GB2098730A - Immunolocalisation - Google Patents

Abstract

A method of detecting an antigenic substance, in animal tissue, in which a primary antibody to the substance is raised, labelled with a selected hapten such as DNP and contacted with the substance, after unwanted binding sites on the substance have been blocked by applying a wide spectrum of antibodies obtained from a different animal. Subsequently the primary antibodies are contacted with a bridging antibody e.g. Monoclonal IgM anti-DNP antibody, raised in a different species to be specific to the primary antibody. A detecting agent consisting of an enzyme with the same hapten label, e.g. peroxidase labelled with DNP, is then contacted with and binds to the bridging antibody. Finally the enzyme is reacted with, e.g. diaminobenzidine, to cause visible coloration, by which the antigenic substance is detected.

Description

SPECIFICATION Immunolocalisation This invention relates to methods or procedures and prepared biochemical materials for use in detecting a substance of interest, in an environment of biological cells, for example animal tissue. The term "detecting" as used herein is intended to include locating, identifying or quantifying, and in general any method or technique which involves a response to the presence of the substance.

Immunological methods of detection are well known and in general rely on the ability of an animal to produce antibodies to a particular antigen and the specific binding properties of the antibody to the antigen.

It is well known that an antibody is an immunoglobulin molecule which has two (or more) identical sites capable of binding to a part of an antigen molecule referred to as an antigenic determinant. The binding is so strong that it can be regarded as for practial purposes irreversible. Antibodies capable of binding to a particular antigen (antigen-specific antibodies) are produced in an animal by immunisation. This results in the appearance of several groups of antibodies in the animal's serum. Each is capable of binding to a different antigenic determinant. Such a determinant can be natural to the antigenic protein or added artificially. The artificially linked antigenic determinant is referred to as a hapten. Antibodies themselves are known to be antigens possessing their own unique set of natural determinants, and antibodies to these determinants can be produced by immunisation.

Alternatively, several molecules of a given hapten can be chemically attached to an antibody molecule (a hapten-labelled antibody) and antibodies specific for this hapten can be produced again by the process of immunisation. These hapten-specific antibodies will then bind to hapten-labelled antibodies, but not to their unlabelled counterparts. Serum from an immunised animal, in addition to having antigen-specific antibodies, contains naturally occurring antibodies which may bind to a wide spectrum of molecules present in a tissue section. These may represent either the corresponding antigens or specific receptors for immunoglobulins. Hence it is desirable in immunological procedures that tissue binding of the natural antibodies should be either differentiated from that of antigen-specific antibodies or alternatively prevented from taking place.The antigen-specific antibodies are termed either polyspecific or monospecific depending on whether they bind to a number of structurally different antigenic determinants or a single unique determinant respectively. Both polyspecific and monospecific antibodies may arise from several unique groups of clones of antibody-forming cells and are hence polyclonal in origin. However, it is possible to raise a monospecific antibody from a single clone of cells using Köhler-Milstein hydridoma technique and the resulting antibodies are known as monoclonal hybridoma antibodies. Although the majority of monoclonal hybridoma antibodies have only two identical antigen binding sites per molecule of antibody, it is possible to produce a special class of antibodies called IgM monoclonal antibodies, which have as many as ten identical binding sites per molecule of antibody.

Existing techniques suffer from a number of limitations and problems. For example, it is not easy to maintain a storage bank of antibodies for all possible antigenic determinants and for some uncommon antigens antibodies can only be raised in rare exotic animals. There is also the general problem of non-specific binding of antibody to antigen, which occurs for a number of reasons. Indeed at all stages in the procedure there is a chance of error occurring and it is an object of the invention to reduce the overall risk of error, and to improve the specificity and sensitivity of the process.

Broadly stated, the invention consists in a method of detecting a substance of interest in an environment of biological cells, in which a primary antibody to the substance is caused to bind to the substance, a bridging antibody is bound to the primary antibody, and a detecting agent is bound to the bridging antibody, thereby specifically linking the detecting agent to the substance of interest.

