WO1989006362A1

WO1989006362A1 - Intensification of polymerised chromogens in immunochemical analysis

Info

Publication number: WO1989006362A1
Application number: PCT/GB1989/000015
Authority: WO
Inventors: John Michael Morgan
Original assignee: University Of Wales College Of Medicine
Priority date: 1988-01-08
Filing date: 1989-01-03
Publication date: 1989-07-13
Also published as: AU2938789A; EP0440611A1; GB8800373D0

Abstract

The intensification process in an immunoperoxidase analytical procedure, comprises marking an analytical sample with a peroxidase; contacting the marked sample with diaminobenzidine or similar chromogen so as to polymerise the latter as a precipitate on the sample; treating the sample with a water-soluble halogen complex of a Group VIII metal of Period 5 or 6 of the Periodic Table (such as a tetrachloroplatinate II); and further treating the sample with a physical silver developer containing dissolved Ag (I) so as to precipitate reduced silver where said diaminobenzidine has been polymerised and precipitated.

Description

Intensification of Polymerised Chromogens In Immunochemical analysis

The present invention is concerned vith the intensification of polymerised chromogens, and kits therefor, for use in immunohistochemical and immunocytochemical analytical techniques.

Biological substances (such as antigens) can be marked ("visualised") in tissue sections by reacting them with labelled antibodies. A. commonly used label is a peroxidase (such as horseradish peroxidase), in which case the procedure is known as immunoperoxidase analysis. Various chromogens, when in solution in the presence of such a peroxidase and hydrogen peroxide, can be caused to polymerise; a particularly preferred such chromogen is diaminobenzidine (hereinafter referred to as DAB). Polymerised DAB, which is a reddish brown in colour, can sometimes be visualised microscopically (using, for example, an electron microscope or a light microscope), so that sites of antigen/antibody binding can be identified. However, the sensitivity of the visualisation is limited by the low optical density of polymerised DAE, and various methods have therefore been proposed to amplify or intensify the polymerised DAB. One particularly advantageous method involves the use of physical silver developers so as to produce a black stain of metallic silver; such a method is described in Journal of Histochemistry and Cytochemistry, Vol 30, No.2, pp 163-184, 1992 (F Gallyas, T. Gores and I. Merchenthaler).

A subsequent method (as described in, for example, U.S. Patent 4687736 and PCT Specification WC87/C3009), involves the combined use of heavy metal salts and physical silver developers. In this method, polymerised DAB is contacted with a salt of a heavy metal, such as sodium tetrachlereaurate or osmium. tetroxide, and the resulting bound heavy metal is used to initiate deposition of silver from a physical silver developer containing Ag(I) ions, such that the presence of a polymerised DAB (hence the peroxidase enzyme, the antibody and ultimately the target enzyme) is visualised as a black silver precipitate.

It has now been established that certain Group VIII metal complexes enhance the precipitation of silver from the physical silver developer.

According to the present invention, therefore, there is provided a method of intensification of a polymerised chromogen in an immunoperoxidase analytical procedure, which comprises marking an analytical sample with a peroxidase and contacting the marked sample with a chromogen so as to cause polymerisation thereof in the form of a precipitate on said sample; treating said sample with a halogen complex of a Group VIII metal of Period 5 or 6 of the periodic table which is soluble in an aqueoυs medium; and further treating said sample with a physical developer containing dissolved Ag (I) ions, so as to cause precipitation of silver.

The sample can, in some embodiments, be treated with the chromogen and the halogen complex simultaneously; however, it is generally preferred to treat with the halogen complex subsequent to treatment with the chromogen.

The chromogen used according to the invention is preferably BAB, although it is sometimes possible to use other compounds which act in a similar way, such as 4-chloro-l-naphthol or 3-amino 9-ethyl carbazole (which are disclosed in the above mentioned U.S. Patent 4687736), o-dianisidine, or an y of the chromogens referred to by L. Scopsi and L. Larsson in Histochemistry (1986) 84:221-230 (Springer-Verlag).

The present invention further comprises a kit for use in the method according to the invention, the kit comprising a peroxidase; DAB (or a similarly acting chromogen, such as described above); a chromogen image-intensification system comprising a halogen complex of a Group VIII metal of Period 5 or 6 of the Periodic Table, which halogen complex is soluble in an acueous medium; and a physical silver developer containing dissolved Ag (I) ions.

