GB2333358A

GB2333358A - Reagent delivery to a sample site

Info

Publication number: GB2333358A
Application number: GB9800724A
Authority: GB
Inventors: David Charles Mangham; Mark Trehane Drayson
Original assignee: Binding Site Ltd
Current assignee: Binding Site Ltd
Priority date: 1998-01-15
Filing date: 1998-01-15
Publication date: 1999-07-21
Also published as: DE19901166A1; GB9800724D0

Abstract

A method of delivering a reagent 1 to a biological sample 2 to be tested for the presence or absence of a substance comprises providing the reagent 1 on a carrier 6 which is placed on the sample 2 ensuring that liquid is present at the sample/carrier interface. The reagent 1 leaves the carrier 6 and migrates to the sample 2 where it interacts with the substance. The carrier 6 delivers reagents, e.g. antibodies, antibody complexes, enzyme substrates, staining substrates in a dried form to a reaction site. The carrier 6 can be used to deliver reagents 1, to a reaction site for the purposes of detecting the presence or absence of a substance, for example in a diagnostic test kit. Also claimed is a method of increasing the contrast in a diagnostic test between a signal indicating the presence or absence of a substance and a background signal and a method for reducing the need for critical control over the concentration of reagent applied to a test sample e.g. in histological testing.

Description

IMPROVEMENTS TO THE DELIVERY OF REAGENTS TO A REACTION SITE The present invention relates to an improved method of, and apparatus for, delivering reagents, such as for example antibodies, nucleic acids, enzyme substrates or stains, to a reaction site, such as a tissue sample.

Background Immunohistochemistry is a known technique for detecting the presence or absence of specific molecules in a biological sample and their distribution within the sample (typically a tissue sample).

Immunocytochemistry is a very similar known field working with cells.

These techniques take advantage of a specific antibody-antigen binding reaction.

We will take immunohistochemistry as an example, but the present invention is relevant to other fields as discussed later.

In order to detect an antibody bound to a specific antigen (i.e. primary antibody/antigen complex) it is necessary to use a detection system to enable the presence of the bound antibodies and their physical distribution to be determined. The detection system can be a direct system (with the antibody conjugated directly to the detection system) or it can be an indirect detection system where the readily-detected substance is associated with a molecule that binds directly or indirectly to the antibody. An example of a direct detection system is radio-labelling the primary antibody. An example of an indirect detection system is the wellknown avidin-biotin complex method.

Some detection systems use enzymes and require the application of a substrate of the enzyme used so as to generate a precipitate or coloured product (or to generate something that can be coloured subsequently, or to generate something which will cause a precipitate to form in a later step).

From the results of immunohistochemistry tests the presence of a target antigen can be detected as well as its localisation, or position between the cells or inside the cells.

Prior Art By way of example, and in order to put the present invention into context, we shall discuss one prior art way of applying reagents in immunohistochemistry. One known way of delivering the antibodies and the detection system to the tissue sample is to use liquid phase application which entails applying drops of antibody solution or components of the detection system at a specific concentration to the tissue sample mounted on a slide using a pipette. A three step liquid phase application may be used. In such a three step system a primary antibody is, as a first step, applied to the tissue sample, the sample and antibody are incubated for about 30 minutes and then washed, followed by application of the detection system comprising a second step of applying via a pipette a secondary antibody conjugated with an enzyme (to amplify the signal), the tissue sample and secondary antibody conjugate is incubated for 30 minutes and then washed, and then taking the third step of applying by hand, via a pipette and one or more reagents which react with enzymes to give a visible result. The sample is then washed (or incubated and washed) and the insoluble precipitate produced by the enzyme acting on the third reagent can be seen by eye or through a microscope.

It will be appreciated that there are many other known similar immunohistochemical techniques, and that the one discussed above is purely an example.

The Present Invention The object of the present invention is to improve the known method of delivering reagents, such as antibodies, to reaction sites. Some embodiments of the invention reduce the cost, skill and time involved while maintaining the accuracy of the results.

Although we worked initially in the fields of immunohistochemistry and immunocytochemistry when we thought of the invention, we have realised that it also has wider application to, for example, nucleic acid probe delivery systems, enzyme delivery systems, and stain delivery systems. In these areas the presence or absence of a specific molecule in a sample is being sought, and its distribution within the sample. These techniques also use detection systems/visualisation systems.

According to a first aspect of the invention we provide a method delivering a reagent to a biological sample to be tested for the presence or absence of a substance, the method comprising providing the reagent on a carrier; placing the carrier on the sample; ensuring that liquid is available at the carrier/sample interface; the arrangement being such that the reagent leaves the carrier and migrates to the sample where it interacts with said substance.

This method removes the need to use liquid phase application of the reagent which can be inaccurate, due to careless pipetting and requires skilled operators to pipette, and to prepare liquid reagents. The delivery means of the present invention is easy to apply to the reaction site.