From one aspect the invention is based on the concept of artificially linking a hapten to the primary antibody. Thus from this aspect, the invention consists in a detecting method in which the primary antibody used in the first stage of the process is artificially labelled with a selected hapten, and the briging antibody, used in the second stage of the process, is specific to said hapten, and binds thereto.

Conveniently the detecting agent used in the third stage of the process is labelled with the same hapten, and binds to the bridging antibody, and in a particular preferred example the hapten is of the dinitrophenyl group (DNP).

From another aspect, the invention is concerned with an improved procedure for blocking unwanted binding of the primary antibody to the various components of the tissue section. From this aspect the invention consists in a detecting method of the type referred to in which the environment of cells is first selectively blocked by contacting therewith a wide spectrum of blocking antibodies which designedly excludes antibodies to the substance of interest, the blocking and primary antibodies being derived from either animals of different species, the same species, or from the same animal.

This is a useful procedure in any immunological staining or other detection process but is a particularly versatile manoeuvre in conjunction with the hapten labelling technique mentioned above.

From another aspect of the invention the bridging antibody is selected to be polyvalent, ie having more than two identical binding sites. At best only one site will bind with the primary antibody, leaving available a plurality of binding sites for the detecting agent(s), which in effect provides a magnification factor in the detection procedure. This is particularly valuable if a monoclonal polyvalent bridging antibody is used, since it maintains the selective specificity of the linking.

When used with the hapten labelling technique the detecting agent(s) will be labelled with the same hapten as the primary antibody.

The invention is also concerned to provide an improved performance in the final detecting step of the procedure, and from this aspect the invention is based on the simultaneous use of two different detecting agents both bound through a bridging antibody to the origianl antigen. From this aspect, the invention consists in a method of the type referred to in which two different enzymes or other detecting agents are used, both bound to the bridging antibody, and such that one enzyme or agent will produce the substrate for or otherwise activate the other to produce a detectable product.

In one form of the invention, the two detecting agents are linked to the same polyvalent bridging antibody and it follows that the two agents must be closely apposed. The reaction of the second agent will only occur if the first agent is itself activated and it follows that no reaction occurs if only one of the two agents binds.

In an alternative procedure, likewise using two related but different detecting agents, the two agents are linked via two distinct bridging systems to different but closely apposed antigens of the original cell environment. This is of particular value in providing a more precise identification of an antigen by binding of two different antibodies simultaneously to different but closely juxtaposed antigen determinants,.

From yet another aspect, the invention consists in a method of the type referred to in which the bridging antibody does not bind to any natural determinants on the primary antibody, other than the artificially linked determinant.

The invention may be performed in various ways and some specific embodiments will now be described by way of example, with reference to the accompanying drawings, in which: Figure 1(a) is a diagram illustrating the binding of a specific antibody to one of a number of determinants of an antigen; Figure 1(b) illustrates the binding of different antibodies to different determinants:: Figure 1(c) illustrates a natural unlabelled antibody having natural determinants; Figure 1(d) illustrates an antibody having hapten artificially linked thereto, binding anti-hapten antibodies; Figure 2 illustrates diagrammatically a technique for linking a visually detectable agent to a selected antigen of a base tissue; Figure 3 is a diagram illustrating one of a number of possible detecting methods according to the invention, with a polyvalent bridging antibody; Figure 4 is a diagram illustrating the generation of a visual pigment by a detecting agent, in this case an enzyme called peroxidase; Figure 5 is a diagram illustrating production of a pigment by a pair of interacting detecting agents, in this case enzymes peroxidase and glucose oxidase;; Figure 6 is a diagram illustrating a pair of interacting detecting agents (eg peroxidase and glucose oxidase) located separately to different antigen determinants.

