Use of monoclonal antibodies in immunocytochemistry with special reference to the central nervous system

Brain Research Bulletin,

Vol. 5, pp. 575-587. Printed in the

U.S.A.

Use of Monoclonal Antibodies in Immunocytochemistry with Special Reference to the Central Nervous System A. C. CUELLO,*’ ~~e~urt~e~ts

C. MILSTEIN,t

AND J. V. PRIESTLEY”

of P~ar~acalogy

and ~u~un Anatu~y, University of Oxford, South Parks Road Oxford, England and TMRC Molecular Biology, Cambridge, England Received

2 April

1980

A. C., C. MXLSTEIN AND J. Y. PRIESTLEY. Use of mono&ma1 antibodies in immunncytochemist~ with antibodies to the central nervous system. BRAIN RES. BULL, S(5) 575-587, 1%0.-Monoclonal produced by hybrid myelomas have been successfully applied in immunocytochemistry. Here we discuss the results obtained when applying one of these antibodies, to substance P, on tissue sections in conjunction with immunofluorescence and unlabelled immunoenzyme procedures. In addition we discuss a novel approach to the localisation of tissue antigens by using internally labelled, tritiated monoclonal antibodies. CUELLO,

special

reference

Monoclonal antibodies Central nervous system

Histochemistry Immunocytochemistry Tritiated monoctonal antibodies

techniques are based on the visualisation of the binding of antibodies to specific tissue antigens. In most cases such visualisation is accomplished by developing antibodies (indirect techniques) which bind the first antibody. Until recently the required antibodies have been of polyclonal origin; that is to say that they have been raised in laboratory animals by the repeated injection of immunogen with adjuvant substances. The sera of such animals contain a mixture of a large variety of secreted antibody molecules sythesised within the animal by an equally large variety of clones of cells [2] (see Fig. 1). Each element of such a complex mixture recognises independent as well as overlapping determinants of the immunogen. In addition the mixture includes a large number of immunoglobulin carrier molecules e.g., those recognising the adjuvant substances. The cha~cte~sti~s and a.&ity as well as the relative abundance of individual antibody molecules in these conventional sera will vary considerably from animal to animal and even from bleed to bleed. Some of these problems can be solved by the isolation of the more specific antibodies by affinity columns and related procedures. A more radical solution can be accomplished by the hybrid myeloma strategy. This technique has two very valuable advantages: (1) the derivation of highly specific antibodies from unpurified immunogens, and (2) the continuous supply of unlimited quantity of the same specific antibody. Furthermore, because the method involves the in IMMUNOHIST~CHEMICAL

‘Send reprint requests to Dr. A. C. Cueilo, Departments Oxford OX1 3QT, England (UK).

Copyright

o 1980 ANKHO

P

vitro production of antibody, it can be easily adapted to the preparation of inte~ally labelled antibody of a single molecular species. As discussed below, this property is proving to be of particular value to immunocytochemistry. HYBRID MYELOMA STRATEGY OF MONOCLONAL ANTIBODY PRODUCTION

The hybrid myeloma strategy for the production of monoclonal antibodies consists of (a) “immo~~isation” of the antibody producing cells by cell fusion with myeloma cells, (b) elimination of non-fused parental cells, and (c) the selection and isolation of the desired clones [12,14] (Fig. 1). ImmortaIisation

of Antibody Producing CeNs

This is accomplished by the fusion of spleen lymphocytes of the hyperimmune animal with a rapidly dividing, especially derived line of myeloma cells in permanent growth. For the present studies we have used rat spleen lymphocytes from adult Wistar rats immunised with repeated doses of 3 fig substance P-bovine sera albumin conjugate. These spleen cells were mixed in suspension with the mouse myeloma cell line NSlIl-Ag 4-l in ~lyethylene glycol 1500 (50%) as described by Galfrk et al. [lo]. Elimination of Non-fused

Parental Cells

After cell fusion the cell suspension

of Pha~acology

International

Anti-substance

was divided in a large

and Human Anatomy, University of Oxford, South Parks Road,

Inc .-0361-9230/80/050575-13$01.80/O

CUELLO.