The reagent may be provided on the carrier in dried or immobile form. A dried form of the reagent may be far more robust than a wet form. For example antibodies in solution stored in a vial have to be kept at about 40C and have a shelf-life of weeks or months. Dried antibodies can survive temperatures up to 20"C or 30"C (or even higher) and have a shelf life that is significantly longer.

The carrier will have an affinity for the reagent that it delivers, and preferably has a high affinity for the reagent that it delivers. The affinity of the carrier for the reagent is less than that of the reagent for its specific target but higher than that of the reagent for other components at the reaction site.

The carrier may be porous or non-porous.

The carrier may be flexible. This enables it to hug a tissue sample when there is a thin film of liquid between them: surface tension effects can be used to draw a thin film to a tissue sample, reducing the carrier sample distance and reducing the amounts of antibody (or other reagent) needed to ensure a satisfactory concentration of reagent at the sample site.

The carrier may be a membrane, or film. It may be a tab of material. Alternatively, it may have a substantial thickness and may be a sponge.

The carrier is preferably manufactured material, which may be cellulose based, such as nitro-cellulose, cellulose acetate or other treated form (e.g. paper), or nylon. The carrier is preferably hydrophilic.

The reagent is preferably provided on the surface of the carrier.

Nitro-cellulose and nylon are well known for their affinity for protein and they are used in many applications as a protein gatherer (e.g. to filter out protein). It is dogma in the art that nitro-cellulose (and nylon) binds proteins and never lets them go: we use it to deliver proteins, going directly against this dogma (which is not the whole truth).

We have found that we can use nitro-cellulose to deliver reagents in the controlled manner.

The method may comprise having a reagent that binds to the carrier and ensuring that the carrier does not have too many free binding sites to which a disassociated reagent can bind, so that a reagent that disassociates from the carrier tends to bind to the substance in the sample and not re-bind to the carrier.

The reagent is preferably provided impregnated in or bound to the carrier (e.g. membrane) and the carrier provided to the user with active chemicals pre-provided in it.

We can provide different reagents carried by different carriers, each being applied to the sample in use. Alternatively or additionally we can provide more than one active reagent on a single carrier.

If more than one reagent is provided on a carrier they may react with each other at the sample so as to produce a complex, or some other useful product at the sample, or they may be specific to different target molecules. If they are specific to different target molecules the reagents may have different detection/visualisation systems and may (or may not) require the application of different further substances to visualise their presence and location.

The carrier may carry, in addition to the reagent that is specific to the molecules being sought, a preservative. The carrier may alternatively or additionally carry an enhancing agent which makes the operation of the reagent more effective. For example, the carrier may have a high affinity for the reagent and may carry (in addition to the reagent) a blocking substance which blocks many binding sites on the carrier to make it more difficult for a disassociated reagent molecule to re-bind to the carrier in use.

The carrier (e.g. membrane) can be provided to the user (or stored) with the reagent in a functional (e.g. wet) state, or a potentially functional (e.g. dry) state.

The method preferably comprises applying the carrier to a reaction site where the reagent - target molecule specific reaction is to occur. The reaction site is typically a tissue section, or cytological preparation mounted on a slide.

Before, at, or after the stage of applying the carrier (e.g. membrane), the carrier may be altered by a process, such as, by rehydration.

The delivery means containing the reagent in dried form may be rehydrated by applying a drop of a solution to the reaction site prior to the application of the delivery means. Alternatively the reaction site may already be wet (for example when it is a tissue sample) and no separate application of liquid may be necessary. Liquid may, in some circumstances, be applied to the delivery means itself.

Preferably the solution is water, an aqueous solution, a buffer solution or organic solution.

The delivery means is preferably applied to the reaction site such that it covers or substantially covers the reaction site and is in contact therewith.

After the carrier has been applied to the sample the method preferably comprises having an incubation period to allow release of the reagent (and any other useful substances) from the carrier to the reaction site (e.g. tissue section).

The method may (or may not) further comprise applying one or more other carriers (e.g. membranes) carrying different reagents (e.g. proteins or enzyme substrates) for the purpose of detecting either the reagent that has been delivered to the reaction site or pre-existing substrates in the reaction site (e.g. tissue sample). The purpose of the subsequent application of another carrier (or other carriers) is to achieve visualisation of the targeted molecules (although an appropriate single carrier can also achieve visualisation) by delivering a detection system or components thereof.

One component of the detection system may be a reagent delivered to identify a specific molecule/substance and may comprise an antibody (e.g. immunohistochemistry or immunocytochemistry); or a nucleic acid probe (in-situ hybridisation); or an enzyme substrate (enzyme histochemistry); or a stain.

Another component of the delivery system may be a means of visualising the presence/absence of the above identification reagent. Such visualisation techniques include the use of enzymes (e.g. peroxidase, or alkaline phosphatase); or fluorescent compounds (e.g. FITC); or particulate material (e.g. gold); or radiolabelled compounds.

If the enzyme visualisation method is used a substrate of that enzyme must be applied (and this may be applied on a membrane in a similar way as are other reagents as previously discussed).