It will be appreciated from Figures 1 (a) to (d) that antibodies specific to different determinants on a common antigen tissue can bind simultaneously to the different determinants, the specific binding properties being illustrated by matching symbols. By artificially attaching selected determinants such as haptens to the antibody, anti-hapten antibodies can then be linked to the primary antibody (Figure 1 (d)).

In the procedure illustrated in Figure 2, the tissue section to be investigated is illustrated at 20 with the antigenic determinants under investigation at 21. A primary antibody (22) to this determinant is raised, for example in a rabbit, and before this is applied various unwanted immunoglobulin sites on the tissue are "blocked" by applying a wide sepctrum of antibodies (23) obtained from a different species of animal eg goat. These blocking antibodies may be the serum from a goat which has not been immunised with the antigen in question and therefore does not include the specific antibody. When the primary antibody (22) is applied, it therefore binds selectively to the determinant (21 ) unless there are any sites capable of binding rabbit antibody which have not been adequately blocked by the goat serum.

Subsequently a bridging antibody (24) is applied, this antibody having been raised in a different species, for example a goat, to be specific to the primary antibody (22). Next a visualising or "detecting" agent is linked to the bridging antibody. In this case it consists of enzyme labels (25) both binding to an antibody (26) raised in the same species as the primary antibody (2) (ie a rabbit), and therefore capable of binding selectively to the bridging antibody (24).

In this way it will be seen that the detecting agents, for example the enzyme peroxidase, are linked specifically to the determinant (21) of interest. By generating a suitable reaction with the peroxidase, for example by adding diaminobenzidine (DAB), an insoluble coloured pigment is produced, thus forming the visually detectable stain.

The technique illustrated in Figure 3 also produces a visual staining but provides improved efficiency and accuracy. In this case the tissue (30) is taken for example from rats' pituitary in which the presence of the antigen in question (31) (thyroid stimulating hormone or TSH) is to be visualised. The section is exposed firstly to serum from an animal such as a guinea pig prior to immunisation with TSH. This serum, referred to as non-immune serum, contains natural antibodies but no anti-TSH antibodies. Any natural antibody that has an affinity for any of the myriad of antigens, other than TSH, present in the rat pituitary section will bind virtually irreversibly at this stage. In addition, any of these antibodies, including the antibody to TSH, may bind to naturally occurring immuoglobuiin receptors. In this manoeuvre substantially all the natural antibody binding sites are virtually permanently blocked. Serum obtained from the same animal or an animal of the same species, after its immunisation with TSH, is added next. This serum has been labelled with a suitable hapten (dinitrophenyl group, DNP) such that all the proteins present in it, including all the antibodies, natural and anti-TSH, are known to possess on them a number of chemically linked DNP groups. Since the natural antibody binding sites are already occupied by unlabelled antibodies, the only DNP labelled antibodies that will bind irreversibly at this second stage will be the anti-TSH antibodies. After an appropriate degree of washing, only the TSH-containing sites on the tissue sections should remain covered with DNP-labelled antibodies.The DNP on these antibodies is then allowed to interact with monoclonal IgM anti-DNP antibodies. Since this antibody has as many as 10 combining sites, for every antibody molecule binding to a DNP group in the section there will be left a certain number of free sites, which are then used as highly avid and selective receiving units for a suitable form of DNP labelled peroxidase. Since the anti-DNP antibodies are monoclonal in nature, it can be assumed within reasonable limits that they will only bind to DNP groups and no other constituent of either the tissue or the peroxidase enzyme preparation.

The last step in the staining sequence is that of formation and deposition of an insoluble brown product at the peroxidase containing sites in the tissue section by use of a substance which will react with the enzyme to produce a visually detectable insoluble coloured product. For this, a solution containing diaminobenzidine is added onto the section for a period long enough to allow deposition of clearly visible amounts of the coloured product. The final step of producing a visible stain may be enhanced by means of the novel double enzyme technique of this invention.

