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Acta Bot. Need. 40(1), March 1991, p. 1-27 Review Antibodies in plant science M. D. Dewey D. Horsley Evans and C. Coleman J. R. Priestley R. Hull Hawes ;* Department ofPlant Sciences, University of Oxford, South Parks Road, Oxford 0X1 * School of Biological Oxford 0X30BP. 3RA UK and and Molecular Sciences, Oxford Polytechnic, Gipsy Lane, Headington, UK CONTENTS Introduction Antibodies in 1 plant biochemistry Preparation of and molecular antigens 2 Native protein, synthetic peptides expressed in bacterial Monoclonal versus Purification of 2 biology plant proteins or 3 systems? polyclonal antigens, 3 criteria of purity and hapten 3 conjugation Screening antibodies 6 Purification of antibodies 7 Uses of antibodies in molecular Immunodiagnostic assays for Immunodetection of plant biology and biochemistry 8 plant pathogens 8 viruses 9 Immunodetectionof phytopathogenic bacteria and 9 phloem-restricted prokaryotes Immunodetectionof Commercial plant-invading fungi 10 potential 15 Immunocytochemistry 15 17 Light microscope immunocytochemistry Whole cell techniques 17 Sectioning techniques 18 Visualizing Future the 19 antigen developments 21 Immuno-electron microscopy Post-embedding Other 21 labelling 21 23 techniques Conclusions 23 Key-words: antibodies, immunochemistry, immunocytochemistry, immunodiagnostics. INTRODUCTION Antibodies are useful for the extracellular constituents of detection, plants measurement and of their and purification pathogens. 1 Their of intracellularand high sensitivity coupled 2 M. DEWEY with potential for high specificity, a in whole given antigen Antibodies useful in are cellular constituents other changes growing disease where niques be cell Antibodies of or is also plant pathology and to tech- immunological outline to We do problems. and and techniques seek not to an reviews general texts, be useful in research in to and provide plant pathology. AND method for specific very They also be used can not binding the identification and specific inhibitors, as surprising also on and consideration of the antibodies in basis for a are and molecular biology. biochemistry a provides that antibodies in Table 1 and include work deal with use problems likely of use and have found we biology Antibody-antigen presented will of variety hydrates, growth regulators, signal receptors produce localize key because frequently results in steric effects which inhibit the activity of the tool in both powerful scientists BIOCHEMISTRY constituents. It is therefore antigens. discussion a tool for identificationof specific where the areas plant a techniques molecular sensitive a antigen to an those to with PLANT plant of interest. cation are and to of material. BIOLOGY provide measurement and biology amount plant quantify developmental to Their usage in powerful a of extracts of the literaturebut instead have included those IN MOLECULAR cation of identify dealing biology, ANTIBODIES antigen to invaluable aid which illustrate the biochemistry, binding seek we an coverage papers proteins, etc.), gene products. detection provides available for approaches exhaustive identify specific antigen review, can structural purified cell and molecular plant biochemistry, (enzymes, to indicate the presence and to and in crude and early stage. at an In this and and be used can cells, tissues, ETAL. plant to be a useful of their appli- proteins, carbo- other cell constituents. Further strategies biochemistry be encountered in their Examples enzymes, structural many affinity purifi- proving and which can molecular be employed to and the biology will biology, use. Preparation of antigens In general, be a the cation of yields) a antigen for point for permit mixed An (although will be for workers in antibody possible and (providing alternative preparation Where the from approach an this does homology the problems less likely to possible of antigens of contaminant be present in use of purification antigens antibodiesraised between antigens, plant from alternative the such that a alternative source, perhaps species as fractions possible so of that a purified to prohibitively also a purify higher possible are small starting starting available antibodies from a to a plant tissue is its from another possible, kingdom. it eliminates many of mammalian contaminants much higher yields litre antibodies may mixed from tissue makes it species or are antigen from (for instance) from another against result in monospecific antibody is obtained. cells. In many instances, sources, not screening procedures it is also interest; careful to and molecular number of different criteria) is the best production. Antigen-enriched polyclonal antibody preparation purifying a monoclonal antibodies if subsequent selection of antibodies of Similarly, plant biochemistry necessarily completely pure) antigen. Frequently, purifi- not antigen (determined using pure point are starting point well defined can well be useful in of are antigen be obtained. screening plant antigen preparation. and ANTIBODIES IN PLANT SCIENCE Native The protein, synthetic peptides techniques of plant biology immunogen in addition to protein!) available, peptide sequencing tion of are These will antigen. which produced be a but proteins. the of plant proteins expressed ability expression libraries, generally and Sambrook identifying al. et 1989) is expressed protein are available; the and the plant it will as tertiary will antigen use of the required Monoclonal versus A good other of native to methods, forms and ease production as of amount production and a new which do are not be obtained, pure as a to several: serum animals vary in permitting study selective of and/or be to are the be to may prove advantages over extracted/purified polyclonal antibody and quite large (20-50 ml) litre. The goats) yet of further production well result in amounts the available can but few laboratories have the and molecular of of of antibodies is antibody may to just biology require polyclonal possible be obtained one epitope screening protocol to cost one if that ofpurity techniques Affinity chromatography time in epitope epitope It may also lead be used. can and to a may is over Their many years of the antigen Monoclonal antibodies have production, the lead to lack of fact that a given apparent specificity altered, for instance by fixation a can need not be litre apparently lower than for recognize and signifi- a antisera. structure/function relationships; immunogen in addition criteria equivalent antibodies directed antigen recognition variety many ideal for are Affinity purification than resources properties; Purification ofantigens, wide likely expressed protein. two in many available is limited and plant biochemistry in antiserum which contains antibodies that nature. the last antigen Rabbits (e.g. sheep, large quantities eletrophoretic procedures. A plant protein but this is specific high-titre preparation. however, loss of (e.g. bacterially small. However, unlike monoclonal antibodies, the antibody preparation only recognizes or of of native series into another rabbit and may injection investment highly drawbacks of available is serum handle such animals. to very greater advantages handling. of relatively are Monoclonal antibodies for cantly and quantity by using larger a use may differ from that in the affords frequently litre, recognition of antibody-containing needed yield can of of antibodies with differentproperties be increased resources terms preparation the total antibody requires The elsewhere impossible. proves antiserum polyclonal least in immunization doses total plant protein of such preparation and authenticate the using; a permits Drawbacks of the method expressed protein produce of interest polyclonal monospecific at for amounts as are obtained from cDNA are require further purification excess. used which is present in presented are produc- synthetic peptides antigen These small only sufficient then be can not one the protein Given sufficient resources, all three methods are worth successful ifthe and encoding the be present in structure and effort cost that ensure be further considered here. not useful alternative when a that the possibilities will which coli; methodologies clones that express and to in bacterial systems. in Escherichia protein produced straightforward more of the obtaining (and sufficient informationfor the yield cDNA identify to for routes cells. If facilities plant synthetic peptides unique region of libraries will alternative provided from is needed care variety use of amounts pure, represent The have purification directly comparatively large