In the method according to the invention, the use of sulphur-containing sensitisers (in particular, sulphides such es sodium sulphide) can be avoided, so as to alleviate the possibility of evolution of noxious hydrogen sulphide as an unwanted by-product. The avoidance of the use of such sensitisers means that the method according to the invention can be simpler, with fewer steps, than certain known intensification procedures, so that it can be performed more rapidly than such known procedures. The method according to the invention is, in any case, reproducible, reliable end at least as sensitive as currently employed techniques.

It may, nevertheless, sometimes be desirable to employ a sensitiser, such as a sulphur-containing sensitiser (as outlined above), or a selenium or tellurium-analogue of such a sensitiser. Examples of suitable sulphur-containing sensitisers include simple sulphides (such as sodium sulphide), a sulphur-containing compound of a Group VIII metal (such as platinum thiosulphate, platinum thiocyanate or the like), or sr organic sulphur-containing sulphide precursor (such as allyl thiourea, thioures, thioacetamide or the like).

The Group VIII metal present in the halogen complex employed according to the invention is typically of iridiυm, palladium or platinum (the latter being preferred). The halogen present in the complex may be chlorine end/or bromine; chlorine is preferred.

The complex may be a simple halide, in which case it may be Pt(II)Cl_2. Pt(IV)Cl₂ or Pd(II)Cl₄, all of which are water-soluble; other simple halides of the Group VIII metals of Period 5 and 6 of the periodic table are less preferred. Such a simple halide may be further complexed with neutral ligands, such as CO or NH ₃ ; examples of such a complexes are PtCl₂(NH₃)₂ and Pt(COCl₂)Cl₂. When the complex is other that a simple halide, it is typically of iridium, palladium or platinum; the Group VIII metal in such a complex is preferably present in a complex anion such as (PdCl₄)^2-, (IrCl₆)^3-, (IrCl₆)^2-, or (PtCl₄)^2-. The complex may typically have souere planar, tetrahedral or octahedral geometry. When it has squere planar geometry, the anion thereof is preferably tetrachloroplatinate II; the cation may be, for example, sodium, potassium and/or ammonium.

Tetrachloroplatinate II complexes are stable and may be provided in solution in distilled water or in dilute hydrochloric acid, or as a solid.

In the method according to the present invention, the peroxidase is brought into close proximity with the target biological substance (generally an antigen) using an antibody specific to the biological substance of interest (such an antibody is known as the primary antibody). The primary antibody and the peroxidase enzyme can be brought together on a sample in one of two well known ways, either direct or indirect.

In the direct system, the peroxidase may be chemically bonded (conjugated) to the primary antibody, whereas in indirect systems, the peroxidase is conjugated to a second antibody which is itself directed at the primary antibody. Other indirect systems may use so-called bridging technioυes, such as the peroxidase-antiperoxidase method described in Sternberger, L.A. (1986) Immunocytochemistry, Third edition, John Wiley & Sons Inc., New York, pages 90-204; or the anti-hapten bridge method described in Jasani, B., Thomas, M.D., Newman, G.R. and Williams, E.D. (1983), DMP-hapten sandwich (DRSS) procedure, design, sensitivity, versatility and application, Immunol. Commun. 12:51; and also Jassni, B., Wynford-Thomas, D., and Williams, F.D (1981), Use of monoclonal anti-hapten antibodies for immunolocalisation of tissue antigens, J. Clin.Pathol. 34: 1000-1002.

Further possibi lities include the Avidin-Biotin system and the use of lectins as described in Polak, J. M. and ΪOorden, S.V (1986) Immunocytochemistry, Wright & Ser Limited, Bristol pages 54-70 and 172. In all of these, the peroxidase is brought into close proximity with the target biological substance; the method according to the invention is used to visualise the precise location of complexes between the target substance and the peroxidase. The target biological substance may be, for example, a nucleic acid fixed to e synthetic substrate (such as nitrocellulose), or an antigen susceptible to antigen-antibody reactions.

The analytical sample employed in the method according to the invention may be, for example, a tissue section mounted on a glass slide, a plastics microtitre plate (typically of polystyrene), a biological substance fixed on nitrocellulose paper, or any suitable solid phase substrate. The tissue section mounted on a glass slide may be a frozen section, or tissue embedded in plastics or paraffin; suitable plastics for such embedding may be LR White, which is commercially available from London Resin Company Limited of Woking, Surrey, England.