In the single enzyme technique, the final localisation of the site of the antigen depends upon the use of a histochemical technique, whereby the substrate of the enzyme used as detecting agent is added to the medium, together with a reagent which is converted to a coloured insoluble product as a consquence of the reaction involving enzyme and substrate. Thus with reference to Figure 4, the substrate H202 is added, together with DAB, and these react with the peroxidase enzyme to produce the brown pigment.

In the double detecting agent technique as shown in Figure 5, both agents (in this case the enzymes glucose oxidase and peroxidase) are labelled with the hapten DNP, and the use of polyvalent IgM as the bridging antibody allows both enzymes to bind to one antibody molecule. When the substrate (glucose) of glucose oxidase is added, together with a reagent such as DAB, a coloured insoluble product is formed as a consequence of the reaction involving peroxidase and its substrate H202, which in this instance is not added to the medium, but produced by the first enzyme.

As H202 is rapidly destroyed, it can be seen that the reaction will only take place when the two enzymes are closely apposed. Either of them binding aione would not give rise to a reaction. This has several advantages: 1. It will virtually abolish non-specific staining due to natural peroxidase within the tissue; 2. It will lower "background" due to non-specific binding of small numbers of enzyme molecules, as isolated enzyme molecules will not give any reaction; 3. The local generation of H202 from the first enzyme, glucose oxidase, is a very efficient method of provision of substrate for the second enzyme peroxidase.

The double detecting agent technique may also be used in a more complex way by labelling for example the two enzymes with different haptens, and using the two enzymes as the final steps in the localisation of two separate antigens, as shown in Figure 6. It can be seen that this would only lead to a positive result if the two enzymes were sufficiently close for the H202 produced by the first to diffuse to the second. The distance through which this diffusion may lead to significant reaction may be controlled by controlling the rate at which H202 is destroyed in the medium. The uses of this double enzyme technique can therefore be extended to: (a) More precise identificationof antigen. There is increasing interest in the possibility that antibodies may not be as specific as was once thought, because of a variety of cross reactions.With this technique, two antibodies directed against different parts of the molecule of the antigen can be used.

A positive result indicates the presence of both parts of the antigen, thus increasing very greatly the degree of specificity of the reaction; (b) Identification of proximity of two separate antigens. This technique can be used to demonstrate that two hormones are present in a cell, or that two hormones are present in one granule in a cell without, as at present, using serial semi-thin sections; (c) Identification of a unique structure by virtue of its content of two antigens, both of which occurred separately elsewhere. This application may be of particular value in automated measurements.

Methods and Materials Dinitrophenylating reagent 3-Aminopropionitrile was prepared from acrylonitrile by the method of Buc (1947). 3-(2,4 Dinitrophenylamino) proprionitrile was prepared by reaction of 3-amino-propionitrile (Aldrich Chemical Company, England) with 1-fluoro-2,4-dinitrobenzene (Sigma Chemical Company, England) in ethanol. After recrystallisation from ethanol it was obtained as bright yellow needles, m p 137 , (c.f. Schramm, 1967; 135 ).

3-(2,4-Dinitrophenylamino) proprionitrile (1 9) was suspended in absolute methanol (40 ml) and cooled to 0 under an atmosphere of dry nitrogen. A vigorous stream of dry hydrogen chloride was bubbled through the mixture keeping the temperature constant at 0 . After 1 hour the hydrogen chloride stream was stopped and the mixture was allowed to stand a further 2 hours at 00. A small amount (typically 0.03 to 0.06 g) of starting material was removed by filtration while still under dry nitrogen. Anhydrous ether (50 ml) was added and the product methyl 3-(2,4-dinitrophenylamino) propionimidate hydrochloride (DNP imidate-HCI)* allowed to crystallise out slowly.The solid was filtered off rapidly, washed with cold anhydrous ether, allowed to stand in a dessiccator under vacuum and stored at 0 under dry nitrogen. The DNP- imidate-HCI salt (final yield 0.8 g = 62%) was obtained as very fine bright yellow needles (m.p. 1 16-8" decomp.) with a molecular composition of C1oH13N405CI (found C,39.1; H,4.5; N,18.3% as compared to the calulated C,39.4; H,4.3; N,18.4%) At 2"C the solubility of DNP-imidate-HCI in deionised H2O (pH 5.5) was found to be 10 mg/ml.However, it fell to 1 mg/ml on raising the pH to 9.0 (triethanolamine-HCI buffer; 0.3M) ie. at conditions which were found to be necessary for efficient dinitrophenylationof proteins (see below)