in bacterial systems? plant proteins expressed or molecular 3 number of sites in the or in polyclonal antigen. hapten conjugation are available is particularly to purify useful as antigens, a depending method for rapid on their and efficient 4 M, Table 1. Some of uses of antibodies in examples Antigen plant biochemistry/molecular biology Antibody Plant growth substances (indole- Reference type Monoclonaland 3-acetic acid, abscisic acid, DEWEY ETAL. See Weiler et al. 1986 for references Polyclonal cytokinins, gibberellins) Cell wall polysaccharides Isocitrate Polyclonal Northcote et al. 1989 lyase Polyclonal Martin & Northcote 1982 Endopolygalacturonase Polyclonal Grierson Auxin-bindingprotein Monoclonaland Napier et et al. 1985 al. 1988 Polyclonal Phytochrome Polyclonal Ldbler & Klambt 1985 Monoclonal and See refs in: Thomas et al. 1986 Polyclonal Plasma membrane H + Plasma Monoclonal with other purification (possibly coupled yield relatively preparative step antigen acts to antigen pure for the from polacrylamide present gels are which and are keyhole the experience prior on the nature permit the to to of the stances. to that may Weiler have been added antigen procedures are such as of the hapten; to be prior to Bodanszky & el al. which conjugated bovine linked is a be used and to a presented cut acrylamide relatively pure techniques large, with to use. highly more known are as strongly antigenic as haptens albumin, ovalbumin, used for hapten conju—NH as 2 , —COOH, protein (Bauminger If the Bodanszky 1984; Tijssen 1985). may high to introduce a and hapten functional have been used in plant number of the techniques plant growth used are sub- described (1986). (PBS) preferably be immunized using antigen and in the absence of toxins should be avoided if weakly immunogenic are to a serum to the and are not (e.g. during purification. Adjuvants (compounds response) as injection; here, functional groups such covalently a useful final a major chromatographic thyroglobulin. The techniques hapten animals should buffered saline strong immune the 5,000), they plant growth substances involved Experimental also will indicate the best raise antisera and monoclonal antibodies extensively by phosphate any of the permit conjugation. Hapten conjugation techniques The usually produces after initial purification; bands determine purity investigator lacks such groups then derivatization procedures science it as al. 1989 experimental animal) purified by large proteins, and Wilchek 1980; Kabakoff 1980; group and et immunization. Such small molecules to limpet haemocyanin —SH al. 1988; electrophoresis is antigens small molecules (< to generally conjugated or may be rapidly degraded, requires molecule gation depend —OH of peptide which may also be used are preparative steps) short time. Gel a et frequently strongly antigenic (especially antigens Production of antibodies antigenic Briars Evans stimulate the immunesystem of the peptide antigens, or in preparation is present. Other techniques, and Polyclonal transporter of Oleski & Bennett 1987 Polyclonal transporter 2+ membrane Ca or in small in Harlow & Lane at all possible quantities. (1988). sodium dialysed azide), intended but may be to into which provoke necessary a if Full details of immunization ANTIBODIES IN Fig. The I. Flow chart of amounts and the antigen PLANT SCIENCE of a amounts induce an production to be raised. Our intervals of 0-1-1 0 mg monthly membrane calcium total in amounts pump at into mice primary as a to screening (see as guide and will need antigen evoke an further a contaminant antibodies. 1% of the has into rabbits 2-week intervals of 6-60 pg gave are, the on experience plant protein a weak figures and be amended possible depending own however, from immunization depending seldom effects of any antigen response the on required. contaminants; would be injected in a immune response. Immunization schedules should be pre-immune below and injection as to injection per contaminant present carefully, by taking obtained a of 1 mg of antigen will also increase the sufficient boost, monitored excess of antibodies. immune response vary are while about three injections be taken only for instance 10 pg of is to antibodies in the viable monoclonal antibodies could be raised. These antigen; 1 mg at good antibody litre, schedules should Larger which injections purified plant plasma which outlining the major steps antigen required animal in indicated that three produced 5 is Fig. 1). likely The to bleed and trial bleeds injection increase the and carrying should stop when a high out litre likelihood of the presence of 6 M. DEWEY Table 2. Uses of antibodies in plant biochemistry Method Label Immunosorbent assays and molecular biology second antibody on Suitable for measurement? ELISA (enzyme conjugates) RIA (radiolabelled antibody Yes by comparison Yes or preferably antigen) blotting/Western (competition assay very specific) conjugate Fluorescent Dot ETAL. Yes Enzyme conjugate Yes Gold conjugate/silver Yes Biotin/avidin/streptavidin Yes by comparison blotting Chemiluminescence Yes Radio-isotope Yes Radiolabelledantigen Immunoprecipitation Yes or secondary antibody Gel Table 3. Yes electrophoresis Applications of antibody/antigen detection systems in plant biochemistry/molecular biology Technique Application ELISA Primary screening of antibodies. Measurement of antigens; usual to perform ELISA against dilution series of antigen; comparison between RIA Western particularly useful where Measurement of dilutionseries with standard comparison between Measurement of blotting assay comparison Screening expression lift/blotting that available Primary screening of antibodies. blotting Filter antigen note always valid Measurementof antigens; competition radiolabelledpure Dot assays not assays not always of antigens by set. Note that valid libraries Measurement difficult antigens. (requires scanning densitometer; values obtained not absolute and vary between experiments). Gives absolute identification of antigen Measurementof Immunoprecipitation or second antigens. Preferably requires antibody. Use either labelled of gel electrophoresis to antigen identify antigen advisable Screening antibodies Techniques for Johnstone and techniques screening antibodies Thorpe 1982; available immuno-sorbent are assay) is are detailed in Harlow and Lane summarized probably in Tables 2 most a number of 1988) useful as and will and a 3. laboratory not The be manuals presented ELISA primary screening (e.g. here. The (enzyme-linked method as most ANTIBODIES IN PLANT SCIENCE antigens be can supernatants one coated however, assay so results. Plates verify the onto be screened can wells rapidly. absolute immunize the animals and the in which blotting’, nitrocellulose (or After does test used in commonly and the (1979), electric given are incubated with mary antibody a bound antibody specific the to phosphatase reaction product, or because Western a for preparation the of diffusion double into use agar an this assay antibody-antigen followed by analysis of for which antigen prior to is the the is is onto supernatants ‘Western’ which be (or similar) by appliTowbin sites some to antigens antibody fails antibody quantity are used, technique 1968), antigen and has is affinity silver intensifi- of secondary the can is Other to the In into placed site. give or In the anti- required; are any small too antigenic suggested. is recognize to antigen but if this fails antibody. a clear Ouchterlony circular techniques wells that cut yield followed by SDS-PAGE of the pre- preparation which bind insoluble coloured using studying antibody specificity containing pri- secondary (commonly enzyme an identified has masked immunoprecipitation complex, an a indicate the location of the cases, non-denaturing gels the of radio-iodinationof antibodiesand use be because the for al. et on the presence of the paper in produce subsequently is albuminand the paper serum to blotting SDS PAGE separated by paper either conjugated labelling, In may central well cipitated bovine or also available (Ouchterlony a antibody unbound antibody, peroxidase), can are procedures additional information induce material(s) powder immunoprecipitation surrounding to screen, dotted antigen technique, developed by electrophoresis/transfer may be retained if genicity to used as rapid primary to antibody. nitrocellulose to washing which systems. Blot; latter case, alternative result after which which techniques, of the antigen is indicated by incubating primary chemi-luminescent assay on be included must Thorpe 1982). Non-specific antibody binding solution of milk gold, to a monoclonal or details