Journal oflrnmunological Methods, 50 (1992) 193-198
193
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.110
JIM06364
Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure A 10 year review of its principle reagents
and applications
B~ J a s a n i ~, N.D. T h o m a s ~, H. N a v a b i 1, D.M. Millar J, G . R . N e w m a n 2, j. G e e 3 a n d E.D. Willk..as t t Department of Pathology, 2 EM Unit, and "~TenoL'us Institute, Unit'ersity of Wales College of Medicine, Cardiff CF4 4XN, Wales, UK
(Accepted 12 February 1992)
Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labe!!ed primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or ant:-DNP linked marker enzyme. In this article the development of these reagents and various modificatit~,s of the b~sic technique are reviewed in conjunction with the special applications accruing from their use. Key words: Dinitrophenyl; lmmunoperoxidase;Dinitrophenyl-ilaptenprocedure; Dinitrophenyl localization system: Immunolocal-
ization; Immunoassay
Introduction The prototype, three-layer DNP hapten sandwich staining (DHSS) procedure (see Fig. 1 for a
Correspondence to: B. Jasani, Head of Immunopatholo~ Unit, Department of Pathology, University of Wales College of Medicine, Cardiff CF4 4XN, Wales UK (Tel.: 0222-743523; Fax: 0222-744276). Abbreviations: DNP, dinitrophenyl; DHSS, dinitrophenyl hapten sandwich staining; PAP, peroxidase-anti-peroxidase; TSH, thyroid stimulating hormone; DNBS, dini'~robenzene sulphonic acid; FDNB, fluorodinitrobenzene; TNF, tumour necrosis factor; DAB, diaminobenzidine; DNP-CC-peroxidase, dinitrophcayl-cytochromeC-peroxidase.
schematic representation) was developed some 10 years ago as a modification of the PAP method of Sternberger (Sternberger et al., 1970) primarily to provide a versatile secondary detection system for high specificity immunoperoxidase localisation of tissue antigens and hormone receptors using DNP-labelled primary probes (Jasani et al., 1981). The technique has since been optimised and modified to suit a diverse variety of immunocytochemical and immunoassay applications. The object of this article is to give a brief review of the development of the principle reagents, various modifications of the prototype technique and the special applications resulting from their use.
l ¢}4
Principle reagents, variations of the technique and applications The key reagents of the various DNP haptcn based techniques to be described include: (1) a non-deleterious DNP hapten labelling compound, (2) a multivalcnt igM monoclonal, antiDNP bridge reagent; and (3) DNP hapten substituted or anti-DNP linked marker enzymes, respectively.
DNP hapten labelling compound A novcl DNP labelling reagent, namely methyl-3-(2,4-dinitrophenylamino) propionimidate hydrochloride (DNP-N-IE) capable of producing adequate substitution ratios (10-15 per immunoglobulin molecule) under relatively mild alkaline (pH < 9.0) conditions and without causing significant protein insolubility, was primarily developed to replace the less suitable conventional reagents, viz., fluorodinitrobenzene (FDNB) and dinitrobenzene sulphonic acid (DNBS). The non-deleterious character of the compound was furthcr confirmed by its capacity to substitute up to 15 DNP ~roups per antibody molecule without causing a significant change in
I
I~
ONP-Locallsanon
OflBIGIUCOSe
its antigen binding capacity or affinity (Hewlins et al., 1984). The compound has since been applied to a wide range of monoclonal and polyclonal (primary and secondary) antibodies as well as non-antibody probes, e.g., lgE myeloma protein, thyroid stimulating hormone (TSH), and tumour necrosis factor (TNF), without significant alteration in their respective specificity or sensitivity (Jasani et al., 1982, 1986; Schmid et al., 1988; and C. Sarraf, N. Matthews and B. Jasani, unpublished data). The compound is effective over a wide range of concentrations (e.g., 100 /xg-10 mg/ml) of immunoglobulins whether present in their affinity purified, unfractionated or whole serum form. The use of DNP hapten labelled secondary antibodies in the modified form of the DHSS procedure, the DI']P localisation system (see Fig. 1), has facilitated high sensitivity and versatile immunocytochemical and immnnoenzyme assay screening of hybrid monoclonal antibodzy s,~pernatants. Thus all three major classes of mouse monoclonal antibodies - IgG, IgA and lgM - are capable of being screened with equal efficacy and at a much higher overall signal to noise ratio than that afforded by the commercially available indirect immunoperoxidase conjugates in the EL1SA mode (Jasani et al., 1986). The ELISA approach has also proved highly effective for serological screening of light chain restricted human autoantibodies (Morgan et al., 1988).