Dintrophenylated peroxidase marker Directly DNP-labelled peroxidase failed to yield any staining using the hapten sandwich procedure.

Indirect labelling of peroxidase e.g. in form of DNP-labelled peroxidase anti-peroxidase complexes (for details see Jasani, et al 1981) gave positive staining but the marker was found to be unstable since it lost its activity within a few days of storage at 4"C. Stable conjugates of peroxidase with proteins obtained using glutaraldehyde as the coupiing agent were also found to be inactive (c.f. Sternberger, 1980). On the other hand conjugates synthesised using a modified periodate method of Nakane (see Boorsma and Streefkerk, 1971 for detaiis) were found to be both active and stable.A method was used to produce various conjugates of horseraddish peroxidase (H.R.P.; Boehringer Grade IV) with either bovine serum albumin (BSA; Sigma Chemical Company) or Cytochrome C (CYT-C; Sigma Chemical Company) in the following manner: HRP (8 mglmi in deionised H2O) was treated with a freshly prepared sodium periodate solution (0.8 ml; 0.1 M) over 20 minutes at room temperature with intermittent gentle mixing. The reaction was stopped by addition of concentrated ethylene glycol (3 drops) and mixing for 5 minutes. The mixture was then passed down a column of Sephadex G-25 equilibrated with sodium acetate buffer (0.001 M; pH 4.2). To the eluate (2.5 ml) was added either BSA (4 mg) or CYT-C (4,8 or 16 mg).The pH of the solution was adjusted to 9.5 by addition of 1 ml of sodium bicarbonate buffer (pH 9.5; 1 M) and mixed intermittently over a period of 2 hours at room temperature. Freshly prepared potassium borohydride solution (0.2 ml; 8 mg/ml in deionised H2)) was then added in order to stabilise HRP-protein conjugates. After 2 hours incubation at 4"C, the reaction mixture was dialysed overnight against PBS at room temperature.

The dialysed conjugates solution was then DNP-labelled for varying lengths of time (2,4 or 24 hours) using conditions described below for the DNP labelling of antiserum. Any unreacted protein or DNP reagent were separated from the conjugates by ACA 44 column chromatography (1 x 10cm; PBS).

Of all the conjugates examined, the most active conjugate finally chosen for the staining purpose was the one formed between HRP {8 mg) and CYT-C (4 mg) and DNP-labelled for 2 hours. This agent was found to be stable for at least 1 year at 40C in the presence of thiomerosal (0.002% w/v).

Preparation of DNP-glucose oxidase Glucose oxidase (740 units/0.5 ml; Sigma Type V; 2"C) was mixed with 450 pl of distilled water (2"C) and 50 ml of triethanolamine-Hel buffer (3 M; pH 9.0,2 C). The reaction was initiated by adding DNP-N-IE suspension (0.5 mg in 50 Mgl of distilled water, 2" C), and terminated after 2 hours incubation at 20 C by separation of the dinitrophenylated glucose oxidase on a Sephadex G-25 column (equilibrated with PBS).

DNP-glucose oxidase was stored at -70" C until used.