of this horse-radish or as binding procedure; antigen transferred (full include biotin/streptavidin antibodies, material are the to by its specificity for this primary antibody; alkaline cation. Other gel current using or within only reproducible antigen preparation which is also used titre antisera high in Johnstone & blocked are paper an same of antisera variety a is difficultand standards is assayed demonstrate to laboratory of the proteins cation of our are paper. screening, be screened further must and differentiateantibodies to contaminantsfrom not ELISA, the antibody similar) primary plates coated with the those of interest. An alternative to ‘dot of ELISA The results obtained comparison generally are 7 of an antibody affinity both demonstrate the without involving column nature of the denaturation of the antibody-antigen binding. Purification of antibodies Both monoclonal supernatants and use. the polyclonal Purification methods may be divided into serum/medium, HPLC, gel body class (see of the Harlow & Lane 1988 for details); and are purification given in Johnstone & immunoprecipitation (see above) and the A of A, Protein depending one antibody (1981) antibody is immunoblotted against antigen and the portion of nitrocellulose ing the buffers which disrupt the cut out. Antibody antibody-antigen is then washed from in which contain- off the nitrocellulose interaction. While G, anti- Alternative methods of Olmsted the antibody/antigen on thorough consideration Thorpe (1982). technique of antibodies from Protein all used serum/medium by affinity chromatography. before further purified purification precipitation, ion-exchange chromatography methods available is include areas; for which ammonium sulphate filtration and others within the antisera may be two only yielding very using small M. DEWEY 8 Fig. 2. Flow chart for the steps taken in the purification of antibodies. See section on amounts of Purification Uses The in molecular basis: the need to same and is coated centre where antigen of antibody; antibody on and and effective and has been used rapid biology is successfully biochemistry and molecular quantify antigens. The around three basic methods; ELISA, or identify ELISA applied and to wells and identified or 3 used essentially the broadly are the similar techniques, where antigen subsequently quantified; immunoblotting, where quantified. governed by indicate the have biology techniques similar and its indicated paper presence or immunoprecipitation circumstances and will be and plate nitrocellulose subsequently used. Tables 2 and biochemistry and plastic onto is procedure of antibodies in both same an this laboratory (S. Montgomery, personal communication). of antibodies uses technique of Olmsted (1981). of Antibodies p. 7. antibody, in the authors’ ‘Purification ETAL. the the antigen is by use precipitated using an The selection of success of the initial the basic methodologies techniques depends screening procedures applicable to variety a of problems. A number of these preparation techniques of antibodies to ion pumps and enzymes. calcium to pump, a In several plant actin, and antigenic homology between enced. This was have been number of used to a were Raising antibodies cation required protocols to advantage cases plant-coated to actin for actin did in the authors’ laboratories in the proteins, including (antibodies when in an antibody general our for immunization and plant cell the to vesicles) plant structural plasma problems proved not exist. to a be to a mammalianprotein experience low litre Use of a very were was from experi- used for indicated that larger doses of antibody production particularly proteins, membrane resulting plant protein and native mammalian proteins subsequent experiments. However, antigen employed plant difficult because resulted. good purifi- simple procedure resulting in an ANTIBODIES IN PLANT SCIENCE followed preparation, impure polyacrylamide gel detection of the enzyme serine: aration was by Olmsted bound problem. Talian et This gave al. 1986). This excellent an to permit an (1983) for preparation of the desired was clathrin, they column affinity prepared against clathrin as it case Immunogdiagnostic The also useful required was diagnostics field. provide Immunoassays detection of disease. an important role in needed for the methods of same as which early detect are unique detection and the large for in to the ‘visualizing pathogen, as samples review of to where screening plant in pathogens easy correct fungal visual diseases. be either upon actin and plant In the protein. horticulture and from the adapted relatively inexpensive. symptoms and permit early pathogens Immunoassays on have These on or the detector or known plates. Such assayed ELISAs, assays are see to cover are easy are upon antigen, organism, or the are antibody enzyme simultaneously. They all of the or as an are assays techniques within the target played based are page 18 of this review. The tagging usually assays, will be made in this section of the review highlight been and complexes. can recent plant of each agriculture, have identificationof antigen’ depend be pure PATHOGENS replicate to confirming plant immunoassay techniques Instead this review will PLANT the some not are used for immunizationand was fluorescein (immunofluorescence), conjugate numbers of mass FOR specific antigen-antibody to 1986). method of choice a of viral and bacterial diseases. Similar spread Current methods extract. general antibody preparation of monospecific antibodies. sensitive, detection of many Enzyme-antibody sively in which Monoclonal antibodies key epitopes to impure antigen as for micro-titre wells linked together in strips enable prepof the purification antigen preparations membrane calcium pump, fast method of fluorescent molecule such ticle. They limiting visualizing be to a but increasingly important used Early those referred must soluble the a for the methods described and is prepared successfully. ASSAYS are assays techniques be to raise antibodies specific IMMUNODIAGNOSTIC medical by (1984), immunocytochemistry plant plasma to were selection after screening forestry. achieved was and from prepared were here, although produced and Smith & Fisher when antiserum of sufficient titre is available, were Blue stained band antibodies Polyclonal nitrocellulose after Western Blotting is eluted and used (Montgomery to enough Coomassie a glyoxylate aminotransferase; required (Montgomery (1981), of pure, contaminantantibodies were relatively antiserum was excision by this overcame 9 or a with gold performed now all the aspects advances and review of (1986). in handle and to used exten- steps have been automated. For Barbara & Clark a par- a No attempt plant immunoassays. plant-fungal diagnostics in greater depth. Immunodetection ofplant Over the years viruses. They (ISEM), a number of immunological (1984). There is assay systems that For lose immunosorbent enzyme-linked dot-blot or can practical guides an dip-stick increasing a assays the by to immunosorption-electron (ELISAs) subject towards and consult dot-blot Cooper development of detect dip-stick or & Edwards quick ‘user the farmer or food processor, such plant microscopy as (1986) friendly’ dip-stick, assays. and dot-blot e.g. on move be used ‘on site’ agglutination surface, methods have been used include immunofluorescence (IMF), assays. For reviews and and Hill viruses flexible assays plastic. the is pre-coated capture antibody When sap is squeezed onto onto a nitrocellu- the coated surface the viral 10 M. if present, particles, in between. If colourless. It is series. For the positive that important of such example an detected are the substrate and the antibody-enzyme conjugate, washings and trapped are dip-sticks and positive an incubation by stopping controls negative assay system if are Mitchell et al. see with the successively with solution, coloured, appear DEWEY ETAL. appropriate remain negative they included in each who (1988) test developed a system, initially for the detection of potato viruses in leaves of standing crops. The majority of viral diagnostic antibodies. In viral antibodies is able. In necessarily of the viral same only recognizes against missing one antisera useful in nate a such leafroll disease the as (Gugerli al. et polyclonal studies, and ingenious to developed on/in Vuurde (ISDP) an have in which bacterial as well by incubating form. Colonies cence. can Several outer pathogens extracted and to than that whole phloem-restricted using raise antisera specific pathogens, Preparation impossible. but also of the to plant not easy. pathogenic incu- bacteria. Van