, ONA-Labolled Glucose 0.idase
T
Indirect
[INP-Method
One/Xydrogen Peroxide
OABIAyarogen Peroxide
ONP-tabollea Peroxldase
T
T
T
Conjugate
IOM Rntl-bNP Ortdge
ONP-lebelled Anti-Re,bit/Mouse
RnII-DI~PPeroxldase DNP-tabell~d nnUnody or tlgzn~
T
~ ONP-LaGoIlod Antlboay or tlga~d
Section
0NP-Labelled Peroxiaaso
T
IoM Rnn-nNP
Oft(loG
Immun°ol°bullns Unlabolled Antibody
Surface
Fig. l. Schematic representation of various modifications of the prototype DNP hapten sandwich staining (DHSS) procedure LM and EM. DAB = diaminobenzidine; LM = lighi microscopic: EM = electron microscopic.
Multivalent rnonoclonal anti-DNP bridge antibody This reagent is derived from one of the hyoridoma clones generated from mice immunised with DNP-labelled keyhole limpet haemocyanin (clone K7 generously provided by Dr. M. Kennedy, Institute of Medical Research, Mill Hill, London, UK). The antibody has proved to be remarkably carrier independent in that it is able to detect DNP hapten groups on monoclonal and polyclonal antibodies from a diverse variety of animal and immunoglobulin species (e.g., rabbit, guineapig, sheep, horse, human, mouse and rat antibodies of IgG, IgA, and IgM classes of immunoglobulins) as well as on non-antibody ligands such as TSH, TNF and rat IgE myeloma proteins. The bridge antibody's efficiency appears to derive
195 largely from its exquisite specificity and its extremely high affinity/avidity for the DNP hapten (Dr. J. M. Parkhouse, personal communication). The multivalent character of ~he bridge antibody has imparted two special advantages to the DHSS procedure when compared with its parent technique the PAP method of Sternberger, and its nearest competitor the avidin-biotin bridge method (Guesdon et al., 19791. Thus, the method has been shown to generate a higher amplification ratio at each antibody concentration (e.g., anti-ealeitonin tested on rat thyroid C cells; Jasani et al., 1983a) or each antigen dilution level (e.g., in form of human |gG diluted in a gelatin stepwedge constructed according to the method of Millar and Williams, 1982), when tested by the three methods applied in tandem as illustrated in Fig. 2 (see also Table I and Fig. 3, respectively, for the corresponding quantitative data) (Jasani et al., 1983b). Secondly, it has allowed an effid e n t coupling of peroxidase and glucose oxidase enzyme reactions resulting in a highly focussed and intense deposition of the diaminobenzidine (DAB) product at the site of the primary antibody binding (Jasard and Williams, 1985; Jasani et al., 1985). This point is discu3~,~,d further under the description of the DNP-labelled glucose oxidase reagent.
DNP hapten labelled and anti-DNP linked marker enzymes In the prototype DHSS method based upon the PAP procedure (Jasani et al., 1981), use was
PAP
I/IP-peroxldase conjugate R~-hb I ~atlhulro~I IKG
Norr~ rabbit
3=
-PAP
Biotin-Sandwlch .......
~ . . . .
conjugate
i Biotin ~b anti~u anti-huimm) IgG h u ~ IgG lk~rma2 goat Normal rabbit
T
Serial step v~edge secticms containing d i f f e r i n g concentrations of htman IgG (20 to 0.04 mg/ml)
Fig. 2. A schematicrepresentationof the DHSS, the PAP and the Biotin sandwich procedures as applied to serial sections taken from a gelatin step-wegdecontainingdifferent dilutions of human lgG.
TABLE I MICRODENSITOMETRIC COMPARISON OF IMMUNOPEROXIDASE STAINING DUE TO THE DHSS. TIlE PAP AND TIlE BIOTIN SANDWICH PROCEDURES ON SERIAL RAT TIIYROID SECTIONS USING UNLABELLED OR APPROPRIATETLY LABELLED RABBIT ANTI-CALCITONIN PRIMARY ANTIBODY APPLIED AT SET DILUTIONS Thc biotin sandwichmethodproducedno signalbeyond 1/320 dilution, ~hilst the PAP and the DHSS techniques were positive upto 1/160() and 1/6400 dilutions of the primary antibody, respectively Anti-calcitonin (dilution)
n
DHSS
PAP
p
1/800 I/1,600 1/3,200 I/6,4(10 1/12,800
10 10 10 1(1 I0
5.4_+0.9 4.4_+1.0 3.3,+0.6 2.2 _+0.5 0
3.6-+0.4 2.4,+11.3 0 (I 0
< 0.(ll)l < 0.001
1/20
I0
7.0,+2.0
1/40 1/80
10 I0
5.2__+0,6 3.2-+ 1.0
1/160
I0
2.0+0.6
1/32() 1/640
I0 I0
2.0__+0.6 (l
Biotin sandwich
