INTENSIFICATION OF POLYMERISED CHROMOGENS IN IMMUNOCHEMICAL ANALYSIS

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 chro ogenε , and kits therefor, for use in immunohiεtochemicai and immunocytochemical analytical techniαues. Biological substances (such as antigens) car. be marked ("visualised") in tissue sections by reacting therr vith labelled antibodies. A. commonly used label is a peroxidase (such as horseradish peroxidase), in vhich case the procedure is knovn as immunoperoxidase analysis. \ariouε chromogenε, 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 iε limited by the low optical density of polymerised AE, and various methods have therefore been proposed to amplify or intensify the polymerised DAB. One particularly advantageous method involves the use of phyεicel silver developer? εo εε tc produce a black stain of metallic silver; such ε method iε described in Journal of Hiεtochemistry and Cytochemistry, \ ol 30, I^o_2, pp 163-184, 15&2 (F Gallyaε, _ . Gores and I. Merchenthθler) . A subεecuent method (aε deεcribed in, for example, U.S. Patent 4687736 and PCT Specification C87/C3009), involves the combined use of heavy metal salts and physical silver developers. In this method, polymerised DAB iε contacted vith a salt of a heavy metal, such aε sodium tetrεchlcroεurεte cr oεmium. tetroxide, and the resulting bound heavy metal iε used to initiate deposition of silver from a physical εilver developer containing Ag(I) ions, such that the presence of a polymerised DAB (hence the peroxidase enzyme, the antibody and ultimately the target enzyme) iε visualised aε a black silver precipitate. It hβs now been established that certain Group VIII metal complexes enhance the precipitation of silver from the physical εilver developer. According to the present invention, therefore, there iε provided a method of intensification of a polymerised chromogen in an i munoperoxidaεe analytical procedure, which comprises marking an analytical sample vith a peroxidase and contacting the marked sample vith a chromogen so aε 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 iε soluble in an aσueoυs medium: and further treating said sample with a physical developer containing dissolved Ag (I) ions, so as to cause precipitation of εilver. The sample can, in some embodiments, be treated with the chromogen and the halogen complex simultaneously; however, it iε generally preferred to treat with the halogen complex εubεequent to treatment with the chromogen. The chromogen used according to the invention iε preferably BAB, although it iε sometimes possible to use other compounds which act in ε similar way, such as 4-chloro-l- naphthol or 3-amino 9-ethyl carbazole (which are disclosed ir the above mentioned U.S. Patent 46S7736), o-dianiεidine , or εrr of the chromogens referred to by L. Scopsi and L. Larsεon in Histochemistry (1986) 84:221-230 (Springer- erlag) . The present invention further comprises a kit fc; use in the method according to the invention, the kit comprising a peroxidase; DAB (or a similarly acting chrcmoger. , such as deεcribed above); a chromogen image-intensification system comprising a halogen complex of a Group 1111 metal cf Period 5 or 6 of the Periodic Table, which halogen complex iε soluble in an acueouε medium; and a physical silver developer containing dissolved Ag (I) ions. In the method according to the invention, the uεe of sulphur-containing sensitiεerε (in particular, sulphides such es sodium sulphide) can be avoided, so as to alleviate the possibility of evolution of noxious hydrogen sulphide aε an unwanted by-product. The avoidance of the uεe of such sensitiserε means that the method according to the invention can be simpler, with fewer εtepε, than certain known intenεification procedures, so that it can be performed more rapidly than such known procedures . The method according tc the invention iε , in any caεe, reproducible, reliable end at leaεt aε εenεitive as currently employed techniques. It may, nevertheleεε , εometimeε be desirable to employ a senεitiser, such as a sulphur-containing εensitiser (as outlined above), or a selenium or tellurium-analogue of such a sensitiεer. Examples of suitable sulphur-containing senεitisers include simple sulphides (such as εodium εulphide) , a sulphur-containing compound of a Group VIII metal (such aε platinum thioεulphate, platinum thiocyanate or the like), or εr organic εulphur-containing εulphide precurεor (εuch eε allyl thiourea , thioureε , thioaceta ide or the like). The Group \III metal preεent in the halogen complex employed according to the invention iε typically