technique Petri dishes and those that remain or are LPS been strains of used level of immunogen (1986) are easily of LPS is purposes (Lyons more and & for Taylor against 1989). can be grown in culture. Bove spiroplasmas is serogroups. It very only present in the is with antisera raised mycoplasma-like organisms, partially they and spiroplasmas, mycoplasma (MLO) number of intovarious syringae etal. spirolplasmas a diagnostic specificity pathogens: for for (Wyatt the organisms rehydration. (LPS) present highly antigenic Pseudomonas of bacteria specific is to immunofluores- detected and, if desired, the stained colonies after against purified preparations has high are staining by A have been extracts are dilution-plating by washing (BLO), only because Lin & Chen 1985). common. antibodies, antibody-coated onto endocellular antisera detect and differentiate the to of lipopolysaccharide bacteria. cells and cultivar-specific the to Antiserum raised polysaccharide (EPS) and others trapped fluorescently raised antisera and bacteria-like organisms (1984) different grapevine definitive signals molecules is of monoclonal down the agar and by drying Other workers have obtained a the extracellular Of the host and/or use removed are proved discrimi- cause cause & De Boer give strong to immunosorbent an are Gram-negative purified. differentiation of 1990). low that only viable bacteria ensures of but that not trapped bacteria with selective media and allowing colonies be re-isolated from the membranes specific the workers have monoclonal phloem-restricted prokaryotes are too the as developed detected are This method bacteria IV) (Halk problems. Commonly aliquots inoculation rods. Unbound bacteria detected and enrichment medium to increase the numbers of (1987) the viruses have physical identity same contaminant bacteria methods, circumvent these al. et to antibody safeguard assays by desir- different other prokaryotes, directly from infected plant tissue is with cross-reactivity number of bated or the numbers of bacteria present Frequently monoclonal antibody detected luteoviruses and others 1990), a rarely recognizing where antisera do (GLRaV I,II,III Immunodetectionof phytopathogenic bacteria and Detection of bacteria, many antibodies higher signal than and taxonomic closteroviruses rather than monoclonal be achieved with monoclonal monoclonal antibodies between different strains that have the symptoms, can target epitope one However, aetiological that Detection of individual strains is Furthermore, strains where the new employ antisera containing protein give coat epitope. antibodies may have changed. particularly assays still high specificity advantage. an general, monospecific epitopes that not the diagnostics in have been able to difficult, however, because they are obligate small numbers in infected plants. without contamination from host molecules is almost and Clark et al. (1989) have found monoclonal antibodies ANTIBODIES IN be to PLANT SCIENCE useful in particularly 11 Aster Yellows and differentiating Primula Yellows MLOs respectively. Immunodetection of The development plants are plant-invading fungi of When tested specific. extracts from appear to be tions of antigens when tested non-specific antigens material and fungal producing culture or by fungi diluting are may be useful in non-specific out rarely satisfactory, competition and others present in both the insoluble and soluble frac- (Dewey al. et antibodies but there is assays filtrates, extracts, but or by immunodiffusion.Chard (1985a,b) are have shown we antisera that against mycelial fragments, of solid cultures washings in diseased fungi 1990) the that converse: present in both the soluble and insoluble fractions. Attempts are specificity related surface difficulty antisera raised IMF, or in with both related and unrelated fungi and host tissues species-specific have shown that due to the partly ELISA by lyophilized mycelia, widely cross-react for the detection of immunodiagnostic techniques has been slow. This has been al. et with evidence that fungal antisera increasing (Kitagawa antisera cross-absorbing or specific improve to N. S. 1989; Lyons al. et personal communication). A number of workers have shown that reasonablelevels ofspecificity may be obtained by of culture filtrates using protein precipitates Examples include antisera raised in Phomopsis longicolla tissues root (Gerik Antisera raised et al. 1987) against carbohydrates, generally raised antisera to antisera are have ing far, relatively results with et al. aphanidermatum and specificity. not and factors. the of Many antibodies P. megasperma species on plants or fungus fungus, immunfluorescence, al. 1989). soluble (< 1987) have that such food stocks raw are the against fungi. Disappoint- animal house facilities monoclonal antibodies been raised for diagnostic purposes. However, var. al. et raised against Phytophthora glycinea (Wycoff 1989) clearly some, have et al. such have raised as the cinnamomi 1987), and Pythium diagnostic potential. It is aphanidermatum the basis of zoospores. Specific assays for the detection of soil would be more Wright et useful. al. (1987) have successfully (dot-blot) raised and used assays for the detection of Sirococcus strobilinus and sporocarps of the vesicular-arbuscular Glomus occultum. wilt et or 1988); cotton (Mohan and showed the monoclonal antibodies for P. cinnamomi and P. in infected banana fungi determine if to & Pitt toxins enzymes, Notermans need for monoclonal antibodies in ELISA and membrane fungus as from several be used can costs Mitchell & Sutherland (1986) and the seedborne in strawberries such immunogen. processing. to (Estradia-Garcia differentiate mycelia discouraging 1986), unfortunate that only prior monoclonal species-specific of high degree fungal antisera, against fungi have (Hardham a carbohydrates the as (Barker Verticillium dahliaein 1987); few monoclonal antibodieshave been raised been undoubtedly al. et Phytophthora fragariae genus-specific contaminated with moulds So and (Gleason extracts anemone corms specific fungal fractions, heat-stable almost seeds soybean mycelial or Colletotrichumin to Wong Fusarium et al. (1988) have raised oxysporum the thick-walled f. sp. a monoclonal cubense chlamydospores that will to the differentiate, by antibody of strain 4 from those of strains 1 and 2. Our own experiences species of causes Dutch Elm are fungi involved in raising have been varied. in monoclonal antibodies Development disease, Ophiostoma post-harvest spoilage of ulmi (Dewey of rice that diagnostic et grains al. and are specific assays for the 1989a) to various pathogen and three fungi, copra, Humicola that which lanuginosa 12 M. (Dewey et al. Pencillium islandicum 1989b), (F.M. Dewey unpublished) have had for level, 1988). difficulty the The sponse. The site and by our but immunogen that the non-specific, adjuvant the as antigen and IgG, IgG 2a ti tre wells coated with whereas species-specific with readings example between distinguished raised we were the tested healthy We extracts have and we fragments, to specific response. with and by found species- were IgGs belonging were ELISA to mostly IgM micro- against correlation no was infected from non-infected tissue whereas tissue healthy to clearly another give higher Another monoclonal anti- Ceratocystis vitro but in not all mycelial we monoclonal antibodies extracts. It did 1989a), and three were healthy produced plants. secrete raise soluble a gave low absorwhen high readings with cross-react washings or from extracts P. islandicum, H. This high were and fungi lanuginosa methodof simple of the directly the as the addition of Freund’s adjuvant only and A. flavus antigen preparation fusions one or two monoclonalantibodies with the monoclonal extracts All these response points freezing or to antisera titres to and specificity suitable for the with 1 in 200,000 for H. antibodies specific etal. and were were to (Dewey was very needed needed to to the by carbohydrate were detection of the surface fractions or has been weak and hyphal fragments against lanuginosa. Higher pathogen 1990). washings ELISA including to develop only surface washings Eyespot washings, mycelial (F.M. Dewey tried with and without Freund’s these different immunogens for antisera tested (Dewey antibody protein immunogens from mice immunized with surface used diseased have used several differentimmunogens unpublished). pared of assays. trying pathogen end and cross-reacted cell-free surface unpublished). cases cell lines that diagnostic general source ulmi using al. 30w/v), distinguish to et antibodies in (1 species-specific against