made of DNP-labelled PAP complexes to represent the enzyme marker in the third layer. This reagent, however, was found to ilz,,e a very shert shelf life. Attempts were made, therefore, to oroduce a more stable DNP-enzyme marker. Direct or indirect co-valent coupling of DNP to the peroxidase protein moiety led to an inefficient conjugate similar to the observations of Sternberger and Petrali (1977) and Mason and Sammons (1979). This was most probably due to steric inhibition caused by the bridge antibody binding to DNP groups attached closely to the active site of the enzyme. Since then, an indirect linking of the DNP groups through the enzyme's carbohydrate component using cytochrome C (CC) as the linker protein has resolved the problem (Newman and Jasani, 19841. The optimum DNP-CC-peroxidase conjugate is a relatively small marker with an average molecular weight in the region of 100 kDa. This
considcrably smaller conjugate size compared to the PAP complexes (300-~)00 kDa) is largely responsible for the DHSS procedure's high resolution immunopcroxidasc !abelling observed at the elcctron microscopic level (Jasani et al., 1983a; Newman and Jasani, 1984a,b). This has permitted vc~, precise double immunolabelling based on a combination of thc DHSS method and an indirect immunocol!oidal technique at the electron microscopic level (Newman et al., 1986, 1989) as well as very powerful silver intensification of light mtcio.~copic DAB deposits (Newman et al., 1983; McRae ct al.. 1986; Schmid et al., 1988; Gee et al.. 1990, 1991). These variations of the DHSS are schematically represented in Fig. 4. The idea of using DNP-glucose o×idase in conjunction with DNP-peroxidase as discussed above, has arisen directly from the multivalent character of the IgM bridge reagent (Jasani and Williams, 1985). Titration of the DNP-labelled glucose oxidase prepared using the highly pure Sigma V glucose oxidase solution and added sequentially after the
DNP
150
loo
~'~ 12~
20
I0
S
25
125 O6
O-3
015
OO8
HUMA,-, ,gGCm~/m0
Fig. 3. Microdensitometriccu,=7,'~risonof the staining intensities generated by the DHSS, the PAP ano the biotin sandwich procedures on serial sections laken from a gelatin step-wedge containing a set seriesof human lgG concentrations.
Double ImmunoenzymeTechnique
;k ~]TIGEN A
ANTIGIfl I SECTION SURFACE
Fig. 4. Schematic representation of the simultaneous double immunolabelling technique.
DNP-CC-peroxidase conjugate on the human lgG step-wedge sections (Millar and Williams, 1979), showed the optimum concentration to be between 1-2 p.g/ml. Using these conditions, the glucose oxidase based DHSS system has been found to yield higher amplification ratio at every antigen concentration included in the human IgG step-wedge compared to the DHSS procedure utilising the standard preformed H 2 0 2 as the peroxidase substrate (Fig. 5). The DAB staining produced in the presence of nascent H 2 0 2 has been shown to be highly intense and consistently free of non-specific background staining in tissue section procedures (Kumar et al., 1985). For this reason the DNPlabelled glucose oxidase variation has been included in the commercial analogue of the DHSS procedure, the DNP localisation system (see Fig. 1 for a schematic representation). The recent commercial availability of the horseradish-linked polyclonal anti-DNP secondary detection system (Dakopatts) has allowed the development of simple and versatile single (see Fig. 1) and a powerful simultaneous double immunoenzyme (Fig. 4) labelling methods (Navabi et al., 1990; Nair et al., 1991). The latter technique is intrinsically more versatile and economi-
30(3
)
f
C~
u ~, ~o(
1oo
HulgG rngtml Step-wedge
Fig. 5. Microdensitometric comparison of the staining intensities achieved at different human IgG concentrations in serial sections of a gelatin step-wedge stained with the DHSS/H202 (o), and DHSS/glucose oxidase (e) imm~ nopero:ddase procedures, respectively. cal c o m p a r e d t o t h a t d e s i g n e d b y B o o r s m a e t al. (1986).
Conclusion T h e D H S S p r o c e d u r e a n d its m o d i f i c a t i o n s collectively offer a highly versatile and efficient approach to immunolocalisation and immunoassay d e t e c t i o n a n d s c r e e n i n g o f a n t i g e n s , a n t i b o d ies a n d r e c e p t o r s .
References Gee, J.M.W., Nicholson, R.I., Jasani, B., Newman, G.R. and Amselgruber, W.M. (1990) An immunocytochemical method for localization of estrogen receptors in rat tis.,,ue using a dinitrophenyl (DNP)-labelled rat monoclonal primary antibody. J. Histochem. Cytochem. 38, 69.