One or more of the components of the detection system may be delivered by using the carrier of the present invention.

According to a second aspect the present invention provides a method for determining the presence or absence of a substance at a reaction site (such as a tissue sample) comprising the steps of: providing a first reagent on a first delivery means; applying the delivery means to the reaction site; ensuring that the reagent is hydrated at the reaction site; removing the first delivery means; providing at least one component of a detection system adapted to indicate the presence or absence of said first reagent on at least one second delivery means; applying the second delivery means to the reaction site; ensuring the release of the component of the detection system from the second delivery means; and allowing the component of the detection system to react to provide a detectable signal.

The first reagent and/or the detection system components are preferably provided in dried form.

Preferably the substance (e.g. a protein) whose presence is to be determined is an antigen.

The first reagent is preferably a primary antibody specific to the antigen whose presence is to be determined. The primary antibody is preferably chosen from a library of antibodies specific to different antigens but originating from a common source and therefore having a common part of their structure. Preferably the source is a mammal such as a sheep.

The detection system preferably comprises a combination of two components on separate delivery means. The first component of the detection system may be a secondary antibody - enzyme complex (or some other detection/amplification means). The secondary antibody-enzyme complex is preferably specific to the common part of the library of primary antibodies. The enzyme functions to enhance the signal for detection.

When an enzyme detection system is used, the second component of the detection system may be a substrate upon which the enzyme operates to provide a detectable signal (e.g. an insoluble precipitate). Of course, the detection system may be a non-enzymatic system, in which case no substrate is necessary.

The use of a library of primary antibodies having a common source and therefore a common part, but each being specific to a different antigen, together with a secondary component is advantageous (but not essential). Although the library of primary antibodies is cheap to make, there may be many varieties of antibodies in the library and conjugating each one with a second component of a detection system (e.g. an enzyme to give an antibody-enzyme complex) and hence merging the reagent and the first component of the detection system would be costly and time consuming. If the secondary antibody, however, will bind to all the primary antibodies in the library, because of the common part possessed by the library, only the secondary antibody needs to be conjugated to the detection system and this can then be used with all the primary antibodies in the library, thus reducing the cost involved.

As a simplification-of the principle involved with one feature of one embodiment of the present invention consider the (unlikely) position when the delivery means is really 100% fully loaded. An antibody on the delivery means that disassociates itself from the delivery means has a choice of only two bound positions; it can re-bind to the delivery means in its previous position only or it can move to the antigen present on the reaction site. A competition of affinity is therefore created. If the antibody is specific to the antigen on the reaction site the antibody will bind thereto as its affinity for the antigen will be greater than that for the delivery means. If the antibody is not specific to the antigen on the reaction site the affinity with the delivery means will be greater and therefore the antibody will remain on the delivery means.

Of course, in practice we will not 100% load the delivery means because it is too expensive, but we can achieve a similar effect by blocking many potential binding sites on the carrier.

Reduction in Background Signal There is in accordance with another aspect of our invention disclosed herein a three level affinity competition which results in a reduction in background signal caused by the reagent (e.g. antibody) binding weakly to molecules present on the reaction site to which it is not specific. With the present method the affinity with the specific antigen is stronger than that with the delivery means but the affinity with the delivery means is stronger than that with other molecules/antigens to which the reagent (e.g. antibody) is not specific. Thus, as a simplification, the reagent binds to the molecules to which it is specific or remains on the delivery means. Of course affinity reactions/affinity constants are a matter of statistics or probabilities: the system is dynamic with reagents (e.g. antibodies) binding and unbinding continually, but there will be a greater tendency for them to stay bound longer on the specific protein being detected, and the lowest tendency for them to bind/stay on the non-specific tissue sample protein, and a middle affinity for them to bind to the delivery means.

Of course, the same applies when the reagent is not an antibody: nucleic acid probes/enzyme substrates, and stains can all have three-way affinity competition, and this can be used to reduce background staining.

Another advantage of the present method over the prior art is that the concentration of antibodies used is less important due to the competition of affinities. With the prior art methods the antibody solutions used in immunohistochemistry are titrated exactly to suit the sample, apparatus and conditions. This in itself is time consuming and requires more than a little skill. The method of present invention prefers that there be more reagent (e.g. antibodies) on the delivery means than target substance (e.g. antigen) on the reaction site, but the exact concentration is not so important as in the prior art method.

The effect of the delivery means being present as reagent (e.g. antibodies) disassociates is to offer an alternative to binding to the non specific protein in the sample, thus less background protein binding occurs, and thus the contrast between specific protein caused precipitate and background precipitate is increased, making it easier to see the results of the test.

Less Wasted Reagent Furthermore the prior art liquid phase application method results in a lot of waste. When a droplet of antibody solution is placed onto the tissue sample only the reagent (e.g. antibodies) adjacent to the sample contacts it and reacts, the remaining reagent in the droplet is wasted and might as well not be present. The delivery means of the present invention is preferably thin and flexible and therefore the majority of reagent (e.g. antibodies) contained within the delivery means contacts the sample and can react usefully with any antigen present.