antigens without concentration effective. In both In specific a Ophiostoma tested were of both Ophiostoma used successfully 1989b/1990 identify about the best different workers and the by species-specific diseased than with from diseased to genus-specific ability of the from antibody species raise monclonal antibodies al. hyphae by gently washing tissue. immunogen et one extracts when tested against walls of the stimulating genus-specific diseased tissue and the extracts monoclonal these that recognized body bance values from extracts fungal specificity material. For plant in immunogen (Dewey antibodies. When all the monoclonal antibodies found between no consensus re- generally not antigens recognized cross be removed can schedules used about one-third were The monoclonalantibodies that sub-class is attained. with Freund’s most were specific. comparison of the various a and the presence proteins that the (Fig. 5) adjuvant of monoclonalantibodies that panel homogenates same the effectiveness of Freund’s specificity Of the of this solid slant culture with PBS. There is a or See Table 4 for levels of some we the genus non-T cell stimulated a present in the walls and are at herpotrichoides (Dewey sub-species-specific antigens of P. islandicum we have shown case the spores and that the surface of and species- monoclonal antibody specific not of nature In contrast, even immunogenicity of the different that induce glycoproteins or Aspergillus flavus quick. specific, are Pseudocerosporella be related to the to and 1990) forward and reflects both the levels of soluble carbohydrates known but in the of cereals pathogen probably turn of non-specific al. et relatively straight of difficulty appears degree (Dewey monoclonal antibodies that raising Eyespot which in fungi, in has been DEWEY ETAL. non-specific. had low were 1988, adjuvant. The Antisera titres; dilution 1 in 50,000 com- titres and cell lines secreting monoclonal obtainedwhen a specific protein fraction was ANTIBODIES IN PLANT SCIENCE 13 (1990)1990) or species ( Ref rence (1983) al. (1984) (1986) a/. al. al. al. et et et Sutherland (1987) (1987) (1988) al. al. etal . et & & al. al. et llaanneleicl/ai. Banowetz Hardham Mitchel Wrighhtt WWyycocfoeftu/. et et 1986) (1989b) (1990) &HaHahhnnee Estrad-Grcai Dewey Dewey & Wong al. al. (1988) (1989a) Fox Fox al. al. et et Dewey et other al. al. etal. against et et et tested not mAb class NG NG — —— — — — IgM NG IgM NG IgM IgG IgG IgM IgG IgA IgM ND ND = IgG IgM IgM IgG IgM IgM IgG IgG IgM IgG IgM NSG & & Specifcity Genus NSG SpSpecies NSG NSG NS NS genus RaceNSG Race- blot Scre ni ng method Route ELISA-S ELISA-S ELISA-S IMF-zo sp.ELISA-sol.Ag ELISA-S ELISA-sol.Ag western Bo ster i.p. i.p. Early i.p. F i.v. i.v. i.p. i.p. + against raised Injection + 7 mg 7 7 3mg 10 per 3 10 2xl2x0 mg 1 1 i.p. NG NG NG Intrasplenic i.p. i.p. i.p. — ELISA-h.f IMF i.p. i.p. + ± ± ELISA-sol.Ag ELISA-SW ELISA-SW IMF-zo sp. i.v. i.v. i.p. i.p. + + 7 mg mg 10 50,50,0000 015015 protein 8xl8x0 pg 40 NG NG mg mg 3 3 Spores Spore extracts Zoosp. Hyphal fragments Spores Culture filtrate Spores Conida hyphal fragments Hyphal fragmentsHyphpahall fragments i.p. i.p. i.p. i.p. i.p. i.p. — — surface = fragments. SW det rmined, hyphal = h.f. protein pprrotein pg pg SW SW not = ND zo spores, = given, zo sp. not = NG antigens, 7 10 10 x 1 1 Zoosp. 2. with 7 antibodies soluble intravenous, cros-eacts = = i.v. intrapeional, Ag sol. species, genera, other related 18 against from i.p. = oxysporum Monclona 4. Table washings, & i.p. i.p. i.p. i.v. 150 500 Dose Nature i.p. i.p. i.p. i.p. i.p. — fungi plant-ivading Im unogen i.v. i.v. i.p. i.v. i.v. specif c N.S.G. SSppecies ggenusenus Genus* Species Zo spores Cyst;: NSNS Species Fungus Fusarium cinamo i strobilnus lyucopersic sp. f. f. Tiletia meqasperma oxysporum lanuginosa islandicum mel ea oc ult m glycinea cubense Phytophtora Siroc us Glomus Pythophtora Fusarium Armilari Ophiostmoa Humicola Peniclum Pythium ululmmii sp. sp. f. f. adjuvent, tested species not = Freunds = F NS against genera, �Tested 14 M. DEWEY ETAL Most of the monoclonal exception micro-titre wells infected of a plants followed secondary antibody-enzyme We have found, as did Gleason islandicum in rice usual. This method has enabled if or only a friendly’ dip-stick detection of within 5 is fungus proved grains (Dewey et few individual assay that has of harvest. The allowed to coat grains re-wetted incubated with The gold conjugates. in lanuginosa are The wells at and lanuginosa soaked, individually, then are processed as low level of infection in all a Paddy heaps developed a ‘user soaked in situ in the Philippines, membrane Tmmobilon P’ a new in PBS, in individually, dip-sticks overnight. amplified by was Grains made of were The followed hybridoma supernatants signal antibody. that these assays enable detection to infected. We have also are use the surface of the infected near day. of from the successful under field conditions. It has enabled dip-sticks the surface of the the reporter as is sufficient 1989b/1990). (Millipore, UK). Fungal diffusates from grains were on or next coating extracts invaluable in the detection of H. al. proved of H. significant growth days present in gold conjugate present are, with the monoclonal antibody and the determine if there is to us fungi involve direct (1987) using polyclonal antisera, in micro-titre wells and removed the overnight grains or They antigens specific passive release, by overnight soaking, low levels. This method has very al. et well where the particularly tissue and where P. incubationwith the by for developed assays all indirect assays. membranes with the mixture of or commercial work antibody diagnostic of the few commercial assays, exposure to a dip-sticks by Eppendorfs were then commercial commercial silver dried, immuno- enhancing solution. The P and specific fungal antigens to that we have worked with bind micro-titre wells. We have also found that the strongly their respective antibodies. More than 95% of the total monoclonal the is bound within 30 antigen have shown that in the case glycoproteins (Dewey el Only few double a detection The s. most sandwich are Milleret al. monoclonal antibodies and both Immobilon specific antigens is that the lanuginosa bind rapidly known but not to that binds antibody specific antigens to we are 1989b/1990). antibody by of of P. islandicum and H. al. purposes. Most of these of turf grass diseases nature to specific antigens assays (DAS-ELISA) have been commercial and have been (1988, 1990) polyclonal for developed developed for for detection Agri-Diagnostics. They employ both antisera. Commercialpotential The use of antibodies poses, is was now estimated (Klausner to to 1987). viruses but assays detect to be £3 million in At the important detect moment advances spiroplasmas, these assays will necessitate the specificity needed. Use of development plant pathogens, for both research and commercial well established. The commercial value of of 1985 and is this are likely mainly to be made in to use be about £8.5 million in the production of bacteria and of monoclonalantibodies inorder growers and immunoassays by user friendly interpretation of the assay results pur- assays 1995 concerned with the detection of mycoplasma-like organisms, low-cost, on-site, methods of sampling and projected market is plant immunodiagnostic importers kits such to asses as to commercial fungi. Most of attain the level of will depend dip-stick on the assays. Good the levels of infection, will be critical. IMMUNOCYTOCHEMISTRY Over the last decade the tissues has emerged as use one of antibodies of the most as probes for macromolecules within cells and powerful techniques in cell biology. There are, ANTIBODIES IN therefore, many PLANT SCIENCE excellent reviews and cols that have been Varndell 1984). The be said that the light and electron probes by the has finally numerous levels. Most of the 1982, a of problems of proto- However, it routinely at of inaccessibility plant tissues, cells has plant to biology. be used can and 1985, 1989; Polak & techniques in animal cell nature techniques 1983, have been can both the antibody overcome variety of tissue preparative techniques. our intention to review some of the the initialfixation and preparation of the that have been methodologies of permit successful immunocytochemical labelling to on the of age and come due to the cell wall and the development of emphasis rapid developments technique In this section it is developed outlining application of immunocytochemical microscope antigens, to texts Bullock & Petrusz, developed (See somewhat lagged behind the now 15 