Gee, J.M.W., Amselgruber, W.M., Jasani, B. and Nicholson, R.I. (1991) Use of the dioitrophenyl hapten sandwich staining procedure (DHSS) to localize estrogen receptors in paraffin-embed~ed tissues. J. Histochem. Cytochem. 39. 1659. Guesdon, J.L., Ternynck, T. and Avrameas, S. (1979)The use o~ avidin-biofin interaction in immunoenzymatic techniques. J. Histochem. Cytochem., 27, 1131. Hewlins, M.J.E., Weeks, 1. and Jasani, B. (1984) Non-delcteri()us dinitrophenyl (DNP) hapten labelling of antibody protein: Preparation and properties of some short-chain DNP imidoesters. J. lmmunoh Methods 70, 11 I. Jasani, B. and Williams, E.D. (1985) Hapten enzyme labelling. British patent no. 2,008,730B. Jasani, B., Wynford-Thomas, D. and Williams, E.D. (1981) Use of monoclonal anti-hapten antibodies for immunoloealisation of tissue antigens. J. Clin. Pathol. 34, 1000. Jasani, B., Newman, G.R., Stanworth, D.R. and Williams, E.D. (1982) lmmunolocalisation of tissue receptors using dinitrophenyl (DNP) hapten sandwich procedure. J. Pathol., 138, 50. Jasani, B., Thomas, N.D., Newman. G.R. and Williams, E.D. (1983a) DNP-hapten sandwich staining (DHSS) proce(lure: Design, sensitivity, versatility and applications. Immuno!. Commun. 12, 50. Jasani, B., Millar, D.M. and Williams, E.D. (1983b) A quantitative comparison of the sensitivity of immunocytochemical techniques. J. PathoL 140, 162. Jasani, B., Edwards, R.E., Thomas, N.D. and Gibbs, A.R. (1985) The use of vimentin antibodies in the diagnosis of malignant mesothelioma. Virchows Arch. (Pathol. Anat.) 406, 441. Jasani, B., Thomas, N.D., Ludgate, M. and V'/iWams, E.D. (1986) Adaptation of dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure for screening monoclonal antibodies. Proc. R. Microsc. Soc. 20, 1M7. McRac, M.A., Newman, G.R., Walker, S.M. and Jasani, B. (1986) lmmunohistochemical identification of prolactin and 24K protein in secretory endometrium. Fert. Steril. 45, 643. Kun;ar, S., Jasani, B., Hunt, J.S., Moffat, D.B. and Asscher, AW. (1985) A system for accurate immunolocalization of T~mm-Horsfall protein in renal biopsies. Histochem. J. 17, 1251. Mason, D. and Sammons, R.E. (1979) The labeled antigen method of immuooenzymatic staining. J. Histochem. Cytochem. 27, 832. Millaf, D.M. and Williams, E.D. (1979) A step-wedge standard for quantitation of immunoperoxidase techniques. Histochem. J. 14, 609. Morgan, J.M., Phillips, D.I.M., Lazarus, J.F.., Manhews, N. and Jasani, B. (1988) Light chain restriction of thyroglobulin autoantibody (TA) activity in Hashimoto's thyroiditis. Abstract Book, Joint Meeting of Soci6t6 Franejaise D'lmmunologie and British Society for Immunology, Spring, 1988, p. 116, poster 54. Nalr, S., Navabi, H., Williams, G.T. and Jasani, B. (1991) High frequency of light chain restricted plasma cells in
primary B-cell gastric lymphoma demonstrated by double immunolahclling. J. Pathol. 164, 348A. Navabi+ It,, Morgan. J.M. and Jasani, B. (1991)) Simultaneous I- and K isotypc immunl~labellirig of plasma cells (PCs) in Graves' disease (GT) h~r estimation of light chain restrictinn tit individual patient level. J. Pathol. 1611, 152A. Newman, G.R. and Jasani, B. (1984a) Post-embedding immunoenzyme techniques. In: J.M. Polak and I. Varndell (Eds.), Immunolabelling h~r Electron Mncroseopy, Elsevier, Amsterdam, p. 53. Newman, G,R. and Jasani, 13. (1984b) Immunoeleetronmieroscopy: immtinogold and immunoperoxidase compared using a nev.' post-embedding system. Mcd. Lab. Sci. 41, 23S. Newman. G.R.. Jasani, B. and Williams, E.D. (1983) The visualisation of trace amounts of diaminobcnzidine (DAB) polymer by a novel gold-sulphide-silver method, J Microsc. 132. RPI. Newman. G.R., Jasani, B. and Williams. E.D. (1986) Multiple
hormone storage by 'polycrine' cells in the pancreas (from a case of nesidioblastosis), Ilistochem. J. 18, 67. Newman, G.R., Jasani, B. and Williams. E.D. (1989) Multiple hormone stor:~gc by cells of the human pituitary. J. Histochem. Cytochem. 37, 1183. Schmid, K.W.. Jasani. B., Morgan, J.M. and Williams, E.D. (1988) Light microscopic immunocytchemical demonstration of thyroid-stimulating hormone (TSH) receptors on normal rat thyroid cells. J. Histochem. Cytochem. 36, 977. Sternberger, LA. and Petrali, J.P, (1977) The unlabelled antibody enzyme method. Attempted use of peroxidasecgnjugaled antigen as the third layer in the technique. J. Histoehem. Cytochem. 25, 1036. Sternberger, L.A., Hardy, Jr., P.H., Cuculis, J.J. and Meyer, tt.G. (1970) The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complexes (horseradish peroxidaseantiperoxidase) and its use in identification of spirochaetes. J. Histochem. Cytoehem. 18, 315.