Other Features of the Present Invention The primary reagent (e.g. antibody) and a secondary indirect visualisation system reagent (e.g. an antibody-enzyme complex) may be provided on the same delivery means. The storage of these substances together is not possible in the liquid phase as the two substances begin to react and complex within hours. However with one embodiment of the present method the delivery means can be dried before the substances react and no reaction will occur until they are rehydrated for use.

The delivery means of the present invention can be stored for longer periods of time than their liquid counterparts as the predried reagents do not deteriorate as fast.

According to another aspect the present invention provides a means (such as a membrane) for delivering reagents to a reaction site comprising a carrier carrying a reagent which in use is released to a reaction site.

The delivery means is preferably a hydrophilic membrane. The membrane is preferably nitro-cellulose, cellulose acetate, nylon, or the like. The delivery means may be another suitable material such as fabric, paper, or sponge. Preferably the delivery means is flexible to allow good contact with the reaction site, and to create a thin film of liquid between the delivery means and the reaction site, minimising spatial separation, and economising the amount of reagent (e.g. antibodies) needed. The reaction site is preferably a histological or cytological sample such as a tissue or tissue aspirate sample. The reagents may be antibodies, antibody complexes, nucleic acids, enzyme substrates, staining substances or other reagents to be delivered to the reaction site.

According to another aspect the present invention provides a method of making a means for delivering reagents to a reaction site comprising the steps of: providing a physical carrier, such as a membrane or the like, and applying to the carrier a solution of the reagent.

Preferably the method comprises drying the carrier. The carrier may be completely dried, or partially dried. The carrier may be soaked with reagent. The method may comprise incorporating more than one reagent into the carrier. The method may comprise incorporating a blocking substance into the carrier so that the reagent has difficulty in reassociating with the carrier once it has disassociated in use. The carrier is preferably sealed in foil sachets until it is used.

According to another aspect the present invention provides a kit comprising at least one carrier of the present invention containing a reagent which may or may not be in predried form.

The kit may include instructions. The kit may include a carrier (or delivery means) bearing primary reagent (e.g. antibodies) in predried form. The kit may include several carriers each bearing a different type of primary reagent. The primary reagents are preferably selected from a library of reagents having a common source and base sequence. The kit may include one or more carriers bearing components of the detection system of the present invention.

The detection system may comprise a secondary reagent (e.g. an antibody-enzyme complex). The secondary reagent preferably binds with the common base sequence of the library of reagents.

The kit may include one or more carriers bearing primary antibodies (or other reagents) in predried form and one or more delivery means bearing components of the detection system in predried form. The primary antibodies (or other reagents) in each carrier may be different.

The kit may include one or more carriers bearing both reagents, i.e. primary reagent (primary antibodies) and a secondary reagent that allows the primary reagent to be detected (e.g. an antibody-enzyme complex). The kit may include a peroxide, (or other substrate required for enzymatic reaction), which may be in dried form. The carriers may be provided in foil sachets, the foil sachets may be labelled with their contents.

We may provide a carrier with more than one active reagent (e.g. antibodies specific to different antigens). If so, we may prefer to have a first reagent (e.g. antibody) produced from an animal of a first species (e.g. sheep), and a second reagent (e.g. antibody) produced from an animal of a different species (e.g. rabbit). This can enable us to use simultaneously two different detection systems which do not interfere with each other (e.g. detection system 1 specific to material from species 1, and detection system 2 specific to material from species 2).

Third and subsequent different reagents possibly from a third and different species may be provided on the same carrier. Alternatively the kit may have different carriers with reagents from different species on them so as to simplify having different visualisation systems. A carrier having substances involved in more than one visualisation system may be provided. A kit may have a carrier with a plurality of independently different specific reagents and a carrier with a plurality of detection systems on it each complementary to and specific to a respective reagent.

Another way of looking at the present invention is as a diagnostic test. Whilst diagnostic tests practised on the human body are not patentable in some countries, diagnostic tests practised in vitro, not on the human body, are patentable practically everywhere.

According to another aspect of the invention we provide an in vitro diagnostic test comprising taking a biological sample from a subject (e.g. human, plant, animal, or microbial); providing a carrier which carries a reagent (e.g. an antibody) specific to a substance for which the test is looking; putting the carrier and the sample in close proximity and ensuring that there is a liquid phase between them to enable migration of said reagent from the carrier to the sample; examining the sample to detect the presence of the reagent.

Preferably the method includes the step of waiting for an affinity competition or reaction kinetic between the carrier and the substance for the binding of the reagent to approach equilibrium (or to at least begin sa that reagent is bound to the substance/reacts with the substance).

The diagnostic test will almost invariably involve extrapolating from the results of the examination of the sample to detect the presence of, or effect of the presence of, a substance on the probability of a disease, disorder, or abnormal or altered condition being present in the subject (and usually a specific disease, disorder or the like).