plant antigens with cell/tissues prior to light an or electron microscope immunocytochemistry. Light microscope immunocytochemistry When preparing tissues plant or be taken into consideration. cells for First, is the denatured by fixation and the other steps in the preparative affect the adversely recognizing in prepared probe a a manner the to whole cells, probe to cross-reactivity single epitope in antigen made examine which permeable, or permit gaining are in of both the a has most to are or be selected. tissues have as to whether the employed. Finally, At the popular. However, the be to and the attached be taken to be to often can monoclonal antibody light microscope use of colloidal gold in combinationwith silver enhancement. At the popularity gold a antibody decision has sectioning techniques level colloidal microscope if to be localized is to procedures. This is the fact that the cells access In consequence, still the antigen antibody, especially Second, primary antibody binding LM marker is electron will question. (LM) level fluorescent probes as an with the is used. three main factors have immunocytochemistry the likelihood that has become the marker of choice (Hyatt 1990). However, enzyme-linked secondary antibodies can still be useful with sectioned material. Wholecell to techniques. The cell wall and the the entry of antibodies and associated the specimen has approach may be proved sectionpd or plasma the cell made three-dimensionalorganization of which can permeable in the popular, especially very membraneform the probes into the cell. To staining protocols involving permeabilization (Lloyd el al. 1985; Goodbody pension culture cells was enzymes technique used to was soon if washed or to al. to et such al. work 1980). digested was with et coverslips prior to staining. al. 1981). The 1980; Wick etal. former, 1981; the to mild of the wall. shown that cellulase and the to of this steps use of sus- with However, this roots, under controlled poly-L-lysine technique in Roberts formaldehyde-fixed pectinase the treatment original morphology, major disadvantage on of the various restricted was In the These cells adhered release of cells from the tissue all information plant cytoskeleton, rigours cellulose and the matrix tissues when it could be et al. release whole cells which retained their ‘square protoplasts’ (Wick or 1989). Initially loosen the extended adequately, conditions et protoplasts (Lloyd study these barriers, the antibodies. This latter to of the withstand the barriers major two overcome the so-called coated slides is the fact that spatial organization on of cell types within the tissue is lost. Various approaches have been used cases fixation with practice to to make the paraformaldehyde (3-8%) further treat the cells with a mild plasma membrane permeable. will prove sufficient. detergent such a However, Triton X-100 or In many it is normal Nonidet P40 16 M. Air (0-5-001%). been used To (Wick al. plasts proved 1981; Goodbody popular. osmotically bursting PM to are PM left adhering the Palevitz 1989; Goodbody Sectioning techniques. tissue integrity These can be al. et Cryosections: either 2 pm. Sections by then stuck are a very simple fixation cryo-stub, Sakaguchi tions of shoot tips of system of the apical now common et Vinca whole cell either for the tissue In that al. report from major infusion of the roots was 10 to up and same pm incubated for at of 5 al. staining of pea Wax sections: has not been likely labels. to cryo- be restricted to 1 slides gelatin-subbed or a longitudinal, be able some in specimens to the drop of microtubule complete such produce up to a water on 6-8 pm cryosec- supporting matrix into good cryo- the tissue. It concentrationof 2 to prior M as to support a roots al. of slides prepared by Although a a a strip of were be cryo- (Fig. 3). a 1 -5 m laboratory water a of slides placed face film and and heated to the adhesion useful technique for adhering 3 demon- subsequent processing. Figure monoclonal antibody yeast tubulin in thick to the methods described above. to use paraffin in being dissolved 1985, Hawes 1988). Sections histological studies of animal wax-embedded material in molecular weight the PEG on be sectioning to poly-L-lysine coating markedly strengthened probably popular technique practice the root found further the surface of on then rinsed in ultra-pure slides for of microtubules with common et were treatment a of the tissues Cleaned multiwell slides poly-L-lysine was sucrose, the surface of the slide. the effectiveness of kD glass them up onto it 1990) m of the regions fragmentation this al. et in 2 markedly improved by picking the slide and this will to little very roots pea vacuolated highly manner. mg/ml 390,000 foundthat this embedding matrix, Lammeren to sucrose touching 1990) following cytochemistry. However, high an and least 2 h. The slides was cryosections cryosection antibodies and to for because ultramicrotome with they labelled m sucrose slides glass loop et problematical plant specimens strates the an treated the paraformaldehyde-fixed a wire report (Hush 70°C for 2 h. It of thick 1989), technique cryoprotectant giving better freezing and in thickness with held in drops onto by or freezing tissue with a with 10% gelatine in 1-5 also enhanced in the down al. et of cryo-sections of the authors’ laboratories (Hush one then transferred to were droplet In the is accessible poly-L-lysine however, characteristics of the tissue. This enabled sucrose again proved antigens (Cyr & satisfactory use section thickness is within which rare, both incubation of following Sections the obtained excellent (1988) perfuse to acts as face of the during sectioning (Hawes 1988). a recent sectioned has of ‘footprints’ clathrin (Hawes as and 1988). procedure dome. It is practice this then sectioning, or cytoplasmic technique most material is sections of plant material without the infusion of is such the Probably system onto immunostaining (Hawes to proteins conventional cryostat a with the latter attachment, although Using This 1980). of burst proto- 1989). is maintained and the cut have also min) coated slides patches associated with the al. et labelling of sectioned immunocytochemical the Valk use poly-L-lysine to microtubule-associated proteins and intermediatefilament microtubules, prior protoplasts for the examination of structural popular or (Van der membrane (PM) the distilled water, circular glass. Any antigens then be localized can or ETAL. 1989). plasma By sticking them with buffer 10°C for 6-8 (— treatments al. et associated with the study antigens has and cold methanol drying et DEWEY as plant polyethylene glycol (PEG) out thin of the section as prior 0-5 pm, with this to can tissues, it immunobe used as staining (Van technique can be ANTIBODIES IN PLANT SCIENCE Fig. 3. Immunofluorescent the meristem cells 1-5 of the root m well sucrose slides. Sidney. dry sucrose and on = a staining of 10% pm, glass solution after B bar (8-10 tun) cryosections of pea were were fixed in in orientation, whilst 3% stuck onto heat contained of Julia fish to vacuolate infiltrated with poly-L-lysine gelatine roots. Behind larger paraformaldehyde, activated 1% in coated reduce multi- background Hush, Biology Department, University of 60 pm. an ultramicrotome and transferred to a slide on a drop of 1988). advantage possible block tural localization of etched from the Roots oblique. 