channels thereon in which edge regions of the other said plate member are slidingly received to mount said plate members in face to face sliding relationship.

11. A method of assay comprising capturing a species to be assayed on a first assay zone provided on a solid support component, and (a) sliding thereover a second support component bearing a substrate for an observable reaction mediated by said species to be assayed so as to contact said substrate with said bound conjugate to produce said observable reaction or (b) binding thereto a conjugate between an enzyme and a species capable of binding to the species to be assayed, washing to remove unbound conjugate and sliding thereover a second colour support component bearing a substrate for an observable reaction mediated by said conjugate so as to contact said substrate with said bound conjugate to produce said observable reaction.

12. A method as claimed in Claim 11, conducted using apparatus as claimed in any one of Claims 1 to 9.