193
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.110
JIM06364
Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure A 10 year review of its principle reagents
and applications
B~ J a s a n i ~, N.D. T h o m a s ~, H. N a v a b i 1, D.M. Millar J, G . R . N e w m a n 2, j. G e e 3 a n d E.D. Willk..as t t Department of Pathology, 2 EM Unit, and "~TenoL'us Institute, Unit'ersity of Wales College of Medicine, Cardiff CF4 4XN, Wales, UK
(Accepted 12 February 1992)
Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labe!!ed primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or ant:-DNP linked marker enzyme. In this article the development of these reagents and various modificatit~,s of the b~sic technique are reviewed in conjunction with the special applications accruing from their use. Key words: Dinitrophenyl; lmmunoperoxidase;Dinitrophenyl-ilaptenprocedure; Dinitrophenyl localization system: Immunolocal-
ization; Immunoassay
Introduction The prototype, three-layer DNP hapten sandwich staining (DHSS) procedure (see Fig. 1 for a
Correspondence to: B. Jasani, Head of Immunopatholo~ Unit, Department of Pathology, University of Wales College of Medicine, Cardiff CF4 4XN, Wales UK (Tel.: 0222-743523; Fax: 0222-744276). Abbreviations: DNP, dinitrophenyl; DHSS, dinitrophenyl hapten sandwich staining; PAP, peroxidase-anti-peroxidase; TSH, thyroid stimulating hormone; DNBS, dini'~robenzene sulphonic acid; FDNB, fluorodinitrobenzene; TNF, tumour necrosis factor; DAB, diaminobenzidine; DNP-CC-peroxidase, dinitrophcayl-cytochromeC-peroxidase.
schematic representation) was developed some 10 years ago as a modification of the PAP method of Sternberger (Sternberger et al., 1970) primarily to provide a versatile secondary detection system for high specificity immunoperoxidase localisation of tissue antigens and hormone receptors using DNP-labelled primary probes (Jasani et al., 1981). The technique has since been optimised and modified to suit a diverse variety of immunocytochemical and immunoassay applications. The object of this article is to give a brief review of the development of the principle reagents, various modifications of the prototype technique and the special applications resulting from their use.
l ¢}4
Principle reagents, variations of the technique and applications The key reagents of the various DNP haptcn based techniques to be described include: (1) a non-deleterious DNP hapten labelling compound, (2) a multivalcnt igM monoclonal, antiDNP bridge reagent; and (3) DNP hapten substituted or anti-DNP linked marker enzymes, respectively.
DNP hapten labelling compound A novcl DNP labelling reagent, namely methyl-3-(2,4-dinitrophenylamino) propionimidate hydrochloride (DNP-N-IE) capable of producing adequate substitution ratios (10-15 per immunoglobulin molecule) under relatively mild alkaline (pH < 9.0) conditions and without causing significant protein insolubility, was primarily developed to replace the less suitable conventional reagents, viz., fluorodinitrobenzene (FDNB) and dinitrobenzene sulphonic acid (DNBS). The non-deleterious character of the compound was furthcr confirmed by its capacity to substitute up to 15 DNP ~roups per antibody molecule without causing a significant change in
I
I~
ONP-Locallsanon
OflBIGIUCOSe
its antigen binding capacity or affinity (Hewlins et al., 1984). The compound has since been applied to a wide range of monoclonal and polyclonal (primary and secondary) antibodies as well as non-antibody probes, e.g., lgE myeloma protein, thyroid stimulating hormone (TSH), and tumour necrosis factor (TNF), without significant alteration in their respective specificity or sensitivity (Jasani et al., 1982, 1986; Schmid et al., 1988; and C. Sarraf, N. Matthews and B. Jasani, unpublished data). The compound is effective over a wide range of concentrations (e.g., 100 /xg-10 mg/ml) of immunoglobulins whether present in their affinity purified, unfractionated or whole serum form. The use of DNP hapten labelled secondary antibodies in the modified form of the DHSS procedure, the DI']P localisation system (see Fig. 1), has facilitated high sensitivity and versatile immunocytochemical and immnnoenzyme assay screening of hybrid monoclonal antibodzy s,~pernatants. Thus all three major classes of mouse monoclonal antibodies - IgG, IgA and lgM - are capable of being screened with equal efficacy and at a much higher overall signal to noise ratio than that afforded by the commercially available indirect immunoperoxidase conjugates in the EL1SA mode (Jasani et al., 1986). The ELISA approach has also proved highly effective for serological screening of light chain restricted human autoantibodies (Morgan et al., 1988).
