Immunology in Weed Science

Weed Science, 1991. Volume 39:514-520

Immunology in Weed Science1

TIMOTHY D. SHERMAN and KEVIN C. VAUGHN2

Abstract. Many problems in weed science may be solved by utilizing immunochemical techniques, although these techniques are currently underutilized by weed scientists. In this review, we describe some of the methods of greatest use to weed scientists. Antibodies may be raised against proteins, such as the targets of herbicide action or against the herbicide itself which can be linked to a larger molecule so that antibodies are elicited. Once specific antibodies are obtained, several techniques may be performed utilizing the binding phenomena of antibody- antigen interactions. Immunofluorescence and immunogold electron microscopy are utilized to obtain tissue and subcellular distributions of the protein or herbicide of interest. Quantitation of either protein or herbicide in a given sample may be performed by ELISA or "slot" and "dot" blotting. These protocols are less costly, more sensitive, and much less labor-intensive than most analytical methods. Molecular mass or charge alterations may be determined by electrophoresis and subsequent immunoblotting. With the increased exposure to immunological techniques by weed scientists and their potential utility, we predict that many more weed science problems will be addressed using these protocols. Additional index words. Herbicide, immunoblotting, immunogold, immunocytochemistry.

INTRODUCTION

Immunological techniques have been underutilized by plant scientists in general and weed scientists in particular. For example, immunofluorescence microscopy was pioneered as a technique in the 1940s and had widespread use by animal scientists in the 1950s. Not umtil the early 1970s, however, did research utilizing specific antibodies appear in the botanical literature (6), although these techniques are frequently used by plant scienitists today (1, 5, 9). Few plant science programs 10 yr ago (other than plant virology) offered immunology as a part of their undergraduate or graduate curriculum. Today, plant scientists are exposed to immunological techniques in plant cell or molecular biology programs and the use of these techniques has become more widespread. In this review, we will briefly describe those immunological techniques of greatest use to weed scientists with the exception of enzynme-linked immunosorbant assay (ELISA) which has been reviewed recently (2). General texts concerning use of immunological techniques in plant sciences are also available (12, 24).

1Received for publication April 5, 1991, and in revised form August 15, 1991.

2Plant Physiol's., U.S. Dep. Agric., Agric. Res. Serv., South. Weed Sci. Lab., P.O. Box 350, Stoneville, MS 38776.

TYPES OF ANTIBODIES

Central to all of these immunological techniques is the antibody. This is a protein generated by fish, birds, and mammals in response to foreign substances that have entered their bodies. Although these foreign substances are often other proteins, antibodies can be elicited to a variety of other compounds, including small molecules such as herbicides, certain carbohydrates, and even the variable regions of other antibody molecules (8, 14, 23). However, smaller molecules (haptens) generally must be attached to a larger molecule, such as a protein, in order to elicit a sufficient immune response (14). When antigen enters the animal, antibodies are made to a number of individual sites on the molecule, including the carbohydrates which are components of many proteins, or haptens that have been attached to a carrier protein. The population of different antibodies produced by the animal injected with the antigen will recognize various sites on the foreign molecule. Collectively, these antibodies are referred to as polyclonal antiserum. This is the source of antibodies that is least expensive and easiest to produce. In fact, most larger universities will have facilities to produce polyclonal antisera. They also are available commercially.

One of the greatest difficulties in using polyclonal antisera results from the fact that many proteims share common structural characteristics and secondary modifications. Poly- clonal antisera, therefore, may contain antibodies that will recognize proteins that are not their primary target. Another problem is that antisera are produced usually by herbivorous animals that have been exposed to many plant antigens as a result of their diet. This can lead to nonspecific recognition by the antiserum. A third consideration is that a fmite amount of antiserum is obtained from an animal. Subsequent batches of antisera from other animals must be characterized for specificity. Polyclonal antisera are very stable, however, and can be stored frozen for years with little loss of activity.

An altemative to polyclonal antiserum is the monoclonal antibody. As its name implies, this class of antibodies is homogeneous and recognizes a single site on an antigen molecule. Homogeneity is obtained by fractionating the antibody producing cells of the immunized animal and growing them in cell culture. Each cell line produces a single type of specific antibody which can be screened for reactivity to the antigen. Isolated cell lines can be maintained almost indefinitely and will offer a constant source of very pure antibody. Monoclonal antibodies can be selected for such specific targeting as active sites of enzymes or secondary structural features of proteins, thus offering a very precise tool for immunological studies.

The primary drawback to this ype of antibody is cost. Screening and cell maintenance procedures demand extensive technical skill and large labor inputs. Also, cell lines are sometimes unstable and stop producing antibodies. High

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specificity of monoclonal antibodies also can create problems when comparing unrelated plant species, or analyzing proteins that have undergone conformational changes or still are associated with other cellular components that block antigenic sites. Monoclonal antibodies are produced by a number of commercial sources.