MILSTEIN

spleen ce.lls (1*2*3+4---n) ( De I” culture)

lmmortalizsti by fusion to myekma % Myeloma

I line

-_[nssayantaodv) tCloning of Sotnsttc cell hybrids

FIG. 1. Monoclonal antibodies from hybrid myelomas. Animals produce highly heterogeneous mixtures of antibodies, while hybrids between myeloma cells and splenocytes produce monoclonal antibodies directed against simple antigenic determinants, regardless of the complexity of the antigenic stimulus. Once isolated, the hybrid clones can be grown in unlimited quantity to produce a permanent supply of the same monoclonal antibody (from Cuello, Galfre and Milstein [8], with the publishers’ permission).

AND PRIESTLEY

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ANTIBODIES IN IMMUNOCYTOCHEMISTRY

577

FIG. 2. Fluorescence ~un~ytoche~st~ for substance P antigenic sites in the iower medulla oblongata of the rat is revealed by the monsoons antibody NC~/34-HL. ST, spinal tract; SG, substantia gelatinosa; NP, nucleus propius of the trigeminal nerve. Open arrows indicate single varicosities from transversely cut axons in the spinal tract, filled arrow indicates a lon~tudin~~y cut axon running towards the substantia gelatinosa and displaying several varicosities. Scale bar=50 pm.

FIG. 3. Whole mount prep~tion of the rabbit ileum showing substance P ~muno~active cell bodies (asterisks) and i~ividu~ varicose nerve fibres in the myenteric piexus (arrows). indirect immuno~uorescence. Scale bar=50 Mm. (From Costa, Furness and Cuelfo, unpublished).

CUELLO,

578

number of individual cultures and grown in HAT (hypoxantine, aminopterin, thymidine) selective medium [22]. The mouse myeloma cell line used (NSIII-AG 4-l) is azaguanine resistant, thus presumably lacking hy~x~tine guanine phosphoribosyl transferase (HGPRT) activity. These mutants die in HAT medium 1131in which the main biosynthetic pathway for the production of nucleic acids is blocked by aminopterin, and the cells are unable to use the so-called ‘“salvage pathway” because they lack HGPRT activity. Conversely, the rat lymphocyte possesses the HGPRT enzyme but does not grow in tissue culture conditions. The information for the production of the desired antibodies is therefore provided by the cells of the hyperimmune animal. The surviving hybrids grow in HAT medium and produce a diversity of antibodies.

The partially fractionated hybrids can now be grown in normal culture medium, and the spent fluid tested for antibody production. The sensitivity of the assay is of utmost importance since a very diluted antibody solution is at this stage recovered from the individual cultures. For instance, for anti-substance P a radioimmunoassay as described by Powell et at. f 16] was applied. More recently for an anti-SHT (serotonin) monoclonal antibody both hemoagglutination and immunocytochemistry were applied to monitor the cultures. The selected active cuftures were then subjected to a cloning procedure. Individual colonies derived from single ceils were produced by growth on soft agar and separately grown. The clones secreting the derived antibodies can then be identified. In the production of the substance P antibody two clones were obtained and their purity was ensured by repeating the above procedure. These clones secreted a complete immunoglobulin molecule as shown by polyacrilamide gel electrophoresis 181, while the parental myeloma cell line produced only a K light chain which was not secreted. The characteristics of this antibody have been reported elsewhere [71. USE OF MONOCLONAL

ANTIBODIES MICROSCOPY

IN FLUORESCENCE

Most of the examples ofimmunocytochemical application of monoclonal antibodies to be presented below have been performed with the rat-mouse monoclonal anti-substance P antibody code-named NCl/34 HL, in conjunction with the indirect immunofluorescence technique [4]. A similar general strategy is being applied in studies of cell surface antigens (11, where particular emphasis is devoted to fluorescence light microscopy and cytofluorimetry. For the immunocyto~hemical application of NCli34 HL, animals were anaesthetised with equithesin and perfused through the heart initially with approximately 20 ml of 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose, followed by perfusion with 4% freshly prepared parafo~~dehyde in the same phosphate buffer, both at room temperature. The brain was removed rapidly and kept in the same fixative for 2 to 3 hr, after which it was transferred to phosphate buffer-sucrose (as above) at 4’C; the brain remained in this solution overnight (12-16 hr). Sections were obtained with a Deittes cryostat (Heidelberg, Germany) at -20°C and thaw mounted on acid washed subbed slides (0.5% gelatin, 0.05% chromalum). Antibody solutions were made up in phosphate buffer saline (PBS) (1140 up to l/400 dilutions of NCl’34 HL) containing 0.1% Triton X-100 [l I], applied on the slide-