of iridiυm, palladium or platinum (the latter being preferred). The halogen preεent in the complex may be chlorine end/or bromine; chlorine iε preferred. The complex may be a simple halide, in which caεe it may be Pt(II)Cl 2. Pt(I )Cl 2 or P<_(1I)C1_ I , all of which are water-εoluble; other simple halideε of the Group V1I1 metals of Period 5 and- 6 of the periodic table are less preferred. Such a εimple hβlide may be further co plexed with neutral ligancε , such aε CO or I ' V T, . exampleε of εuch a complexes are PtCl2(rK* and Pt (COCl2)Cl2. V. 7 hen the complex iε other thεr ε simple halide, it iε typically of iridiur, palladium cr platinur; the Group VIII metal in such a complex iε preferably present in a complex anion εuch as (PdCl *)^-, (IrClg)*^-, (Irri^)2- J or (PtC14)2"_ The complex may typically have εouere planar, tetrahedral or octahedral geometry. When it has εquere planar geometry, the anion thereof iε 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 aε a solid. In the method according to the present invention, the peroxidase iε brought into close proximity with the target biological substance (generally an antigen) using an antibody specific to the biological substance of interest (εuch an antibody iε known aε 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 iε conjugated to a second antibody which iε itself directed at the primary antibody. Other indirect syεte ε may uεe so-called bridging technioυeε, such aε the peroxidase-antiperoxidase method described in Sternberger, L.A. (1986) Immunocytochemiεtry, Third edition, John Wiley & Sons Inc., lev jork, pages 90-204; or the anti-hapten bridge method described in Jasani, B., Thomas, Ϊ-.D., Newman, G.R. and Williams, E.D. (1983), DΪ ' P-hεpten sandwich (DRSS) procedure, design, senεitivity, verεatility and application, Immunol. Com u . 12:51; and alεo Jaεεπi, E., Wy for -Thomas , D. , and Williams, F.D (1981), Use of monoclonal anti-hapten antibodieε for i munolocaliεation of tiεεue antigens, J. Cl-In.Pathol . 34: 1000-1002. Further posεibilitieε include the Avidin-Eictir system and the use of lectinε aε described in Fclak, ..-'. . su ΪOorden, S.\ (1986) Immunocytochemiεtry, Wright S- Sc Liritec. Bristol pages 54-70 and 172. In all of these, the peroxidase iε brought into close proximity with the target biological substance; the method according to the invention iε 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 (εuch aε 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 glaεε slide, a plasticε microtitre plate (typically of polyεtyrene) , a biological εubstance fixed on nitrocellulose paper, or any suitable solid phase substrate. The tissue section mounted on a glaεε εlide may be a frozen section, or tissue embedded in plastics or paraffin; suitable plastics for such embedding may be LR White, which iε 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 tiεεue sections or nitrocellulose paper in generally known ways for a period of about one hour, followed by waεhing with a buffer to remove unbound antibody. Similar conditionε may be uεed for each antibody εtep and also for reacting the peroxidase conjugate with bound antibody. After the conjugate haε been bonded, and exceεε reagent removed by washing, hydrogen peroxide and DAE are added (typically in a phoεphate buffer) and allowed to react for about ten minutes at room temperature. (Up to this point all the procedureε are conventional). The tissue section or nitrocellulose paper is then preferably waεhed of unreacted hydrogen peroxide and DAB uεing εeveral rinεeε in double distilled water; potaεεiuir tetrachloroplatinate iε 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 thioεulphate fixing stage is optional as the reaction can be stopped by washing the developer away with water) The location of the tβrget antigen iε 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 licuid 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 sectionε. Theεe sections were placed on alcohol-cleaned glass slides and air dried for of several minutes, followed by immersion of the slideε 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 vith the εectionε for 1 hour at rccrr temperature; they were then washed in PBS, aε before. The sections were then treated with 50 to 100 microlitreε of ar appropriate dilution (typically 1:15) of peroxidase conjugated sheep anti-mouse i munoglobulin (containing diluent protein, 0.5% Bovine albumin). This reagent was left in contact with the sectionε for 1 hour at room temperature. After washing in PBS, a DAB/hydrogen peroxide mixture waε added (500 icrogramε DAB per ml