Preparation of 6iotin-labelled glucose oxidase Glucose oxidase (185 units/125 Ri; Sigma Type V) was mixed with NaHCO2 (825 FI; 0.1 M; pH 8.1). To the mixture, biotin (Sigma Chemical Company; 50 yl of 20 mg/ml solution in double distilled dimethyl formamide) was added to initiate the reaction. After 1 hour at room temperature, the reaction mixture was dialysed overnight against phosphate-buffered saline (PBS, 21; 0.01 M; pH 7.4).

Preparation of antibodies to thyroid stimulating hormone (TSll) Seven adult guinea-pigs (Duncan Hartley strain), weighing 600 g on average, were injected intradermally with highly purified bovine TSH (20 Fg/animal) emulsified in complete Freund's adjuvant (1 part TSH solution:1 part adjuvant). Prior to injection, serum from each guinea-pig was obtained and stored at -70" C until used (= pre-immune serum). Four weeks later a further injection of TSH in complete Freund's adjuvant was given and sera obtained from each animal after an interval of another 2 weeks (anti-TSH serum).

Antibodies to human immunoglobulin antigenic determinants Monoclonal antibodies to human a, -A and K immunoglobulin chains were purchased from Seward Laboratory, London.

Dinitrophenylation ofprimary antisera using DNP-N-IE Aqueous suspension (80 pI) of DNP-N-IE (10 mg/ml distilled water, 2" C) was added to 395 pI of triethanolamine-HCI buffer (0.3 M; pH 9.0,20 C) pre-mixed with undiluted antiserum (125 pl; 2" C) in order to initiate the DNP-labelling process. The latter was allowed to proceed for 4 hours at 2" C, the reaction being terminated by separation of the product from the reactants on a column of Sephadex G-25 equlibrated with phosphate-buffered saline (PBS; 0.01 IM; pH 7.2) at room temperature. Two readily visible yellow bands were obtained on the column, the faster of the two representing the DNP-labelled anti-serum (DNPantiserum).The concentration of DNP-antiserum at the end of the preparation was found on average to be one twentieth of the original. The recovery of antiserum was excellent. The labelled anti-serum was stored in small aliquots (50 pI) at -70" C, although it was found to be stable at 4" C over a period of at least a year in the presence of 0.025% (w/v) sodium azide.

Preparation of biotin-labelledprimary antisera The technique used was identical to the one employed for biotin-labelling of glucose oxidase (see above), 1251l1 of antiserum being substituted for glucose oxidase solution in the very first step.

Preparation of pituitary sections Rat pituitary glands were fixed in Bouin's fluid for 24 hours, after which they were subjected to standard processing and paraffin embedding procedures. Sections (5 pthick) were cut from the embedded tissue, mounted on chrom-gel coated slides and left to dry ar 60 C overnight.

Preparation of human tonsil sections Freshly obtained human tonsils from patients undergoing routine surgery were fixed in 10% formal saline for 24 hours, after they had been diced into several small pieces (approximately 1 x 1 x 1 cm3). Following routine processing and paraffin embedding, 5 p thick sections were cut from these and mounted onto glass slides coated with a thin layer of an aqueous emulsion of Dufix (ICI Ltd; 1 part Dufix: 2 parts H2O). The mounted sections were dried for 1 hour in 60 C oven, and then treated with trypsin (Trypsin, Sigma Chemical Company; 0.05% in 0.1% CaCI2 solution, pH 7.8) for 4 hours at 370 C, prior to use.

Staining procedure for single enzyme technique eg for localisation of TSH immuno-reactive cells in the rat pituitary sections All steps to be performed at room temperature.

1. Deparaffinise pituitary sections (5 p thick).

2. Incubate sections in sodium periodate solution (2.28% in distilled water) for 5 minutes.

3. Wash in running tap water for 5 minutes.

4. Incubate in freshly prepared potassium borohydride solutuion (0.028%) for 2 minutes.

5. Wash in running tap water for 5 minutes.

Sections should now be free of any endogenous peroxidase activity.