MILSTEJN AND PRlESTLEY

mounted tissue sections, and incubated for 30-40 min in moist chambers at 37°C. As developing antiserum, anti-rat IgG, made in rabbit (Miles-Yeda, Israel) and conjugated to FITC (tluorescein isothioc~anate) was applied similarly. Prior, or between and after the immuno-incubations, the slides were washed for 15 min in staining jars containing PBS-Triton. The sections were mounted in 3: 1 glycerol/PBS (V./V.). Preparations treated with the indirect immunoffuorescent technique were observed in a Dialux 20 Leitz microscope equipped with epifluorescence optics. Micrographs were taken with the aid of a Leitz Orthomat camera on Kodak T-X film (400 ASA/ Din) or Ektachrome daylight (200 ASAi24 Din). A typical result for the cyrostat sectioned material can be observed in Fig. 2, where the fine network of primary sensory terminals immunoreactive to NC1/34 HL (substance P-containing fibres) is clearfy revealed in the substantia gelatinosa of the spinal nucleus of the trigeminal nerve. Whole mounted preparations of the guinea-pig ileum were successfully prepared in collaboration with Costa, Furness and Franc0 [S], to visualise a rich network of substance P immunoreactive fibres in the myenteric plexus, the submucosa, and surrounding blood vessels. More recently, ceil bodies immunorea~tive to NC1134 HL have been seen in the rabbit myenteric plexus (Fig. 3). As for the procedures described below, control preparations were run with the monoclonal antibody NCl/34 HL preabsorbed (45 min at 37°C) with 200 ,&mI of synthetic substance P. Such controls gave a dark background devoid of ffuorescent structures. USE OF MONOCLONAL ANTIBODIES IN CONJUNCTION PAP UNLABELLED ANTIBODY TECHNIQUE

WITH THE

The mouoclonal antibody NC]/34 HL (aat~“substance P) has been shown to be suitabfe for the application of the PAP unlabelled antibody techniques [ 191. For this technique Wistar rats were anaesthetised with equithesin and perfused through the heart for 2 mm with 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose, followed by 4% paraformaldehyde-0.0% glutaraldehyde in the same phosphate buffer. After 30 min perfusion the brain was rapidly removed and kept in the same fixative for 1 hr. Unless otherwise stated all steps were carried out at room temperature. The brain was then cut into a few 1 mm thick slices, kept in fixative for a further 2 hr, and then transferred to the sucrose phosphate buffer for a minimum of 3 hr and kept at 4°C. Sections 50 pm thick were then cut with the Oxford Vibratome, and small areas were cut from the sections by hand and transferred to PBS, thereby ready for subsequent staining. Antibody incubations were carried out in wells allowing the use of small volumes (150 ~1) of reagent. All solutions were made up in PBS and the incubation steps were as follows: 30 min in l/IO normal rabbit serum (Cappel Lab), 15 min PBS wash, 1 hr in 11100 monoclonal anti-substance P, 1 hr PBS wash, 1 hr in l/l0 rabbit anti-rat IgG (Miles-Yeda Ltd), 1 hr PBS wash, tr/z hr in 1150rat PAP (Cappel Lab) and I hr PBS wash. For the peroxidase reaction, sections were transferred to glass vials and incubated for 15 min in 0.06% 3,3’diaminobenzidine . 4HCl (Sigma) in 0.05 M Tris, pH 7.6. Hydrogen peroxide was then added to give a final concentration ofO.Ol%. The progress of the oxidation reaction was