, ONA-Labolled Glucose 0.idase
T
Indirect
[INP-Method
One/Xydrogen Peroxide
OABIAyarogen Peroxide
ONP-tabollea Peroxldase
T
T
T
Conjugate
IOM Rntl-bNP Ortdge
ONP-lebelled Anti-Re,bit/Mouse
RnII-DI~PPeroxldase DNP-tabell~d nnUnody or tlgzn~
T
~ ONP-LaGoIlod Antlboay or tlga~d
Section
0NP-Labelled Peroxiaaso
T
IoM Rnn-nNP
Oft(loG
Immun°ol°bullns Unlabolled Antibody
Surface
Fig. l. Schematic representation of various modifications of the prototype DNP hapten sandwich staining (DHSS) procedure LM and EM. DAB = diaminobenzidine; LM = lighi microscopic: EM = electron microscopic.
Multivalent rnonoclonal anti-DNP bridge antibody This reagent is derived from one of the hyoridoma clones generated from mice immunised with DNP-labelled keyhole limpet haemocyanin (clone K7 generously provided by Dr. M. Kennedy, Institute of Medical Research, Mill Hill, London, UK). The antibody has proved to be remarkably carrier independent in that it is able to detect DNP hapten groups on monoclonal and polyclonal antibodies from a diverse variety of animal and immunoglobulin species (e.g., rabbit, guineapig, sheep, horse, human, mouse and rat antibodies of IgG, IgA, and IgM classes of immunoglobulins) as well as on non-antibody ligands such as TSH, TNF and rat IgE myeloma proteins. The bridge antibody's efficiency appears to derive
195 largely from its exquisite specificity and its extremely high affinity/avidity for the DNP hapten (Dr. J. M. Parkhouse, personal communication). The multivalent character of ~he bridge antibody has imparted two special advantages to the DHSS procedure when compared with its parent technique the PAP method of Sternberger, and its nearest competitor the avidin-biotin bridge method (Guesdon et al., 19791. Thus, the method has been shown to generate a higher amplification ratio at each antibody concentration (e.g., anti-ealeitonin tested on rat thyroid C cells; Jasani et al., 1983a) or each antigen dilution level (e.g., in form of human |gG diluted in a gelatin stepwedge constructed according to the method of Millar and Williams, 1982), when tested by the three methods applied in tandem as illustrated in Fig. 2 (see also Table I and Fig. 3, respectively, for the corresponding quantitative data) (Jasani et al., 1983b). Secondly, it has allowed an effid e n t coupling of peroxidase and glucose oxidase enzyme reactions resulting in a highly focussed and intense deposition of the diaminobenzidine (DAB) product at the site of the primary antibody binding (Jasard and Williams, 1985; Jasani et al., 1985). This point is discu3~,~,d further under the description of the DNP-labelled glucose oxidase reagent.
DNP hapten labelled and anti-DNP linked marker enzymes In the prototype DHSS method based upon the PAP procedure (Jasani et al., 1981), use was
PAP
I/IP-peroxldase conjugate R~-hb I ~atlhulro~I IKG
Norr~ rabbit
3=
-PAP
Biotin-Sandwlch .......
~ . . . .
conjugate
i Biotin ~b anti~u anti-huimm) IgG h u ~ IgG lk~rma2 goat Normal rabbit
T
Serial step v~edge secticms containing d i f f e r i n g concentrations of htman IgG (20 to 0.04 mg/ml)
Fig. 2. A schematicrepresentationof the DHSS, the PAP and the Biotin sandwich procedures as applied to serial sections taken from a gelatin step-wegdecontainingdifferent dilutions of human lgG.
TABLE I MICRODENSITOMETRIC COMPARISON OF IMMUNOPEROXIDASE STAINING DUE TO THE DHSS. TIlE PAP AND TIlE BIOTIN SANDWICH PROCEDURES ON SERIAL RAT TIIYROID SECTIONS USING UNLABELLED OR APPROPRIATETLY LABELLED RABBIT ANTI-CALCITONIN PRIMARY ANTIBODY APPLIED AT SET DILUTIONS Thc biotin sandwichmethodproducedno signalbeyond 1/320 dilution, ~hilst the PAP and the DHSS techniques were positive upto 1/160() and 1/6400 dilutions of the primary antibody, respectively Anti-calcitonin (dilution)
n
DHSS
PAP
p
1/800 I/1,600 1/3,200 I/6,4(10 1/12,800
10 10 10 1(1 I0
5.4_+0.9 4.4_+1.0 3.3,+0.6 2.2 _+0.5 0
3.6-+0.4 2.4,+11.3 0 (I 0
< 0.(ll)l < 0.001
1/20
I0
7.0,+2.0
1/40 1/80
10 I0
5.2__+0,6 3.2-+ 1.0
1/160
I0
2.0+0.6
1/32() 1/640
I0 I0
2.0__+0.6 (l
Biotin sandwich