Recently, a great improvement in production of monoclonal antibodies was described by Huse et al. (4). This technique allows for the cloning of functional portions antibodies into bacteria, with subsequent expression of these antibody fragments. Screening and maintenance of these bacterial cultures is much simpler than for the animal cell lines presently employed for production. This will allow individual researchers or departments to produce monoclonal antibodies. This method also allows for antibody production and screening in a period of 2 wk after primary antibody formation versus 2 mo for traditional protocols. Monoclonal antibodies generated by this technique will soon be available commercially.

TYPES OF IMMUNOREACTIONS

Antibodies are Y-shaped molecules in which the upper arms of the "Y" are the portions responsible for antigen recognition. Each arm has the potential to recognize a unique site on the antigen. Each antibody molecule can then theoretically bind two antigen molecules. Triads of antibody: antigens can then be cross-linked by other antibody molecules to form large complexes. With sufficient antibody and antigen concentration, these complexes can become large enough to precipitate. Not all antibodies have two arms that will bind to the antigen or two with sufficient affinity to stabilize an antibody:antigen complex, and therefore bind only a single antigen molecule. The antigen used for immunization will often determine whether or not an antiserum is precipitating. Antibodies to proteins usually are precipitating, although this may depend on the type of protein and degree of purity. Generally, antibodies raised to haptens recognize only a single site on the hapten and do not precipitate. Monoclonal antibodies also are nonprecipitating. Ouchterlony double diffusion. The most common precipitating reaction, Ouchterlony double diffusion (13, 16, 17), often is used as a primary screen to determine the specificity and titer (relative concentration of a specific antibody in the serum) of antisera because of its simplicity. In the double diffusion assay, a gel composed of agar or agarose is used as a solid support, with wells cut into the gel in one of several pattems. Generally, antisera to be tested are placed in the central well and antigen in the peripheral wells. The Ouchterlony plate is then incubated in a moist chamber so that both the antigen and antibody may diffuse radially into the gel. At the zone of antibody and antigen equilibrium, a precipitation of the complex occurs (precipitin line). To enhance the detection (and subsequent photography) of the complex, the gel is washed to remove unprecipitated antigen and antibody, and then stained for protein with Coomassie blue or Crowle's stain.

In the Ouchterlony shown in Figure 1, antibodies to the urate oxidase purified from soybean [Glycine max (L.) Merr.]

are reacted against extracts from a number of crop and weed species (17). A precipitin line is formed between sample and antibody wells only in those species containing a urate oxidase that is antigenically similar to the soybean urate oxidase [cowpea (Vigna sinensis L.) and mung bean (Phaseolus radiata L.)] but not two weed species [hemp sesbania (Sesbania exaltata Raf.) and Indian jointvetch (Aeschynomene indica L.). Thus, by using a specific antibody, presence of an antigenically similar protein may be detected easily in a variety of plants. Urate oxidase found in the mung bean is immunologically identical to the soybean protein given the distinct precipitin line (Figure 1). If the mung urate oxidase differed in some way from the soybean protein, one would observe a spur in the precipitin line. Interpretation of precipitin patterns has been described extensively (13).

Monospecificity of the antisera is demonstrated by a single precipitin line when a crude extract is reacted against the antisera. Occasionally, double bands are encountered even in monospecific sera. For example, proteins that form complexes in vivo may give precipitin lines for both the monomer to which the antisera are raised and the complex. Dissociation of the complex by modifying the salt concentration during sample preparation or adding a small amount of Triton X-

M B i.

.. ~ U _X

s~f X

SB, ;20SY

Figure 1. Ouchterlony double diffusion of antisoybean urate oxidase (UOX, center well) against crude homogenate of soybean (SY), mung bean (MB), cowpea (CP), hemp sesbania (SB), and Indian jointvetch (AS). A single precipitin line is found in the soybean, mung bean, and cowpea extracts but not in the hemp sesbania or Indian jointvetch.

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Figure 2. Radial diffusion of crude homogenate of triazine-resistant (R) and - susceptible (S) biotypes of black nightshade in a gel containing antiribulose bisphosphate carboxylase (RuBisCO). When equal protein concentrations are added to each well, the radius of the inmunodiffusion ring of the two biotypes is equal, indicating no difference in RuBisCO concentration between the two biotypes.