1990). Micrographs courtesy = knife with Resin sections: The same are in thick predominantly transverse primary antibody staining (Hawes lies in the fact that it is and that the are (w/v) gelatine. Cryosections Hush et al. 30 cortical microtubules microtubules the microtubules washes (see A bar cortical (B) All fluorescence cut (A) 17 can using cut resin-embedded material for sections of tissue that are be used for both thick and thin antigens. surface of to In of the most section cases to the resin expose the sectioning must be antigenic immunochemistry difficult to cryo-section for the ultrastruc- chemically sites prior removed to or staining 18 M. & (Perrot-Rechenmann sodium ethoxide of resins is family retained. Gubler Gadal suitable not (1989) With epoxy 1986). methoxide or (Hawes 1988). levels of satisfactory as reported on the resins In many successful this polymerized by long with Resin acetone. embedded sections is preparation Visualizing removal (Gubler may al. et however, cases, be not The antigen. each An should be taken preparative techniques air-dried protoplasts However, primary antibody if the is stained One background In our 1 % fish second antibody conjugated antibody and schedules have demonstratedin with carrots a be to localization of to a an be to a recent be can paper same antigen & Duniec by Goodbody was only for dependent dipped could set no developed optimized (Wick antigen concen- Therefore, monoclonal antibody be constructed for each to with be used can as with fluorescence, compounds pre-immune on et al. achieved in in methanol. be observed in the control as the control with as which should well polyclonal with the tissue not cross-react of tissues which should staining as substitution experiment. Commonly is used serum antibody, an gelatine greatly immunofluorescence is the latter due found in many in addition laboratories, reduce tissue to not antigen. major problem fluorescent a is immunostaining elegantly antibodies. With monoclonals contain the Lowicryl- approaches which had been treated with Triton X-100 and Suitable controls have being was without methanol treatment. preparations of the low-temperature blocking agents staining microtubule-like distribution of the a This resin tips. root with different antibodies. standard and as of fibrillar bundles in (1989). Staining not are LM immunochemical localizations. most buffer types, of how the successful example for between tissues and particular experiment 1986). the greatly vary procedure with necessary two-layer technique, using protocols used, including exact trations, of this immunofluorescent localization of A summary of these various fluorochrome, is still the method of choice The in the tissues antigenicity with out use of the section temperatureand dissolved out room 1987). the in Table 5. given the ultraviolet at be carried can microtubules in sections of buty-methyl methacrylate-embedded wave DEWEY ETAL. to the to a wide of blocking all solutions after incubationin the to the level of of variety such plant cells, use non-specific staining as general naturally occurring various agents and such and phenols as BSA, primary antibody auto- pigments. the addition of has been found to fluorescence (B. Satiat-Jeunemaitre personnal background communication). It should be remembered that other markers to visualize the second sectioned material can (Van arise in tissues with linked to the second The use binding and endosperm 1987). A own as more gold as With a al. 1981; the of Scadoxus to to at gold as fluorescent probes labelling techniques Bullock & Petrusz levels of have marker right, whereby available be used on 1982) although problems peroxidase activity the LM level is is seen as and other enzymes a increasing dark red stain in popularity. over It may sites of antibody great effect in the study of microtubules in the liquid (Haemanthus) 1981; Holgate (De sp. is et al. to Mey et amplify al. the 1982; Morejohn signal from the this technique reaction monitored microscopically and stopped et al. gold by 1983; Van den Bosch 1986). With this shell of silver is grown, under controlled conditions, around the recently developed reagents are can be used. satisfactory technique, however, silver enhancement (Danscher procedure may a such has been used cells et naturally high antibody of colloidal be used in its Driessche well as Peroxidase layer antibody. can be carried when the out gold particles. in the required density light of and the staining is ANTIBODIES IN PLANT SCIENCE 19 1986 1980 al. Ref rence 1980 1989 al. et Valk etal. et der Lloyd Hawes Van 1985 1989 1989 al. et Schindler 1980 & al. 1988 1986 1981 etal. Duniec 1989 1990 1990 al. et al. Go dbody Meiners Lloyd Wick Wick Knox et & et etal. etal. Stacy Hush Sak guchi Gadal & al. et Lam er n Perot-Rchnma Van 1985 1987 al. et 1989 1989 etal. Gubler Gubler Roberts etal. Wick antigens of localiz ton imunoflresc filaments Antigen Tubulin carboxylase A r a b i n o g l c t p e s A r a b i n o g l c t p r e i n C a l m o d u i n / t b l Clathrin Tubulin Interm diate Con exin Tubulin Tubulin Tubulin Tubulin Tubulin a- mylase PEP for prot plast S p e c i m e n techniques cultures cultures cultures cell cell cells cells Car ot Car ot Tobac o Car ot bean cell tips tips root apices root Onion roots embryos & roots shoot Soy Car ot Onion Pea Car ot Car ot Pea Vinca Tubulin Tubulin Tubulin spp.) cells tips strips pollen leaves aleurone (various Epidermal Leaves root Gasteria SorghumBarley Onion pre ar tive tissue cells various culture pm) (0-5-1 of Examples 5. Table prot plast ghosts Technique Permeable Prot plast su pension Permeable —conf cal prot plast cryosections cryosections embed ing embed ing strips tis ues Square PEG Resin Tis ue Thick Thin Thick 20 M. DEWEY ETAL. Fig. 4. Immunogold localization pumpkin cotyledon 1% paraformaldehyde and SGAT polyclonal (pH 8-2) with of Sue 0-5 serum of serine glyoxylate amino transferase in LR White resin and embedded 1% glutaraldehydein followed by MNaCl,01% (w/v) 15 gold nm 01 polymerized at M PIPES labelled Tween-20 and 1% (SGAT) buffer, pH anti-rabbit on a 50°C for 24 h. 6-9. Staining was IgG. Staining globulin-freeBSA. Bar peroxisome Specimens = buffer 200 nm. give the best signal, conjugated with the second for of the silver growth material or naked deposits. cells. small a antibody This giving technique B. Recently, size colloidal particle thus the is optimum a section of fixed with rabbit anti- 20 mM Tris-HCl (Micrograph courtesy not permeable labelling with colloidal gold with enzymes gold 5nm and and probes immunoglobulin fragments procedure available The main to a advantage of the tissue is (1989) a use to recent fragments plant can be deposited silver, the staining to successfully sectioned immunothe cell by making prior introduction of (papain cleaved should make this to 1-nm 45 kD staining cell types. of silver-enhanced seen In combinationwith the applicable Fab should be communication) single antigen binding site), within cells possible. to personal enhancement. The gold-conjugated wide range of silver-blue fluorescence and should prove methylamine silver with of the the distribution of antigen staining (or (5 pm) restricted necessarily Satiat-Jeunemaitre sol gold number of nucleation sites localized microtubules in epidermal strips of mung bean hypocotyls a with was a Montgomery, Plant Sciences, Oxford.) achieved. To walls in were of gold staining at the LM level is that with transmitted light and further epipolarized light, this becomes plant cells as a very well which will impart impressive technique as animal tissues (Ellis etal. 1989). Future developments. In addition to the introduction of ultra-small nation with silver enhancement and refinements protocols, it is likely that the most to the various significant developments gold probes embedding in LM in combi- and staining immunochemistry ANTIBODIES IN PLANT SCIENCE Fig. 10 5. species specific antigen in the Material gold-conjugated mousemonoclonal antibody. nm maldehyde in will of a Immunogoldlocalization 21 be in 01 the PIPES buffer imaging which microscope, processing m will dimensional can make and low (pH 7-2) was hyphal fixed in temperatureembedded apparatus. The introduction of be epifluorescence used in significant a organization of cells the contribution to wall of Penicillium 1% in LR White resin. the laser Bar 1% = scanning and mode, our islandicum glutaraldehyde and with 400 nm. confocal associated image of three- understanding the With the removal of out-of-focus (Shotton 1989). a parafor- blur, resolution in immunofluorescenceimages will be improved and three-dimensional reconstructions from serial use of this microscope optical sections can is that relatively easily be made. Another large strips of tissue, more be stained and the distribution of antigens below the surface cell potentially important than one cell thick, layer probed (Wick can et al. 1989). Immuno-electron Post the microscopy embedding labelling. variety of plant as cytoskeletal proteins cells due antigens can to only has in level the on biology cells proved very of sectioning, popular due available in terms of pre-embedding labelling locate structural antigens 1986), cannot be used in plasma membrane-bound surface labelling has been used to to (Lawson protoplasts, the cell surface. The in resin and thin not preparation options be limited. The detergent-treated be located after of molecules re-embedding technique appear to the presence of the cell wall. Likewise to rearrangement microscope with such great effect in animal cell procedures, used such At the electron preparative techniques the lack of which undoubtedly of thick (Raikhel penetration the thick cryosection and the technical difficulties of resectioning. causes cryosections, prior et al. 1984), of gold but this probes into 22 M. DEWEY Due these constraints to far the by post-embedding