A consideration of the affinity competition between the carrier and the tissue sample for the antibodies leads us to conclude that another feature of the invention is the increase in contrast between specific signal that we are looking for to indicate the substance for which we are looking" and background signal.

According to a further aspect of the invention we provide a method of increasing the contrast in a diagnostic test between a signal indicative of the presence or absence of a substance of interest and background signal not indicative of the presence or absence of a substance of interest, the method comprising providing a sample being tested and having said substance and other substances; providing a reagent which reacts with, or binds to, said substance specifically, but which also reacts with or binds to said other substances to a lesser degree; providing competition means which react with, or bind to, said reagent to a lesser degree than does said substance, but to a greater degree than at least a significant proportion of said other substances; the arrangement being such that the competition means mops up excess reagent beyond that which reacts with/binds to said substance, so that the contrast between the result of applying the reagent to said substance and to said other substances is significantly greater than it would be if no competition means is provided.

Preferably the competition means is removed and the sample washed before a further step of providing a detectable signal from the said substance - reagent product is taken. The competition means may be the vehicle for delivering said reagent to said sample.

There is another technical effect which provides another way of looking at the invention. The effect having a balance due to competition between the substance to be detected and competition means (e.g. the carrier for the reagent), means that the concentration of antibodies seen by the sample varies less if the concentration of reagent in the carrier is varied then it would do if an equivalent change in concentration were made to a liquid pipetted onto the sample, with no competition means.

Thus there is less need to be too worried about getting the concentration of reagent that is applied exactly right.

According to another aspect of the invention we provide a method of reducing the need for critical control over the concentration of reagent applied to a test sample being tested in a histological or cytological test, the method comprising having a sample to be tested which has, or may have, a substance that is the subject of the test; applying to said sample a reagent which reacts with or binds to said substance; and ensuring that competition means is present, said competition means competing with said substance for reaction with/binding to said reagent, and said competition means having a reaction constant/affinity for said reagent that is significant, but that is less than that of said substance.

Thus this may effectively be thought to be a kind of buffer system, but in an unusual and new field.

Certain embodiments of the present invention will now be described in greater detail, but are not to be taken as limiting the invention in any'way, with reference to the drawings in which: Figure 1 shows the application of reagents to a reaction site by a) prior art liquid - phase application and by b) the method of the present invention; Figure 2a-c shows schematically an example of the prior art e.g. two step liquid phase application of reagents in immunohistochemistry, commonly known as the indirect peroxidase method.

Figure 1 shows the reduction in amount of antibody wasted by using the delivery means of the present invention rather than the prior art liquid phase application method.

In Figure la the reagent, in this case an antibody solution 1, has been delivered to a tissue sample 2 mounted on a slide 12 in the form of a droplet 3 from a pipette 4. The tissue sample has antigens 5 thereon and it can be seen that only the antibodies 1 at the portion of the droplet 3 in contact with the tissue sample 2 bind with the antigen 5, the remaining unreacted antibodies 1 in the droplet are washed away.

In Figure 1b where like numbers indicate like parts, the reagent 1 has been delivered to the tissue sample 2 mounted on slide 12 and having antigen 5 by the carrier (or delivery means) 6 of the present invention.

The carrier 6 is a tab formed of a flexible membrane, for antigens (not shown) on the sample to which it is not specific causing some background noise in the results. After the antibody 1 has been applied to the sample 2 incubation must occur for something of the order of 1 hour at a suitable temperature before the sample is washed to remove any unreacted antibody 1.

The second step of the technique shown in Figure 2b is the application of a secondary antibody 7 conjugated to an enzyme 8 e.g. peroxidase. This conjugate 10 binds to the primary antibody 1 and allows visualisation of the target antigen. The degree of visualisation of the antigen is dependent upon the amount of specifically bound enzyme over and above the amount of non-specifically bound enzyme. Again the conjugate must be incubated for about one hour at a suitable temperature before washing to remove the unbound conjugate.

The final step shown in Figure 2c is the addition of a peroxide, e.g. hydrogen peroxide, plus Diaminobenzidine tetrachloride (DAB). The peroxide reacts with the enzyme 8 of the secondary antibody enzyme conjugate 10 to form oxygen free radicals which oxidise the DAB to give a brown precipitate which is visible by eye or under a microscope.

The reagents are all added in the liquid phase as discussed in relation to Figure la. The overall operation to perform all three steps could take several hours, especially if titration of the antibody solutions to create the correct concentrations is required.

Alternatively, some or all of the reagents can be delivered by the carriers as described in the current invention, similar to that shown in Figure 3.

The carrier of the present invention is made by cutting a 4cm2 tab of nitro-cellulose and soaking it in a solution of the required reagent for 15 minutes at room temperature. The tab may or may not then be dried for subsequent use. For example, the carrier may be soaked in a solution of a primary antibody, secondary antibody-enzyme complex or a solution of Diaminobenzene depending on its intended use.