made of DNP-labelled PAP complexes to represent the enzyme marker in the third layer. This reagent, however, was found to ilz,,e a very shert shelf life. Attempts were made, therefore, to oroduce a more stable DNP-enzyme marker. Direct or indirect co-valent coupling of DNP to the peroxidase protein moiety led to an inefficient conjugate similar to the observations of Sternberger and Petrali (1977) and Mason and Sammons (1979). This was most probably due to steric inhibition caused by the bridge antibody binding to DNP groups attached closely to the active site of the enzyme. Since then, an indirect linking of the DNP groups through the enzyme's carbohydrate component using cytochrome C (CC) as the linker protein has resolved the problem (Newman and Jasani, 19841. The optimum DNP-CC-peroxidase conjugate is a relatively small marker with an average molecular weight in the region of 100 kDa. This
considcrably smaller conjugate size compared to the PAP complexes (300-~)00 kDa) is largely responsible for the DHSS procedure's high resolution immunopcroxidasc !abelling observed at the elcctron microscopic level (Jasani et al., 1983a; Newman and Jasani, 1984a,b). This has permitted vc~, precise double immunolabelling based on a combination of thc DHSS method and an indirect immunocol!oidal technique at the electron microscopic level (Newman et al., 1986, 1989) as well as very powerful silver intensification of light mtcio.~copic DAB deposits (Newman et al., 1983; McRae ct al.. 1986; Schmid et al., 1988; Gee et al.. 1990, 1991). These variations of the DHSS are schematically represented in Fig. 4. The idea of using DNP-glucose o×idase in conjunction with DNP-peroxidase as discussed above, has arisen directly from the multivalent character of the IgM bridge reagent (Jasani and Williams, 1985). Titration of the DNP-labelled glucose oxidase prepared using the highly pure Sigma V glucose oxidase solution and added sequentially after the
DNP
150
loo
~'~ 12~
20
I0
S
25
125 O6
O-3
015
OO8
HUMA,-, ,gGCm~/m0
Fig. 3. Microdensitometriccu,=7,'~risonof the staining intensities generated by the DHSS, the PAP ano the biotin sandwich procedures on serial sections laken from a gelatin step-wedge containing a set seriesof human lgG concentrations.
Double ImmunoenzymeTechnique
;k ~]TIGEN A
ANTIGIfl I SECTION SURFACE
Fig. 4. Schematic representation of the simultaneous double immunolabelling technique.
DNP-CC-peroxidase conjugate on the human lgG step-wedge sections (Millar and Williams, 1979), showed the optimum concentration to be between 1-2 p.g/ml. Using these conditions, the glucose oxidase based DHSS system has been found to yield higher amplification ratio at every antigen concentration included in the human IgG step-wedge compared to the DHSS procedure utilising the standard preformed H 2 0 2 as the peroxidase substrate (Fig. 5). The DAB staining produced in the presence of nascent H 2 0 2 has been shown to be highly intense and consistently free of non-specific background staining in tissue section procedures (Kumar et al., 1985). For this reason the DNPlabelled glucose oxidase variation has been included in the commercial analogue of the DHSS procedure, the DNP localisation system (see Fig. 1 for a schematic representation). The recent commercial availability of the horseradish-linked polyclonal anti-DNP secondary detection system (Dakopatts) has allowed the development of simple and versatile single (see Fig. 1) and a powerful simultaneous double immunoenzyme (Fig. 4) labelling methods (Navabi et al., 1990; Nair et al., 1991). The latter technique is intrinsically more versatile and economi-
30(3
)
f
C~
u ~, ~o(
1oo
HulgG rngtml Step-wedge
Fig. 5. Microdensitometric comparison of the staining intensities achieved at different human IgG concentrations in serial sections of a gelatin step-wedge stained with the DHSS/H202 (o), and DHSS/glucose oxidase (e) imm~ nopero:ddase procedures, respectively. cal c o m p a r e d t o t h a t d e s i g n e d b y B o o r s m a e t al. (1986).
Conclusion T h e D H S S p r o c e d u r e a n d its m o d i f i c a t i o n s collectively offer a highly versatile and efficient approach to immunolocalisation and immunoassay d e t e c t i o n a n d s c r e e n i n g o f a n t i g e n s , a n t i b o d ies a n d r e c e p t o r s .
References Gee, J.M.W., Nicholson, R.I., Jasani, B., Newman, G.R. and Amselgruber, W.M. (1990) An immunocytochemical method for localization of estrogen receptors in rat tis.,,ue using a dinitrophenyl (DNP)-labelled rat monoclonal primary antibody. J. Histochem. Cytochem. 38, 69.