An exemplary sequence of steps in the method according to the invention will now be described. Specifically, the antibody (in a buffered solution with protein diluent) may be reacted with tissue sections or nitrocellulose paper in generally known ways for a period of about one hour, followed by washing with a buffer to remove unbound antibody. Similar conditions may be used for each antibody step and also for reacting the peroxidase conjugate with bound antibody.

After the conjugate has been bonded, and excess reagent removed by washing, hydrogen peroxide and DAB are added (typically in a phosphate buffer) and allowed to react for about ten minutes at room temperature. (Up to this point all the procedures are conventional).

The tissue section or nitrocellulose paper is then preferably washed of unreacted hydrogen peroxide and DAB using several rinses in double distilled water; potassium tetrachloroplatinate is then added (either in distilled water or as a solution in dilute hydrochloric acid) for a contact period of up to fifteen minutes, followed by further rinsing with double distilled water.

Then the physical silver developer may be added for a reaction period of up to 15 or 20 minutes, followed by rinsing with water or buffer, optionally fixing with thiosulphate, followed by a final rinse. (The thiosulphate fixing stage is optional as the reaction can be stopped by washing the developer away with water)

The location of the target antigen is then indicated by the presence of a black silver precipitate. Tissue sections with the antigen thus visualised may be mounted in conventional manner; nitrocellulose paper may be simply dried and stored.

The following Examples are given, by way of illustration only, to describe how the method of intensification of polymerised chromogens according to the invention can be put into operation. Example 1 Visualisation of DAB deposits on frozen sections.

Fresh human tonsil tissue was dissected and frozen in isopentane cooled in liquid nitrogen. The tissue was then stored at -70°C until required. A sample of the tissue was then sectioned using a refrigerated microtome to cut 4 micrometre sections. These sections were placed on alcohol-cleaned glass slides and air dried for of several minutes, followed by immersion of the slides in acetone for several minutes (usually 5 minutes) at room temperature. The acetone was then removed by several washes in phosphate buffered saline (PBS), 2 to 3 minutes per wash.

The sections were then treated with monoclonal antibody to human lambda light chain, the working dilutior cf this reagent being typically about 1:100. This reagent was left in contact with the sections for 1 hour at room temperature; they were then washed in PBS, as before. The sections were then treated with 50 to 100 microlitres of an appropriate dilution (typically 1:15) of peroxidase conjugated sheep anti-mouse immunoglobulin (containing diluent protein, 0.5% Bovine albumin). This reagent was left in contact with the sections for 1 hour at room temperature. After washing in PBS, a DAB/hydrogen peroxide mixture was added (500 micrograms DAB per ml and 0.3 microlitres hydrogen peroxide per ml, in 0.1M PBS), and left in contact with the sections for 10 minutes, followed by washing in distilled water. The foregoing procedure is conventional.

The sections were then contacted with a 1.0mM solution of potassium tetrachloroplatinate in 1mM hydrochloric acid (or in distilled water) for up to 15 minutes (a shorter time can be employed, if desired, with satisfactory results). The sections were then washed in distilled water and a physical developer applied (using the technique described in Gallyas, E. (1979), Light Insensitive Physical Developers; Stain Technology Vol 54: pp.173-176). The sections could be washed in distilled water and prepared for mounting in a conventional manner or treated with a 37- solution of sodium thiosulphate, washed in distilled water, and then prepared for mounting. Prior to mounting, the sections can be stained, e.g. with haemetoxylin.

This procedure improved the visibility of trace deposits of DAB on tissue sections which could be seen as a black precipitate of silver. The strength of the reducing agent in Gallyas's physical developers can be varied to control the rate of development of the silver precipitate and also virtually eliminate background deposition of silver. Typically the range may be 0.2% to 1% in one developer solution.

When used on a comparative basis, using identical procedures, the method described could detect levels of DAB deposition which are consistently below the limits of detection. achieved using a gold-sulphide system (US patent 4687736 and PCT specification WO87/03009). Example 2

Visualisation of DAB deposits on paraffin sections.

Fresh human thyroid tissue was prepared for sectioning by a standard histological procedure which involved fixing in formol-saline and then embedding in paraffin blocks.