6. Incubate in undiluted pre-immune serum (25 pal per section) for 45 minutes.

7. Wash off excess serum by a brief (1 minute) immersion in PBS (50 ml).

8. Incubate in DN P-anti-TSH (25 p/section in dilutions ranging from 1:200 to 1:20,000 in 0.6% Bovine Serum Albumin in PBS) for 45 minutes.

9. Wash in 3 aliquots of PBS (50 ml) for 1 minute each.

10. Incubate in IgM-anti-DNP (25 pl/section of 1:500 dilution in 0.2% rabit gammaglobulin in PBS of ascitic fluid obtained from a mouse growing K7 hybridoma tumour; initial IgM concentration being > 2 mg/ml) for 45 minutes.

11. Wash as in 9.

12. Incubate in DNP-CYT-C-peroxidase conjugate (25 yl/sectionof 1:800 dilution in 0.2% Bovine Serum Albumin in PBS of stock solution) for 45 minutes.

13. Wash as in 9.

14. Incubate in diaminobenzidine solution (25 mg/50 ml of Tris-HCl buffer 0.2 M, pH 7.6) containing freshly added thoroughly mixed H202 (50 pal) for 3 minutes.

15. Dehydrate the sections in standard manner and permanently mount them in Terpene.

Staining procedure for double enzyme single bridge technique eg for localisation of TSH immunoreactive cells in the rat pituitary sections See 'Staining procedure for single enzyme technique' for steps 1 to 11.

12. Incubate in a solution containing DNP-CYT-C-peroxidase and DNP-glucose oxidase (1:8 ratio) for 45 minutes.

13. Wash as in 9.

14. Incubate in diaminobenzidine solution (25 mg/50 ml of Tris-HCI buffer, 0.2 M, pH 7.6) containing D-Glucose (15 mg/ml)for 105 minutes.

15. Dehydrate and mount the sections as in step 15 (for single enzyme technique).

Staining procedure for double enzyme double bridge technique eg forlocalisation of immunoglobulin reactive cells in human tonsil sections 1. Incubatetrypsinised human tonsil sections (see above) in undiluted non-immune bovine serum (SOpl per section) for 45 minutes.

2. Wash off excess serum by a brief (1 minute) immersion in PBS (50 ml).

3. Incubate in a mixture of biotin-labelled and DNP-labelled antibodies to human immunoglobulin determinants, eg anti-human G heavy chain or K light chain or X light chain, respectively, (each at a dilution ranging from 1/5 to 1/80 obtained using 0.6% Bovine Serum Albumin in PBS as the diluent) for 45 minutes.

4. Wash in 3 changes of PBS (50 ml) each 1 minute long.

5. Incubate in a mixture of IgM anti-DNP (1/250 in 0.2% rabit gamma globulin in PBS) and avidin (Sigma; 1/10 in PBS with added NaCI - 27.2 mg/ml) for 45 minutes.

6. Washasin4.

7. Incubate in a mixture of DNP-CYT-C-peroxidase conjugate (1/400 in 0.6% Bovine Serum Albumin in PBS) and biotin labelled glucose oxidase (1/2.5 in 0.6% Bovine Serum Albumin in PBS with added NaCI - 27.2 mg/ml) for 45 minutes.

8. Wash as in 4.

9. Incubate for 1 hour in a mixture of diaminobenzidine (25 mg), Tris-HCI buffer (50 ml - 12 ml Tris 0.2 M + 19 ml 0.1 N HCI + 19 ml deionised H2), p-D (+), glucose (750 mg) and catalase (125 lli; 4 mg/ml in Tris-HCI buffer), pre-equilibrated to 37 C through a 30 minute period.

10. Wash as in 4 but in deionised H2O.

11. Dehydratethesections in a standard manner and permanentlymountthem inTerpene.

Assay kit - broad definition 1. Single enzyme technique.

Reagents a) A series of DNP-labelled antisera.

b) Igm anti-DNP monoclonal antibody.

c) DNP-labelled peroxidase-CYT-C conjugate.