labelling techniques These techniques are discussed here but Hawes & Herman to resins embedding undertaking an both by which use the are 1985; Polak & 1983, 1982, Here (1988). application of these the perhaps two in the tissue is the antigenicity of osmium tetroxide is plant these in cases Thus, in the majority glutaraldehyde Herman nodules leguminous osmication to be made to be achieved when or of vation and reduction in and contrast Shaw to a to on the post-sectioning to to microbody even in in the sections. paraformaldehyde, 1985; Gubler quality of staining most protocols 1986). However, antigenicity & Henwood reduction in the denatured are it has been used in fixation will be restricted (e.g. lead inevitably effect major a investigations, mixtures of both This will 1988). had prior if heat is involved. In den Bosch & Newcomb (Van 1986; Wang cells the reader is Many antigens particularly precluded although undoubtedly be immunocyto- localize storage proteins in developing seeds (Craig & Goodchild 1984) and enzymes not the choice of fixatives and to major goal microscopy. reaction of resins curing techniques to crucial decisions most on study. for immunoelectron specimen employed will protocols Varndell 1984; will limit the discussion we immunoelectronmicroscopy fixatives and the the cases the on retention of Satisfactory preparing Petrusz, Fora review 1988). referred laboratories. General many be found in the many books and review articles can chemistry (Bullock of studies have ultra-thin sections of resin-embedded material. on routine in now overwhelming majority ETAL. al. et 1987; structural preserhave procedures to be modified accordingly. The use of cryofixation will antigenicity 1989). Antigens now techniques and freeze substitution which have been immunostained 1989). extreme at apparatus (Moor fixation, to aid to the popularity during sensitivity such such freezing technology, which will 1987), in unobtainable depth within tissues permit good freezing (Craig & Staehelin as of 1988), in the retention of decade current (Elder have higher plant actin, as the EM level in freeze-substituted cells in developments Recent increase undoubtedly the & (Lancelle high-pressure plant tissues to Hepler freezing hitherto a will allow the localization of fixation-sensitive antigens deep within freeze-substituted tissues. The choice of Although the embedding epoxy resins procedure cations due to loss of merization required resins of many reported to preservation of structural preservation and have generally which is also were applications (Shaw be difficult to use This acrylic resin has the The resin has an polymerized by extremely a Catalytic curing at low viscosity, of methods variety room den Bosch et for conventional electron the most to be of needs no mixing (1) curing an Heat process. low-temperature poly- 1985; tissues 1984; Lowicryl good appli- et Gubler (Herman et al. 1982) al. 1987) 1988). extremely reliable resin for Van den Bosch etal. and many other and is curing not at 60 toxic. It or 1989). advantages. 50°C can also be (Fig. 4),(2) temperature (the reaction is extremely exothermic), (3) temperature ultraviolet polymerization (Fig. 5) (Van plant & Miller follows: as heat procedures (Carlemalm proved hydrophilic properties for the & Henwood with many plant immunocytochemical applications (Craig antigenicity. immunocytochemical exaggerated by the specifically designed the London resin LR White has However, unsuitable for immunocytochemical and have been used in various but have been are antigenicity hydrophilic Lowicryl be critical in the generally give superb sectioning characteristics, they The can al. 1989). Thus, microscopy convenient technique using for or the Lowicryl catalyst the resin can temperature polymerization is carried out at — 20°C in methyl low ether be used with osmicated material with unosmicated retaining antigenicity. benzoin specimens and polymerized by In the authors’ laboratories lowa cryostat fitted with a suitable ANTIBODIES IN PLANT SCIENCE ultraviolet 5 shows the localization of Figure source. 23 walls of Penicillium islandicum after aldehyde in the cell species-specific antigen a fixation and in low-temperature embedding LR White. Other Ultrathin cryosectioning is techniques. tissues (Tokuyasu scientists. It by plant suitable be difficult can infiltrationit is sucrose used this technique with great nin in bean developing by no means success White resin rather than in temperature resin-embedding can be carried cellular thus membranes, cryosections of plant cells, animal ignored but with Chrispeels (1985) cellulose of the in alcohol. polyvinyl or preservation and major shortcomings thin a Unlike osmication of the level of acceptable one Tokyasu technique the labelled cryosection techniques, post labelling overcoming on been immunolocalize phaseolin and phytohemaggluti- methyl an success largely Greenwood & impossible. embedding of This will result in out. that has One modification of the standard cotyledons. that shouldprove useful is the final LR to technique ultra-thin to cut used with great frequently this is another 1986). However, the low cryosections contrast of of layer in the aldehyde-only fixation. of unembeddedcells Labelling a modification of the cortical cells root dehydration and broken were immunolabelled prior to antigens by Goodbody al. One fixing et on area open with under buffer and to be applied Various and to tissues it is of plant cytoskeleton recently plasma by of inter- labelling been described for the localization of membrane and the cortical extensively tissues is that of freeze-fracture plant likely cells labelling on fractured before are has with various techniques (Severs 1989). labelling. As even techniques improve techniques will be and freeze-fracturing These may prove useful for the surface of membranes and that these animal cells on cytochemistry. immunogold labelling methodologies have been developed for labelling the distribution of antigens plant Similar should prove useful face of the cytoplasmic paraformaldehyde-fixed and osmium tetroxide followed Kengen 1986). In protoplasts. microtubule of immunochemicaltechnology that has been used freeze-fracturing if cells the protoplast ‘footprints’ technique This is the combination of freeze-fracture or & of mounts of cells. but which has yet protocols. broken open cells and on glutaraldehyde cleaved This (1989). associated with the cytoskeleton be achieved critical-point drying (Traas mediate filament antigens can dry cleave technique, whole developed within the for the studying lipid bilayer cryopreservation and modified for the of study antigens. CONCLUSIONS Antibodies such are now essential tools in the skills and limited, areas. The resources authors’ experiences number of years need not for instance antibodies success and in by using using be a antibodies in litre; this involves screening of antibodies a science and, indicate that daunting task, purchased plant paying by plant unless research is involved should be available of to given by or such can be severely engaging facilities quickly be of high over in a obtained, other laboratories. The obtaining preparations every to laboratories establishing and useful results science is in attention variety to key to specificity step, including careful and thorough techniques. ACKNOWLEDGEMENTS DE and during CH the were period supported by Royal that some Society 1983 of the work discussed here University was Research carried out. Fellowships 24 M. DEWEY ETAL. REFERENCES Banowetz, G.M., Trione, Krygier, E.J. & of Immunological comparisons wheat bunt fungi, bodies and antisera. J.D. & Barbara, M.F, In: Plant Science. (1984): of two using monoclonal sp anti- T.L. 197-219. in a I, & D. Pitt, of pathogen Cambridge University immunosorbent in (ELISA). assay by corms Plant In; to and E.J.J. S. and & Pathol. 37; A, in Virus M. Wilchek, M. & the old. (eds) Develop179-193. Testing. In: Langone, in Methods (eds) Techniques. A. (1984): 87-150. 70: Part J.M. Wall-less (1984): The Practice Springer-Verlag, Briars, S.A., Kessler, F. a plants. 361-396. & Evans calmodulin-stimulated of prokaryotes Phytopalhol. 22: The (1988): ATPase ofmaize 140,000 M polypeptide. Ptanta, coleoptiles 176:283-285. G.R. & Techniques Academic P. Petrusz, Immunocylochemistry. in Press, Resin Microsc. Chard, 126: R.M. & Villiger, electron microscopy at low W, (1982): and temperature. an Ini. J. 132-143. of Mycena galopus. & antigen an Trans. J.C. Frankland, Br. characteristic Mycol. 84: Soc. 235-241. —, — & — Mycol. Use of an anti -Mycena immunofluorescent Trans. 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