As a modification referring to Figures 1 and 2, the primary antibody 1 and the secondary conjugate 10 are applied using a single tab 6. The primary antibody 1 and the conjugate 10 are loaded onto a single tab 6 as described above and the tab is dried after limited complex formation has occurred. The tab can then be stored for long periods of time without the reagents complexing further as they are immobile on the tab.

Delivering both these reagents in the same tab reduces the number of incubation and washing steps needed.

Figure 3 shows a kit 19 comprising a number of tabs 18 contained in foil sachets 20 having labels 21 indicating their contents. The tabs in this example contain primary antibodies, secondary antibody-enzyme conjugates, DAB or both primary antibodies and secondary antibodyenzyme conjugates on a single tab. The kit also contains a vial 22 of 0.1% hydrogen peroxide solution in TBS and a pipette 23. The kit further contains instructions 24.

Producing Kits Having Carriers Loaded With Reagents Example 1 - Preparation of antibody carrier (or delivery means) A piece of nitro-cellulose (or cellulose acetate, or nylon, or the like) is cut to give 4cm2 pieces (size can vary with application and may be, for example, in the range 1 to 8 cm2) comprising the delivery means or carrier.

The pieces of nitro-cellulose are washed in a solution of antibody diluted in 1-5% BSA. Antibody dilution can vary with application. The nitro-cellulose pieces are soaked in the antibody solution for 15 minutes at room temperature.

The impregnated/soaked nitro-cellulose pieces are dried at 450C in an incubator with a fan for 15 minutes.

The carrier containing dried antibodies are sealed in foil sachets for storage until use.

Example 2 - Preparation of Diaminobenzene (DAB) carrier A piece of nitro-cellulose is cut to give 4cm2 pieces (size can very with application and may, for example, be in the range of 1 to 8cm2) comprising the carrier.

The nitro-cellulose pieces are soaked in a solution of DAB for 15 minutes at room temperature.

The soaked nitro-cellulose pieces are then dried.

The carrier containing the DAB solution in predried form are sealed in foil sachets for storage until use.

Example 3 - General procedure for immunohistochemistry using the carrier of examples 1 and 2 to deliver reagents to the reaction site Sections of the tissue sample to be analysed are mounted on glass slides and put in an incubator over night at 60"C.

The sections are then de-waxed and rehydrated.

A citrate buffer of pH 6.0 (or similar buffer) may be applied to the sample and heated (e.g. in a pressure cooker) for several minutes to retrieve the antigens in the sample.

The sample is washed in a solution of 1% hydrogen peroxide in methanol for 10 minutes to quash endogenous peroxidase activity. The sample is then washed in buffered aqueous solution.

A carrier (or delivery means) containing a primary antibody specific to the antigen whose presence is to be determined is applied to the sample for 1 hour. After removing the carrier the sample is washed with a buffered aqueous solution to remove any unreacted antibodies.

A carrier containing a secondary antibody - enzyme conjugate, wherein the secondary antibody can bind with the primary antibody, is applied to the sample for 1 hour. After removing the carrier the sample is washed with buffered aqueous solution to remove any unreacted conjugate.

A 0.1% solution of hydrogen peroxide in buffered aqueous solution is added to the sample followed by a carrier containing DAB for 10 minutes. Alternatively, the hydrogen peroxide and DAB can be combined in one carrier.

The carrier is removed and the sample is counter stained, dehydrated and a coverslip applied before the results are analysed.

The above specific examples all relate to immunohistochemistry, but it will be appreciated that the advantages of the present invention are not restricted to that field above.

As discussed earlier, the reagent provided on the carrier/tab could be a nucleic acid probe (e.g. we can use impregnated carriers/tabs to detect viral DNA sequences in human tissue, the tabs carrying specifically designed nucleic acid probes for in-situ hybridisation). The nucleic acid probes typically have an associated detection/visualisation system, such as a radiolabelled portion (which can be used to precipitate materials).

An example in another field is in enzyme histochemistry where enzymes of interest already exist in a tissue sample and a suitable enzyme substrate can be applied by a carrier to identify their presence and distribution/location.

The reagent provided on the carrier can be a pre-made complex. If the reagent is dried, the complex is "frozen" and does not complex further. Thus we can store carriers with pre-made complexes. It is not easy to store complexes in liquid form since they continue to complex further and precipitate out.

Although having pre-dried carriers is not essential to the invention in its broadest terms it does have considerable advantages: antibodies or other reagents can be stored dry in a hardy form, and storage at the correct temperature is not so vital; a pack of dry tabs has far less weight than a pack of wet tabs because of the liquid (a factor when we consider shipping kits across the world); and the shelf life of the tabs is increased if they are dry.

The delivery system of the present invention has the advantages of:- increased stability reduced background staining increased reproducibility can use less skilled operators ease of use allows complex interactions to be stabilised for storage.

Typical materials we envisage providing on tabs (but not an exhaustive list) are:- antibodies, conjugated antibodies (e.g. conjugated with biotin, avidin, enzymes, FITC, gold, radiolabelled compounds), lectins, enzymes (for tissue pre-treatment), substrates for endogenous enzymes (histochemistry e.g. alkaline phosphatase in osteosarcoma), general dyes and stains.