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anti-substance P antibody (NC1/34-HL) used in conjunction with the peroxidase-antiperoxidase procedure. 10pm thick Cryostat section of the rat medulla oblongata treated with 0.1% Triton. A band of peroxidase products is present in the substantia gelatinosa (SG) while some individual axons can be noticed in the spinal tract (ST) of the trigeminal nerve. NP, nucleus propius. Scale bar=200 pm. FIG. 4. Monoclonal

followed under a binocular microscope and usually stopped after about 15 min. Sections were then washed for 1 hr in 0.1 M phosphate buffer prior to osmiftcation and flat embedding in plastic resins. One or 2 pm thick sections for light microscopy and silver-gold sections for electron microscopy were obtained with a Reichert ultr~icrotome. After lead staining the material was observed in a Phillips 201 electron microscope. In a series of trial runs we examined various fixation conditions and handling procedures in order to ascertain the conditions for optimal tissue preservation and antibody penetration using the monoclonal antibody. The conditions tried included a range of gluta~dehyde ~oncent~tions (0.05-0.2%), a rapid freeze thaw after fixation, cutting with

either Vibratome or cryostat, and treatment with a range of Triton concentrations (O.l-0.5%) either as a preincubation of various time lengths or added to the antibody dilution and washing solutions. Cryostat sectioning followed by Triton treatment provides optimum access for the reagents to the antigenic sites, and this procedure was adopted for light microscopy. Fig. 4 shows one such preparation. At the light microscopic level the distribution of substance P, revealed by the monoclonal antibody/PAP protocol, agrees with that seen with an indirect immunofluorescent method using the monoclonal antisubstance P antibody or a guinea-pig raised substance P antiserum 191. Of these procedures, the mono~Iona~PAP protocol was the most sensitive and resulted in the lowest

CUELLO,

580

MILSTEIN

AND PRIESTLEY

FIG. 5. Monoclonal anti-substance P antibody (NC1/34-HL) used in conjunction with the peroxidase-antiperoxidase procedure. 2 pm thick section of the surface of the rat medulla obtongata. Individual varicosities with peroxidase reaction products are indicated by arrows. ST, spinal tract; SG, substantia gelatinosa; NP, nucleus propius; c, capillaries. Broken lines indicate approximate limits for posteromarginal layer and substantia gelatinosa. Scale bar=20 pm. Other details as in Fig. 4. nonspecific background staining. With 0.1% glutaraldehyde included in the fixative, such a procedure gave acceptable results at the electron microscope (E.M.) level, although cell membranes were poorly preserved and some break-up of structures was noticed. With higher concentrations of gIut~~dehyde the number of antigenic sites decreased markedly. The most reproducible results at the E.M. level were obtained using the protocol described above with very low glutaraldehyde and staining of Vibratome sections without the addition of any Triton. In these sections there was a

gradient of staining density decreasing towards the centre of the section from each face, the exposed surfaces showing substantial nonspecific staining. Thus, when cutting thin sections for E.M. it was necessary to monitor the progress of staining through the block by examination of semithin 2 g plastic sections (Fig. 5). This also allowed particular profiles examined at the light microscopic level to be studied at the E.M. level. The optimum depth of staining was 5-10 ,u into the block from either face. The 30 min preincubation in l/l0 normal rabbit serum was

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FIG. 6A. Low magnification electron micrograph of the substantia gelatinosa of the trigeminal nerve. Material prepared as described in text. Arrows indicate substance P immunoreactive nerve terminai profiles as depicted by monoclonal antibody NC1/34. N, nuclei of neurones; D, dendritic profiles; asterisks, myelinated axons. Scale bar=5 pm.

routinely, and addition of 1% normal rabbit serum to the antibody solutions did not significantly further decrease nonspecific labelling. A range of incubation and washing times were explored and long washes (1 hr) were found to optimise the specific staining. Although long incubations (48 hr) in the cold are generally recommended for the primary antibody [20], with the l/100 dilution of monoclonal antibody one hour incubation at room temperature was found to be adequate. The concentration and incubation times for the anti-rat and the PAP are similar to those normally used with traditional antibodies. Figure 6a and 6b are low and high power magnification electron micrographs of substance P immunoreactive nerve terminals in the rat CNS, as revealed by monoclonal antibodies combined with the PAP technique. The use of single slot grids allowed, in our experience, the easy examination of serial sections, often necessary when the peroxiadopted

dase reaction products obscured the morphology of the underlying structure. Under the fixation conditions used here, synaptic vesicles and synaptic thickenings were adequately preserved, including in this case prominent post-synaptic specialisation. The osmicated peroxidase reaction product was found both covering and between large and small vesicles, agreeing with the results of other workers using conventional antisera [3, 6, 151. USE OF INTERNALLY LABELLED MONOCLONAL ANTIBODIES IN IMMUNOCYTOCHEMISTRY