The following technique utilises the known hapten-bridge method for depositing polymerised DAB on tissue sections (Jasani et al (1983) as mentioned above).

Sections were cut from one paraffin block to a required thickness of 4 to 5 micrometres and then mounted on chrome-gel coated glass slides. The sections were dried in an oven at 60ºC for 15 hours and then dipped, first in xylene, then In alcohol with 2 to 3 minutes in each. They were then treated with a mixture of methanol and hydrogen peroxide for 30 minutes at 20ºC to eliminate natural peroxidase in the thyroid tissue. The sections were then washed in water for 10 to 15 minutes before immersing in PBS. text, an appropriate dilution of antibody was added, typically a 1:100 dilution of DAKO anti LC monoclonal antibody (directed at a leukocyte antigen), with 0.6% Bovine albumin as diluent. The antibody was contacted with the sections for 45 minutes (this can be varied) at room temperature. After washing in PBS, a haptenated antibody (DMP-labelled sheep anti-mouse immunoglobulin) was added, at an appropriate dilution in 0.6% BSA, and the sections left at ambient temperature for 45 minutes. The sections were then washed again in PBS and a monoclonal antibody with specificity for the DMP hapten added, (in this case the monoclonal antibody was an IgM mouse antibody) and the sections incubated for a further 45 minutes and then again washed in PBS. They were then treated with a haptenstec enzyme conjugate, DEP-peroxidase, for a further 45 minutes at room temperature, washed, and treated with a DAB/hydrogen peroxide mixture for 10 minutes (or less). The sections were then washed in distilled water. The sections were then treated with a 1.0mM solution of potassium tetrachlcroplatinate

II, as in Example 1. The result was the enhanced detection of DAB deposits on paraffin sections. Also, as in Example 1, the platinum system was able to detect lower amounts of DAB on paraffin sections than a gold-sulphide detection system, and resulted in substantiallly lower background staining.

Once again, the strength of the reducing agent in any of Gallyas's developers could be varied to control the rate of development of the silver deposit and virtually eliminate background silver deposits.

Example 3

Visualisation of trace deposits of DAE on nitrocellulose paper. Nitrocellulose paper was prepared by soaking in a Tris-HCl buffer (0.05M Tris, 0.2M sodium chloride and 0.05% Tween 20) and allowed to dry. (Alternatively, distilled water may be used instead of the Tris-HCl buffer.) The paper, cut into 2 × 6cm strips, was then slot-blotted with mouse immunoglobulins in a known way.

One strip of paper accommodated nine slots, such that a range of dilutions of the immunoglobulin (IgG) plus a control slot (buffer only) were included on one strip. The range contained a control slot and slots with the following amounts of IgG per slot: 100ng, 10ng,1ng, 100pg, 10pg, 1pg, 100fg, 10fg.

Nitrocellulose strips prepared in the above manner were dried and stored at room temperature prior to use. The visualisation of DAB deposits proceeded as follows: The strips were soaked in Tris buffer for ten minutes and then soaked in a 1:15 dilution of peroxidase conjugated sheep anti-mouse immunoglobulin in Tris buffer with 0.5% protein diluent (BSA). A 5ml volume was used for each strip in a Petri dish and the reaction allowed to proceed, with occasional shaking, for 1 hour at room temperature. The strips were then washed in buffer five times over s ten minute period (20ml per wash). After s rinse in PBS the strips were then soaked in DAB/hydrogen peroxide for 10 minutes at room temperature. Next, the strips were washed in distilled water and soaked in a 1.0mM solution of potassium tetrachloroplatinate in 1.0mM hydrochloric acid (or in distilled water). The strips were then washed four times in distilled water and a physical developer added (see the abovementioned article by Gallyas 1979) for a period of from 5 to 15 minutes. The strips were then rinsed free of developer with distilled water and air-dried or treated with a 3% solution of sodium thiosulphate for 1 minute, rinsed in distilled water, and then air dried.

The results showed that the use of the platinum-silver system enhances the visibility of DA.E deposits on nitrocellulose paper. Moreover the method according to the invention showed an improvement in sensitivity compared to the gold-sulphide detection system. The platinum-silver system is ten times more sensitive in the detection of the mouse immunoglobulin described in the foregoing procedure, using identical experimental conditions and reagents.