Another area where a pre-loaded tab can be used is to deliver substances necessary to prepare a histological or cytological sample for further steps. For example to deliver enzymes (e.g. trypsin) for the enzymatic treatment of a sample prior to subsequent tests (i.e. immunohistochemistry).

Claims

CLAIMS 1. A method of delivering a reagent to a biological sample to be tested for the presence or absence of a substance, the method comprising providing the reagent on a carrier; placing the carrier on the sample; ensuring that liquid is available at the carrier/sample interface; the arrangement being such that the reagent leaves the carrier and migrates to the sample where it interacts with said substance.
2. The method according to claim 1 wherein the reagent is provided on the carrier in dried or immobile form.
3. The method according to claim 1 or claim 2 wherein the carrier has an affinity for the reagent that it delivers.
4. The method according to any preceding claim wherein the carrier has a high affinity for the reagent that it delivers.
5. The method according to any preceding claim wherein the affinity of the carrier for the reagent is less than that of the reagent for its specific target but higher than that of the reagent for other components in the sample.
6. The method according to any preceding claim wherein the carrier is flexible.
7. The method according to any preceding claim wherein carrier is a membrane, a film, or a tab of material.
8. The method according to any one of claims 1 to 6 wherein the carrier has a substantial thickness and is a sponge.
9. The method according to any one of the preceding claims wherein the carrier is a manufactured material.
10. The method according to claim 9 wherein the material is cellulose based.
11. The method according to claim 9 or claim 10 wherein the material is nitro-cellulose, cellulose acetate or nylon.
12. The method according to any preceding claim wherein the carrier is hydrophilic.
13. The method according to any preceding claim wherein the reagent is provided on the surface of the carrier.
14. The method according to any preceding claim further comprising having a reagent that binds to the carrier and ensuring that the carrier does not have too many free binding sites to which a disassociated reagent can bind (so that a reagent that disassociates from the carrier tends to bind to the substance in the sample and not re-bind to the carrier).
15. The method according to any preceding claim wherein the reagent is provided impregnated in or bound to the carrier.
16. The method according to any preceding claim further comprising providing different reagents carried by different carriers, each being applied to the sample in use.
17. The method according to any preceding claim further comprising providing more than one active reagent on a single carrier.
18. The method of any preceding claim further comprising before, at, or after the stage of applying the carrier to the sample, rehydrates the reagent.
19. The method according to any preceding claim further comprising having an incubation period in which the carrier is in contact with the sample for a period such that the reagent is released from the carrier and reaches the sample.
20. The method according to any preceding claim wherein the method further comprises applying one or more other carriers carrying different reagents for the purpose of detecting either the reagent that has been delivered to the sample or pre-existing substrates in the sample.
21. The method according to claim 20 wherein a first component of the detection system is a reagent delivered to identify a specific molecule/substance .
22. The method according to claim 21 wherein the first component comprises an antibody or a nucleic acid or an enzyme substrate or a stain.
23. The method according to any one of claims 20-22 wherein a second component of the delivery system is a means of visualising the presence/absence of the identification reagent.
24. A method of delivering a reagent to a biological sample substantially as described herein with reference to and as shown in the drawings.
25. A method for determining the presence or absence of a substance at a reaction site (such as a tissue sample) comprising the steps of: providing a first reagent on a first carrier; applying the first carrier to the reaction site; ensuring that the reagent is hydrated at the reaction site; removing the first carrier; providing at least one component of a detection system adapted to indicate the presence or absence of said first reagent on at least one second carrier; applying the second carrier to the reaction site; ensuring the release of the component of the detection system from the second carrier; and allowing the component of the detection system to react, or not reacting with said substance to provide a detectable signal.
26. The method according to claim 25 wherein the first reagent and/or the detection system components are provided in dried form.
27. The method according to claim 25 or claim 26 wherein the substance whose presence is to be determined is an antigen.
28. The method according to any one of claims 25 to 27 wherein the first reagent is a primary antibody specific to the antigen whose presence is to be determined.
29. The method according to claim 28 wherein the primary antibody is chosen from a library of antibodies specific to different antigens but originating from a common source and therefore having a common part of their structure.
30. The method according to claim 29 wherein the source is a mammal.
31. The method according to any one of claims 25 to 30 wherein the detection system comprises a combination of two components on separate delivery means.
32. The method according to claim 31 wherein the first component of the detection system is a secondary antibody - enzyme complex.
33. The method according to claim 30 wherein the first component of the detection system is provided in the same carrier as the first reagent.
34. The method according to claim 32 wherein the secondary antibodyenzyme complex is specific to the common part of the library of primary antibodies.
35. The method according to any one of claims 31 to 34 wherein the second component of the detection system is a substrate upon which the enzyme operates to provide a detectable signal.
36. A method of determining the presence or absence of a substance in a sample substantially as described herein.
37. A means for delivering reagents to a reaction site comprising a carrier carrying a reagent which in use is released when coupled to a reaction site.