One of the potentially more interesting advantages of the use of monoclonal antibodies is that they can be internally labelled with tritium during their biosynthesis by incubating the antibody producing clones in the presence of radioactive aminoacids. This, of course, would be impractical to accomplish in the entire animal where every possible newly

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AND PRIESTLEY

FIG. 6B. High magnification electron micrograph from an area shown in A (square), revealing some ultrastructural features of a substance P immunoreactive nerve terminal (arrows). D, dendritic profile post-synaptic to the immunoreactive nerve terminal. NT, unreactive nerve terminals: N, nucleus; CB, cell body. Asterisks denote myelinated axons. Scale bar= 1pm.

synthetised protein would be labelled as well as any of the different antibody molecules being produced. The procedure is efficient and economical when applied to antibody secreting cells in tissue culture conditions. On previous experience it has been found that when ‘*C-lysine is used the spent tissue culture fluid contains 60% pure antibody in terms of radiolabelled trichloroacetic precipitable material [21]. Internally labelled monoclonal antibodies with 3H-lysine have been used in the experiments which follow. The antibodies were prepared by incubating the hybrid line NC1134 in a Dulbecco modified Eagle medium minus lysine and then several hours in the same medium containing 1 to 5 mCi of :‘H-lysine (sp. act. 75-100 Ciimmol, The Radiochemical Centre, Amersham). After this the cells were removed by centrifugation and the supernatant culture medium recovered and dialised against large volumes of PBS. The 3Hinternally labelled antibodies do not require elaborate purifi-

cation, can be stored for long periods (in contrast to “‘Ilabelled antibody molecules) and applied for autoradiographic purposes. The radioactive reagent obtained had a specific activity ranging from 300 to 2000 Ciimmol of antibody. A diagrammatic representation of this procedure is illustrated in Fig. 7. So far we have applied these tritiated monoclonal antibodies against substance P (:‘H-NC1/34 HL) on the following tissue preparations: (a) 10 pm cryostat sections of tissue fixed as for immunofluorescence were recovered on subbed slides and incubated with several dilutions of :‘H-NC1134 HL with and without Triton for 40 minutes at 37°C. The sections were extensively washed, dried and dipped in previously melted Ilford L-4 emulsion diluted 1:4 in water. The slides were exposed for 4-10 days and developed with Kodak DK 170 solution, fixed in 25% sodium thiosulphate, washed, dehydrated and mounted in DPX mounting medium. The material was ob-

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FIG. 7. Schematic representation of the preparation and application in “radioimmunocytochemistry” of internally labelled monoclonal antibodies. (a), biosynthesis of “cold” antibodies by a hybrid myeloma cell; (b), the addition of 3H-lysine resulting in the biosynthetic incorporation in the antibody molecule of the tritiated amino acid; (c), centrifugation of the culture as to separate cells from medium; (d), dialysis of supematant to eliminate non-incorporated radioactive amino acids; (e), storage of the tritiated monoclonal antibody for long periods as opposed to *45Ilabelled antibodies; (0, application of the internahy labelled antibodies in “radioimmunocytochemistry”. Triangles in middle layer represent antigenic sites in tissue sections. Emission of pparticles from tritiated monoclonal antibodies are represented by irregular arrows. Thick curled traces in upper layer represent silver grains in the photographic emulsion resulting from exposure and development; (g), silver grains indicating the presence of tissular antigenic sites by “~dioimmun~yt~he~st~” at light microscopy (see Fig. 8) or electron microscopy (see Fig. 9) levels.