Gee, J.M.W., Amselgruber, W.M., Jasani, B. and Nicholson, R.I. (1991) Use of the dioitrophenyl hapten sandwich staining procedure (DHSS) to localize estrogen receptors in paraffin-embed~ed tissues. J. Histochem. Cytochem. 39. 1659. Guesdon, J.L., Ternynck, T. and Avrameas, S. (1979)The use o~ avidin-biofin interaction in immunoenzymatic techniques. J. Histochem. Cytochem., 27, 1131. Hewlins, M.J.E., Weeks, 1. and Jasani, B. (1984) Non-delcteri()us dinitrophenyl (DNP) hapten labelling of antibody protein: Preparation and properties of some short-chain DNP imidoesters. J. lmmunoh Methods 70, 11 I. Jasani, B. and Williams, E.D. (1985) Hapten enzyme labelling. British patent no. 2,008,730B. Jasani, B., Wynford-Thomas, D. and Williams, E.D. (1981) Use of monoclonal anti-hapten antibodies for immunoloealisation of tissue antigens. J. Clin. Pathol. 34, 1000. Jasani, B., Newman, G.R., Stanworth, D.R. and Williams, E.D. (1982) lmmunolocalisation of tissue receptors using dinitrophenyl (DNP) hapten sandwich procedure. J. Pathol., 138, 50. Jasani, B., Thomas, N.D., Newman. G.R. and Williams, E.D. (1983a) DNP-hapten sandwich staining (DHSS) proce(lure: Design, sensitivity, versatility and applications. Immuno!. Commun. 12, 50. Jasani, B., Millar, D.M. and Williams, E.D. (1983b) A quantitative comparison of the sensitivity of immunocytochemical techniques. J. PathoL 140, 162. Jasani, B., Edwards, R.E., Thomas, N.D. and Gibbs, A.R. (1985) The use of vimentin antibodies in the diagnosis of malignant mesothelioma. Virchows Arch. (Pathol. Anat.) 406, 441. Jasani, B., Thomas, N.D., Ludgate, M. and V'/iWams, E.D. (1986) Adaptation of dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure for screening monoclonal antibodies. Proc. R. Microsc. Soc. 20, 1M7. McRac, M.A., Newman, G.R., Walker, S.M. and Jasani, B. (1986) lmmunohistochemical identification of prolactin and 24K protein in secretory endometrium. Fert. Steril. 45, 643. Kun;ar, S., Jasani, B., Hunt, J.S., Moffat, D.B. and Asscher, AW. (1985) A system for accurate immunolocalization of T~mm-Horsfall protein in renal biopsies. Histochem. J. 17, 1251. Mason, D. and Sammons, R.E. (1979) The labeled antigen method of immuooenzymatic staining. J. Histochem. Cytochem. 27, 832. Millaf, D.M. and Williams, E.D. (1979) A step-wedge standard for quantitation of immunoperoxidase techniques. Histochem. J. 14, 609. Morgan, J.M., Phillips, D.I.M., Lazarus, J.F.., Manhews, N. and Jasani, B. (1988) Light chain restriction of thyroglobulin autoantibody (TA) activity in Hashimoto's thyroiditis. Abstract Book, Joint Meeting of Soci6t6 Franejaise D'lmmunologie and British Society for Immunology, Spring, 1988, p. 116, poster 54. Nalr, S., Navabi, H., Williams, G.T. and Jasani, B. (1991) High frequency of light chain restricted plasma cells in
primary B-cell gastric lymphoma demonstrated by double immunolahclling. J. Pathol. 164, 348A. Navabi+ It,, Morgan. J.M. and Jasani, B. (1991)) Simultaneous I- and K isotypc immunl~labellirig of plasma cells (PCs) in Graves' disease (GT) h~r estimation of light chain restrictinn tit individual patient level. J. Pathol. 1611, 152A. Newman, G.R. and Jasani, B. (1984a) Post-embedding immunoenzyme techniques. In: J.M. Polak and I. Varndell (Eds.), Immunolabelling h~r Electron Mncroseopy, Elsevier, Amsterdam, p. 53. Newman, G,R. and Jasani, 13. (1984b) Immunoeleetronmieroscopy: immtinogold and immunoperoxidase compared using a nev.' post-embedding system. Mcd. Lab. Sci. 41, 23S. Newman. G.R.. Jasani, B. and Williams, E.D. (1983) The visualisation of trace amounts of diaminobcnzidine (DAB) polymer by a novel gold-sulphide-silver method, J Microsc. 132. RPI. Newman. G.R., Jasani, B. and Williams. E.D. (1986) Multiple
hormone storage by 'polycrine' cells in the pancreas (from a case of nesidioblastosis), Ilistochem. J. 18, 67. Newman, G.R., Jasani, B. and Williams. E.D. (1989) Multiple hormone stor:~gc by cells of the human pituitary. J. Histochem. Cytochem. 37, 1183. Schmid, K.W.. Jasani. B., Morgan, J.M. and Williams, E.D. (1988) Light microscopic immunocytchemical demonstration of thyroid-stimulating hormone (TSH) receptors on normal rat thyroid cells. J. Histochem. Cytochem. 36, 977. Sternberger, LA. and Petrali, J.P, (1977) The unlabelled antibody enzyme method. Attempted use of peroxidasecgnjugaled antigen as the third layer in the technique. J. Histoehem. Cytochem. 25, 1036. Sternberger, L.A., Hardy, Jr., P.H., Cuculis, J.J. and Meyer, tt.G. (1970) The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complexes (horseradish peroxidaseantiperoxidase) and its use in identification of spirochaetes. J. Histochem. Cytoehem. 18, 315.