Each reagent to be individually packaged in a liquid or a freeze-dried state and sold with a full set of instructions for their optimal use and application.

2. Double enzyme single bridge technique Reagents a) A series of DNP-labelled antisera.

b) Igm-anti-DNP monoclonal antibody.

c) DNP-labelled peroxidase-CYT-C conjugate.

d) DNP-labelled glucose oxidase.

Each reagent to be individually packaged in a liquid or a freeze-dried state and sold with a full set of instructions for their optimal use and application.

3. Double enzyme double bridge technique.

Reagents a) A series of mixtures of DNP-labelled antibody and biotin-labelled antibodies to be used in conjunction with each other.

b) A mixture of IgM anti-DNP and avidin.

c) A mixture of DNP-peroxidase-CYT-C conjugate and biotin-labelled glucose oxidase.

Each set of reagents to be packaged in a liquid or a freeze-dried state and sold with a full set of instructions for their optimal use and application.

Claims (23)

1. A method of detecting a substance of interest in an environment of biological cells, in which sequentially a primary antibody to the substance is caused to bind to the substance, a bridging antibody is bound to the primary antibody, and a detecting agent is bound to the bridging antibody, thereby specifically linking the detecting agent to the substance of interest.

2. A method according to claim 1, in which the primary antibody is artificially labelled with a selected hapten, and the bridging antibody is specific to said hapten and binds thereto.

3. A method according to claim 2, in which the detecting agent is marked with the same hapten, and binds to the bridging antibody.

4. A method according to claims 2 or 3, in which the hapten is of the dinitrophenyl group (DNP).

5. A method according to claim 3 or 4 in which the detecting agent is a covalent or a non-covalent conjugate of peroxidase with a protein labelled in part or whole with the hapten.

6. A method according to any one of the preceding claims, in which the environment of cells is first selectively blocked by contacting therewith a wide spectrum of blocking antibodies the blocking and primary antibodies being derived from different or the same animal species, or from the same animal.

7. A method according to claim 6, in which the blocking antibodies designedly exclude antibodies to the substance of interest.

8. A method according to any of the preceding claims, in which the bridging antibody is polyvalent.

9. A method according to claim 8, in which the bridging antibody is monoclonal.

10. A method according to any of the preceding claims, in which two different detecting agents are used, such that the two agents only when acting together simultaneously produce a detectable product, whereas neither will do so separately.

11. A method according to claim 10, in which both detecting agents are bound to the bridging antibody.

12. A method according to claim 10, in which the detecting agents are hapten-labelled peroxidase and glucose oxidase.

13. A method according to claim 10, in which the two detecting agents are linked to the same polyvalent bridging antibody.

14. A method according to claim 10, in which the two detecting agents are linked via two distinct bridging antibodies or combination of a bridging antibody and a non-antibody bridging ligand.

15. A method according to claim 14, in which the bridging ligand has a specific binding activity towards a non-hapten substance.

16. A method according to claim 13, in which the two detecting agents, and the respective primary antibodies are each attached to one or the other of two distinct haptens.

17. A method according to claim 16, in which the two detecting agents and the respective primary antibodies are each attached to one or other of a combination of a hapten and a non-hapten substance.

18. A method according to claim 14, in which the chosen non-antibody bridging ligand is avidin.

19. A method according to claims 16,17, or 18, in which the chosen non-hapten entity is biotin.

20. A method according to any of the preceding claims, in which the bridging antibody or the non-antibody bridging ligand does not bind to any natural determinants on the primary antibody, other than the artificially linked haptens or non-haptens.

21. A detecting "kit" for use in detecting an antigenic substance comprising a primary antibody specific to the antigenic substance, a bridging antibody able to bind with the primary antibody and a detecting agent able to bond to the bridging antibody.

22. Methods of detecting antigenic substances substantially in any of the forms described herein.

23. Detecting kits for use in detecting antigenic substances substantially in any of the forms described herein.