and 0.3 microlitreε 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 iε conventional . The sections were then contacted with a 1.OmK solution of potassium tetrachloroplatinate in lm 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 Inεenεitive 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 thioεulphate, waεhed in diεtilled water, and then prepared for mounting. Prior tc mounting, the εectionε can be εtεined, e.g. with hae etoxylin . This procedure improved the visibility of trace depoεitε of DAB on tiεεue εectionε which could be seen aε a black precipitate of εilver. The εtrength of the reducing agent in Gallyas 'ε physical developers can be varied to control the rate of development of the εilver precipitate and also virtually eliminate background deposition of εilver. Typically the range may be 0.2% to 1% in one developer solution. When used on a comparative basis, uεing identical procedureε, the method described could detect levels of DAE depoεition which are consistently belov the limits of detection. achieved using a gold-εulphide εystem (US patent 4687736 end PCT specification WC£7/03009). Example 2 Visualisation of DAB depositε on paraffin sections. Fresh human thyroid tissue was prepared for sectioning by a standard hiεtological 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 sectionε (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 60 C for 15 hours and then dipped, first in xylene, then In alcohol w τ ith 2 to 3 minutes in each. They were then treated with a mixture of methanol an hydrogen peroxide for 3C minutes at 2C ,ς, C to eliminate natural peroxidase in the thyroid tiεεue. The sections were then washed in water for 10 to 15 minutes before immersin in PBS. text, an appropriate dilution of antibody was added, typically a 1:100 dilution of DA.KC* 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 FES, a haptenated antibody (D P-labelled εheep anti-mouse immunoglobulin) was added, at an appropriate dilution in 0.6% BSA, and the sectionε left at ambient temperature for 45 minutes. The sectionε were then washed again in PPS and a monoclonal antibody with specificity for the DI " P hεpten added, (in this case the monoclonal βntibody was an IgK mouse antibody) and the sections incubated for a further 45 minutes and then again washed in PBS. They were then treated with a haptenεtec enzyme conjugate, DEP-peroxidase, for a further 45 minutes at room temperature, washed, and treated with a DA.E/hycrogen peroxide mixture for 10 minutes (or less) . The εectionε were then washed in diεtilled water. The εectionε were then treated with a l.O K εolution of potεεεiur tetrachlcrorlεtinεte II, aε in Example 1. __ o _ The reεult wεε the enhanced detection of DAE depoεitε on paraffin sections. Also, aε in Example 1, the platinum εyεtem was able to detect lower a ountε of DAE on paraffin sectionε than a gold-εulphide detection εyεtem, and resulted in εubstantiallly lower background staining. Once again, the strength of the reducing agent in any of Gallyas 'ε developers could be varied to control the rate of development of the εilver deposit and virtually eliminate background εilver deposits. Example 3 Visualisation of trace deposits of DAE on nitrocellulose paper. Mtrocelluloεe paper waε prepared by εoεking in a Tris-RCl buffer (0.05M Tris, 0.2K sodium chloride and 0.05% Tween 20) and allowed to dry. (Alternatively, distilled water may be used inεtead of the Triε-KCl buffer.) The paper, cut into 2 x 6cm strips, was then slot-blotted with mouse immunoglobulinε in a known way. One strip of paper accommodated nine slotε, such that a range of dilutions of the im unoglobulin (IgG) plus a control εlot (buffer only) were included on one strip. The range contained a control εlot and slotε with the following amounts of IgG per εlot: lOOng, 10ng,lng, lOOpg, lOpg, lpg, lOOfg, lOfg. Fitrocellulose strips prepared in the above manner were dried and εtored at room temperature prior to uεe. The viεualiεation of DAB depoεitε proceeded eε 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 wεε used for each strip in a Petri diεh and 'the reaction allowed to proceed, with occasional shaking, for 1 hour at room temperature. The strips were then washed in buffer five timeε over ε ten minute period (20ιr.l per waεh) . After ε rinse in PFS the strips were then soaker " in DAB/hydrogen peroxide for 10 inuteε at room, temperature, fcext , the strips were washed in distilled water and soεkec in a l.OiE-*- solution of potassium tetrachloroplatinate in l.O M hydrochloric acid (or in distilled water) . The stripε were then washed four times