38. The means according to claim 37 wherein the carrier is a hydrophilic membrane.
39. The means according to claim 38 wherein the membrane is nitrocellulose, cellulose acetate, nylon, or the like.
40. The means according to claim 37 wherein the carrier is fabric, paper or sponge.
41. The means according to any one of claims 37 to 40 wherein the carrier is flexible.
42. The means according to any one of claims 37 to 41 wherein the reaction site is a histological or cytological sample.
43. The means according to any one of claims 37 to 42 wherein the reagents are antibodies, antibody complexes, nucleic acids, enzyme substrates, staining substances or other reagents to be delivered to the reaction site.
44. A means for delivering reagents to a sample substantially as described herein.
45. A method of making a means for delivering reagents to a reaction site comprising the steps of: providing a physical carrier, and applying to the carrier a solution of the reagent.
46. The method according to claim 45 further comprising completely or partially drying the carrier.
47. The method according to claims 45 or 46 wherein the carrier is soaked with reagent.
48. The method according to any one of claims 45 to 47 further comprising incorporating more than one reagent into the carrier.
49. The method according to any one of claims 45 to 48 further comprising incorporating a blocking substance into the carrier, so that the reagent has difficulty in reassociating with the carrier once it has disassociated in use.
50. A method of making a means for delivering reagents to a sample substantially as described herein.
51. A kit comprising at least one carrier according to claim 37 containing a reagent which may or may not be in predried form.
52. The kit according to claim 51 including at least one carrier bearing a reagent in predried form.
53. The kit according to claim 51 or claim 52 including several carriers each bearing a different type of reagent.
54. The kit according to any one of claims 51 to 53 wherein the reagents are selected from a library of reagents having a common source and base sequence.
55. The kit according to any one of claims 51 to 54 further including one or more carriers bearing components of a detection system.
56. The kit according to claim 55 wherein the detection system comprises a secondary reagent.
57. The kit according to claim 56 wherein the secondary reagent binds with the common base sequence of the library of reagents.
58. The kit according to claims 56 or 57 including one or more carriers bearing both a primary reagent and a secondary reagent.
59. The kit according to any one of claims 56 to 58 including a substrate required for enzymatic reaction.
60. The kit according to any one of claims 51 to 59 including a carrier bearing more than one reagent.
61. The kit according to any one of claims 51 to 60 including a carrier with a plurality of detection systems on it each complementary to and specific to a respective reagent.
62. A kit substantially as described herein with reference to and as shown in the drawings.
63. An in vitro diagnostic test comprising taking a biological sample from a subject; providing a carrier which carries a reagent specific to a substance for which the test is looking; putting the carrier and the sample in close proximity and ensuring that there is a liquid phase between them to enable migration of said reagent from the carrier to the sample; examining the sample to detect the presence of the reagent.
64. The test according to claim 63 wherein the subject is human, plant, animal or microbial.
65. The test according to claims 63 or 64 including the step of waiting for an affinity competition or reaction kinetic between the carrier and the substance for which the test is looking to approach equilibrium.
66. The test according to any one of claims 63 to 65 including extrapolating from the results of the examination of the sample to detect the presence of, or effect of the presence of, a substance on the probability of a disease, disorder, or abnormal or altered condition being present in the subject.
67. An in vitro diagnostic test substantially as described herein.
68. A method of increasing the contrast in a diagnostic test between a signal indicative of the presence or absence of a substance of interest and background signal not indicative of the presence or absence of a substance of interest, the method comprising: providing a sample being tested and having said substance and other substances; providing a reagent which reacts with, or binds to, said substance specifically, but which also reacts with or binds to said other substances to a lesser degree; providing competition means which react with, or bind to, said reagent to a lesser degree than does said substance, but to a greater degree than at least a significant proportion of said other substances; the arrangement being such that the competition means mops up excess reagent beyond that which reacts with/binds to said substance, so that the contrast between the result of applying the reagent to said substance and to said other substances is significantly greater than it would be if no competition means is provided.
69. The method according to claim 68 wherein the competition means is removed and the sample washed before a further step of providing a detectable signal from the said substance - reagent product is taken.
70. The method according to claim 68 or claim 69 wherein the competition means is the carrier for delivering said reagent to said sample.
71. A method of increasing the contrast of a diagnostic test between a signal indicative of the presence or absence of a substance of interest and a background signal substantially as described herein.
72. A method of reducing the need for critical control over the concentration of reagent applied to a test sample being tested in a histological or cytological test, the method comprising: having a sample to be tested which has, or may have, a substance that is the subject of the test; applying to said sample a reagent which reacts with or binds to said substance; and ensuring that competition means is present, said competition means competing with said substance for reaction with/binding to said reagent, and said competition means having a reaction constant/affinity for said reagent that is significant, but that is less than that of said substance.
73. A method of reducing the need for initial control over the concentration of reagent applied to a test sample substantially as described herein.