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MILSTEIN

AND PRIESTLEY

FIG. 8. Micrograph illustrating the application of internally labetled monoclonal antibodies (:%-NCI/34-HL) for light microscopy “radioimmunocytochemistry”. Silver grains are heavily concentrated in the area of the substantia gelatinosa (SC) of the spinal nucleus of the trigeminal nerve, while practically no radioactivity is detected in the nucleus proprius (NP). In the spinal tract (ST) of the trigeminal nerve bands of silver grains denoting underlying tritiated antibodies can be noticed (black arrows) as representation of unmyelinated axons from primary sensory neurones running towards the substantia ge~atinosa. Myeiinated axons remain largely negative. Some of these axons can be seen crossing the substantia gelatinosa (white arrows). Scale bar=50 pm.

served with a Dialux 20 Leitz microscope equipped with phase contrast, bright field and dark field. Black and white micrographs were taken with Kodak Ektachrome (64 ASAll Din). A representative result using this procedure is shown in Fig. 8. (b) Tissue was perfused with the double aldehyde solution as indicated above and Vibratome sections of approximately 50-70 pm were incubated without Triton either at 37°C for 2-3 hr or overnight (12 hr) at 4°C. In both cases after washes in PBS the tissue sections were post-fixed in 1% osmium tetroxide in buffer phosphate 0.1 M pH 7.4, dehydrated and flat embedded in epoxy resins. Sections 1 grn thick were collected on glass slides, dried and dipped either in Iiford L-4 ( 1:4 dilution, w/v) or Ilford K-5 (1: 1 dilution, w/v) photographic emulsion. The material dipped into Ilford K-5 emulsion was developed with Kodak D19 and ftxed with 5% sodium sulphate. After development and fixation, the sections were dried and mounted in DPX. (DePex, mounting medium, Gurr (R), Hopkin and Williams, U.K.). The material was observed and photographed as in (a). (c) Vibratome tissue sections, fixed, incubated and embedded as in (b) were cut in a Reichert ultramicrotome as for electron microscopy and collected by the loop technique on glass slides pre-coated with celloidin. The ultrathin sections

were contrasted by the lead staining procedure [ 171and carbon coated afterwards. The preparations were subsequently dipped in an Ilford L-4 photographic emulsion diluted 1:s in water and exposed at 4°C for 4 to 8 weeks and developed with Kodak Df9B. The material was observed and photographed with a Phillips 201 electron microscope. A representative result on the substantia gelatinosa of the spinal nucleus of the trigeminal nerve is shown in Fig. 9. Silver grains indicating an underlying radiation source were largely concentrated on nerve terminals displaying numerous small clear synaptic vesicles making contact with dendritic profiles. The most outstanding aspect of this new application is the remarkable ultrastructurat preservation of the tissue in contrast with the usual immun~nzyme techniques. GENERAL COMMENTS ON THE APPLICATION OF MONOCLONAL ANTIBODIES IN IMMUNOCYTOCHEMISTRY

Mon~lonal antibodies obtained by the hybridoma strategy have been successfully applied in the immunocytoehemistry of cell suspension preparations. Some of these applications have been illustrated in studies with surface antigens of lymphoid cells detected by monoclonal antibodies, Figure 10 illustrates an example of such an application In this paper we focused our attention on applications

MONOCLONAL

ANTIBODIES

IN IMMUNOCYTOCHEMISTRY

FIG. 9. Microg~ph ilIust~tin~ the application of internally labelled monociona~ antibodies (3H-NCl/3~HL) for Silver grains can be noticed overlying some nerve terminals electron microscopy “~dioimmu~~ytochemist~“. (arrows) synapsing over dendritic profiles (D). Note the degree of ultrastructural preservation which allows the critical analysis of the subcellular features of the underlying immunoreactive Asterisks indicate myelinated axons. Scale bar= 1 pm.

of monoclon~ antibodies in whole organ preparations. Combining monoclonal antibodies with either immunofluorescence or immunoenzyme developing procedures has given most satisfactory results. They yielded remarkably clean “immunostaining” of tissue antigen sites even when used in conjunction with conventional commercial developing antisera. The fact that the monoclonal antibody constitutes a single molecular species contributes to the elimination of an important source of background as represented by the diversity of antibody molecules always present in a conventional serum. The fact that the hybrid myeloma strategy allows a permanent source of the same antibody molecular species will, in time, diminish a major source of contradictory results obtained by different groups of workers using different antisera in the immunohistochemical research of the same tissue

and the non-immunoreactive

sites.