in distilled water and a physical developer added (see the above entioned 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 w τ ith a 3% solution of sodium thiosulphate for 1 minute, rinsed in distilled water, and then air dried. The resultε 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 εyεtem iε ten timeε more εenεitive in the detection of the mouεe immunoglobulin deεcribed in the foregoing procedure, using identical experimental conditions and reagents. Example 4 Visualisation of trace deposits of DAB on nitrocellulose paper. Fitrocellulose paper waε soaked in distilled water for 5 minutes and then allowed to dry. The paper was then slot-blotted v 7 ith biotin conjugated rabbit IgG (biotinylatec IgG) a follows: One strip of paper accommodated nine slots or dots εuch that a range of biotin-conjugated immunoglobulin waε added to one strip (in 5m.M phosphate buffer). The total immunoglobulin content per εlot was kept constant using carrier (non-biotinylated) rabbit IgG, such that the total IgC content per εlot waε lOng . The range used contained the following amounts of biotin-IgG per slot; lng , lOOpg, 10pg, lpg, lOOfg, lOfg, lfg, O.lfg and a control εlot of lOng normal rabbit IgG. Stripε prepared in thiε manner were dried and stored at roorr temperature prior to uεe. The visualisation of DAP deposits proceeded es fcllcvε: The strips were soaked in Tris buffer (0.051. Tris, 0.2M εodiur. chloride and 0.05% Tween 20 detergent) containing 1% bovine serum albumin, for at least 30 inuteε at room temperature. Without further waεhing, the εtripε were placed in a εolution of peroxidase-conjugated streptavidin (Amerεham, 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 hourε at 4^0. The εtripε were then washed several times in 0.1M phosphate buffered saline (PBS, 0.05% Tween 20) for at least 20 minutes. The εtripε were then soaked in DAE/Hydrogen peroxide for 5-10 minuteε at room temperature (DAE in the range 1C to 5C microgramε per ml and hydrogen peroxide at 1 microlitre of 3C% H2O2/IO1DI volume of DAB) . The εtripε were then waεhed in diεtilled water with several changes over 20 minuteε. The stripε were soaked in a ImM solution of potaεεium tetrachloro¬ platinate (up to lOmM can εlεo be uεed) in l.OmM (or diεtilled water), for 10 minuteε at room temperature. The stripε were then washed severβl timeε in distilled water, over 10 to 20 minutes, and a physical developer added (using the technique described in the above-mentioned article by Gallyaε) for from 5 to 16 minuteε. The εtripε were rinεed with diεtilled water, and air-dried or treated with 37- εodiυm thioεulphate for one minute, rinsed in distilled water, and then air dried. The uεe of the. platinu -εilver εyεtem enhanced the viεibility of DAB deposits on nitrocelluloεe paper. The method according to the invention εhowed an improvement in sensitivity (up to ten-fold) compared to the known gold- sulphide εyεtem. Moreover the method according to the invention reεulted in substantially lower background staining and permitted higher amounts of DAB to be uεed than the gold-εulphide procedure. These features, together with the flexibility due to prolonged incubations with developer, allowed routine detection of εε little a s Ipg cuantities of biotin-label led reagents.

Claims: CLAIMS : 1. A method of intensification of a polymerised chromogen in an immunoperoxidaεe analytical procedure vhich co priεes marking an analytical sample with a peroxidase and contacting the marked sample with a chromogen so aε to cause polymerisation thereof in the form of a precipitate on said sample; treating said sample with a halogen complex of a Group III 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 aε to cauεe precipitation of εilver. 2. A method according to claim 1, wherein said chromogen iε diaminobenzidine. 3. A method according to claim 1 or 2, wherein substantially no sulphur-containing sensitiser iε 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 iε platinum. 5. A method according to any of claims 1 to 4. vherein sai 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 viεualiεation of a teεt biological substance, which compriεeε a peroxidaεe; 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 Mil metal of Period 5 or 6 of the Periodic Table, which halogen complex iε soluble in an aqueous medium, and a physical εilver 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.

Inventors: MORGAN JOHN MICHAEL, MORGAN, JOHN, MICHAEL
Applicants: UNIV WALES MEDICINE, UNIVERSITY OF WALES COLLEGE OF MEDICINE
Application year: 1989
Main classification: G01N33
IPC codes: C12Q1, G01N33