Example 4

Visualisation of trace deposits of DAB on nitrocellulose paper. Fitrocellulose paper was soaked in distilled water for 5 minutes and then allowed to dry. The paper was then slot-blotted with biotin conjugated rabbit IgG (biotinylated IgG) as follows:

One strip of paper accommodated nine slots or dots such that a range of biotin-conjugated immunoglobulin was added to one strip (in 5mM phosphate buffer). The total immunoglobulin content per slot was kept constant using carrier (non-biotinylated) rabbit IgG, such that the total IgG content per slot was 10ng. The range used contained the following amounts of biotin-IgG per slot; 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg and a control slot of 10ng normal rabbit IgG.

Strips prepared in this manner were dried and stored at room temperature prior to use.

The visualisation of DAP deposits proceeded es follows: The strips were soaked in Tris buffer (0.051. Tris, 0.2M sodium chloride and 0.05% Tween 20 detergent) containing 1% bovine serum albumin, for at least 30 minutes at room temperature. Without further washing, the strips were placed in a solution of peroxidase-conjugated streptavidin (Amersham, UK) at a dilution of 1:500 of supplied stock solution (in Tris buffer, 0.05% Tween 20, 1% BSA). This reaction was carried out in Petri dishes using working volumes of 15 to 20ml. The reaction was cotinued for 4 hours or 15 hours at 4ºC. The strips were then washed several times in 0.1M phosphate buffered saline (PBS, 0.05% Tween 20) for at least 20 minutes. The strips were then soaked in DAB/Hydrogen peroxide for 5-10 minutes at room temperature (DAB in the range 10 to 50 micrograms per ml and hydrogen peroxide at 1 microlitre of 30% H₂O₂/10mI volume of DAB). The strips were then washed in distilled water with several changes over 20 minutes. The strips were soaked in a 1mM solution of potassium tetrachloroplatinate (up to 10mM can also be used) in 1.0mM (or distilled water), for 10 minutes at room temperature. The strips were then washed several times in distilled water, over 10 to 20 minutes, and a physical developer added (using the technique described in the above-mentioned article by Gallyas) for from 5 to 16 minutes. The strips were rinsed with distilled water, and air-dried or treated with 3% sodium thiosulphate for one minute, rinsed in distilled water, and then air dried.

The use of the. platinum-silver system enhanced the visibility of DAB deposits on nitrocellulose paper. The method according to the invention showed an improvement in sensitivity (up to ten-fold) compared to the known gold-sulphide system. Moreover the method according to the invention resulted in substantially lower background staining and permitted higher amounts of DAB to be used than the gold-sulphide procedure. These features, together with the flexibility due to prolonged incubations with developer, allowed routine detection of ss little a s 1pg cuantities of biotin-label led reagents.

Claims

CLAIMS :

1. A method of intensification of a polymerised chromogen in an immunoperoxidase analytical procedure which comprises marking an analytical sample with a peroxidase and contacting the marked sample with a chromogen so as to cause polymerisation thereof in the form of a precipitate on said sample; treating said sample with a halogen complex of a Group VIII metal of Period 5 or 6 of the Periodic Table, which complex is soluble in an aqueous medium; and further treating said sample with a physical developer containing dissolved Ag (I) ions, so as to cause precipitation of silver.

2. A method according to claim 1, wherein said chromogen is diaminobenzidine.

3. A method according to claim 1 or 2, wherein substantially no sulphur-containing sensitiser is employed during, or subsequent to, treatment with said physical developer.

4. A method according to any of claims 1 to 3, wherein said Group VIII metal is platinum.

5. A method according to any of claims 1 to 4. wherein said halogen is chlorine.

6. A method according to any of claims 1 tyo 5, wherein said complex is a tetrachloroplatinate (II).

7. A kit for use in the visualisation of a test biological substance, which comprises a peroxidase; a chromogen capable of forming a coloured, precipitate by reaction with a peroxidase marked substance; a chromogen image-intensification system comprising a halogen complex of a Group VIII metal of Period 5 or 6 of the Periodic Table, which halogen complex is soluble in an aqueous medium, and a physical silver developer containing dissolved Ag. (I) ions.

8. A kit according to claim 7, wherein said chromogen is diaminobenzidine.

9. A. kit according to claim 7 or 8, wherein said complex is as defined in any of claims 3 to 6.