antigen. This has proved to be possible with the NCli34 HL which is commercially available from Seralab (U.K.) and is currently used by several leading laboratories working on immunocytochemistry of the central nervous system. There are reports in the literature of antisera raised against very small molecules. Very seldom can these “controversial” antibodies be used for the immuno~ytochemic~ investigation of tissue antigens, which require comparatively much larger quantities of antibody than the radioimmunoassays. Their limited production also restricts use so that confirmatory experiments by other laboratories are rare. Such has been the case, for example, of antisera against serotonin used for radioimmunoassays and only recently in immunocytochemist~ [18]. We mention this example because we have now succeeded in raising a rat- x-rat hybrid myeloma antibody against SHT (serotonin) which is capable of detect-

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AND PRIESTLEY

FIG. IO. Lymphocyte surface markers as revealed by a mouse monoclonal antibody (NA1/34), developed for indirect immunofluorescence with an anti-mouse IgG conjugated with rhodamine isothiocyanate (red). Only some lymphocytes are stained while all are immunoreactive to an anti H,TLA conventional antisera raised in rabbit and developed with fluorescein-isothiocyanate (green) conjugated anti-rabbit IgG, (from Bradstock et trl. Ill). Micrograph kindly provided by G. Janossy, of The Royal Free Hospital, London, England. ing immunoreactive neuronal system.

sites

in the

so-called

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The application of internally labelled antibodies for the detection of tissue antigens offers unique possibilities in immunocytochemistry. The internally labelled antibodies offer substantial advantages over other analogous alternatives such as the coupling of ‘7 to purified IgG fractions from conventionally raised antisera. Radioactive antibodies synthesised in vitro do not require purification of the specific antibodies by affinity columns. In addition, monoclonal antibodies can be radioactively labelled with 14C,9, or aH. As the “labelling” is dependent on the biosynthetic incorporation of the isotopically labelled amino acids, a large number of these radioactive units can be incorporated resulting in high specific activity immunoreagents (see above). 3H and “‘C, and even 35S, in contrast to 9 are safer and due to their prolonged half-life allow the storage of the reagent during long periods. Most important is that the biosynthetic incorporation of the label does not affect the antibody activity, a serious problem inherent of the procedures used to label antibodies chemically. The immunocytochemical application of internally labelled antibodies (“radioimmunocytochemistry”) avoids the limitations inherent in a second developing antibody. The most promising applications of radioim-

munocytochemistry are expected in the field of electron microscopy where conventional immunoenzyme methods sacrifice cell preservation to allow the penetration of large complexes of antibody enzyme molecules. Internally labelled antibodies avoid the use of these series of developing antibodies and complexes, thus maximising ultrastructural preservation and allowing a more detailed analysis of immunoreactive structures. No less exciting is the potential of combining immunoenzyme and radioimmunocytochemical methods for the simultaneous detection of more than one antigen site in a single preparation. It is well known that simultaneous detection of two antigenic specificities has not been successful with immunoenzyme methods due to the difficulties in discriminating between different electron dense deposits. With internally labelled antibody it would be theoretically possible to localise one antigenic site by radioautography and a second by an immunoenzyme method. In this manner the electron dense products from the silver precipitation and the enzyme products would be distinctively clear. We are currently exploring this strategy. Several groups are exploring the preparation of monoclonal reagents suitable for developing antibodies in immunocytochemical techniques. These could further diminish the overall background staining. One possible difficulty with

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this approach is that monoclonal antibodies recognise single determinants and may have a lower amplification effect than that of polyclonal antibodies. In the case of the PAP technique, polyclonal antibodies are essential, as the formation of antibody-peroxidase complexes is dependent on the ability of antibodies to recognise different determinants of the enzyme molecule. This difficulty can be easily overcome by the preparation of mixtures of different monoclonal antibodies against peroxidase, greatly simplifying the laborious preparation of these complexes. Monoclonal antibodies are bound to contribute meaningfully to the improvement and expansion of immunocytochemistry. Old problems could be tackled with new and powerful alternatives based on the application of

these unique immunoreagents. It is hoped that the experiments between different laboratories will be more comparable due to standardisation of reagents, thus shortening the time for the solution of problems revolving around the application of immunocytochemistry.

ACKNOWLEDGEMENTS

The skillful technical help of Mr. Bruce Wright, Miss Sara Patel, Mr. Stephen Bramwell and Mr. Michael Richards is acknowledged. Thanks are also due to Mrs. Ella Iles for typing the manuscript. This work has been supported by grants from The Medical Research Council, The Wellcome Trust and The Royal Society.

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