1003 to the gel or sample buffer may ameliorate this problem. As mentioned above, Ouchterlony double diffusion is also

useful in determining antisera titer. Antigen is placed in the central well and serially diluted antibody is placed in the surrounding wells. Antibodies that give a strong precipitin line at antisera dilutions of 1:64 or greater are considered antisera of high titer. However, depending upon the purpose, antisera of lower titer may still be of use in nonprecipitating techniques, such as immunoblotting (see below). Radial diffusion/rocket immunoelectrophoresis. Radial diffusion is similar to Ouchterlony in methodology but is a better quantitative tool. In radial diffusion, antibody is incorporated into the agar or agarose gel matrix while antigen is applied to wells. Antigen diffuses from the wells into the surrounding gel until a precipitin line is formed. Diameter of the ring is proportional to the amount of antigen present in the well. Incubation, washing, and staining are performed as with Ouchterlony double diffusion assay.

A standard curve is prepared by utilizing a purified antigen over a range of concentrations in which a linear response relationship between antigen and precipitin-ring diameter is established. Crude extracts are prepared at various dilutions so that the antigen concentration is in the linear range of the standard curve. From this, antigen concentration may be directly determined.

Radial diffusion offers a quick estimate of antigen concentration. For example, triazine-resistant and -susceptible biotypes of black nightshade (Solanum nigrum L.) that differed greatly in photosynthetic efficiency did not differ in ribulose bisphosphate carboxylase concentration (Figure 2). One problem with the protocol is that large amounts of antiserum are needed. (A 1 to 3% by vol solution of antiserum is incorporated into the gel.)

3Sigma Chemical Co., St. Louis, MO.

(t)? ??(?(i (F(S -t4S Figure 3. Rocket immunoelectrophoresis of nodule extracts from weed and crop species. Extracts from soybean (s), alfalfa (Medicago sativa L.) (f), cowpea (c), hemp sesbania (b), and Indian jointvetch (a) nodules were electrophoresed through a gel containing antiserum raised against soybean nodule extract. Precipitin complexes are present in the shape of rockets, the height of which is proportional to the amount of cross-reacting material present in the extract. The arrow indicates immunoprecipitin of leghae- moglobin which was present as a colored band prior to staining. Note that only some of the antigens found in soybeans are present in other leguminous species.

A variation of radial diffusion is "rocket electrophoresis." Wells are placed at one end of the gel, and an electric field is applied across the length of the gel. This method takes advantage of the fact that at pH 8.3 antibody molecules move toward the anode while most other proteins move toward the cathode. As the two groups move past each other an antibody:antigen complex forms in the shape of a rocket. As more antigen arrives at the trailing end of the rocket, the precipitate redissolves and moves farther toward the cathode to form a new leading edge. When all of the antigen from the well is consumed, the rocket reaches an equilibrium height. Height of the rocket is proportional to the amount of antigen in the well. Using antigen standards, the amount of antigen in the extract can be accurately determined. Because of the sharp precipitin lines formed with rocket electrophoresis, this method is better than radial diffusion for quantitating multiple antigens in a single sample. Figure 3 shows how this technique was used to analyze immunological relationships between nodule extracts from crop and weed legume species (16). Immunoprecipitation. This technique can actually be performed using precipitating or nonprecipitating antibodies. Both methods are very effective in isolating an antigen from a crude extract for subsequent analysis. Classically, im-

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munoprecipitation relied upon the formation of antibody: antigen complexes which could be removed from the solution by centrifugation. A more effective method utilizes molecules such as Protein A or Protein G, bacterial proteins that bind antibodies from a wide variety of species, or secondary antibodies that recognize the constant regions of antibodies found in the antiserum of a given species. These secondary molecules are available in a form conjugated to high molecular mass agarose beads that do not require formation of immune complexes in order to collect the antigen. This allows for rapid isolation and use of nonprecipitating antibodies such as monoclonals and antihaptens. Sensitivity of immunoprecipitation is demonstrated in Figure 4. Anti- body to nitrate reductase and Protein A-Sepharose were used to isolate nitrate reductase precursor protein which had been translated in vitro from total mRNA. In ammonium-grown and nitrate-induced samples, nitrate reductase mRNA was present as 0.08 and 0.44% of total mRNA, respectively, and both show clearly visible bands after denaturing electrophoresis (15). Immunoblotting. This nonprecipitating reaction is probably the most commonly used of all the immunoprotocols. This technique yields information about antigen molecular size and quantity in a single, simple assay. Samples of crude or purified plant extracts are subjected to denaturing gel electrophoresis (7), which separates proteins on the basis of molecular mass. The proteins are then transferred laterally under an electric field from the gel onto a hydrophobic or positively charged membrane (18). This mirror image of the gel becomes the substrate for the immunoreaction. Areas of the blot that have not bound proteins from the gel must be saturated with protein which are unrelated to the antigen in order to prevent the antiserum from producing a reaction across the entire membrane. The blocked membrane can then be exposed to single antiserum or multiple antisera by either cutting the membrane into strips, or by using a commercially available device that clips over the blot to form channels for antibody incubation. Unbound antibodies are washed away and the blot is then incubated in a solution containing Protein A (or G) or a secondary antibody coupled to an enzyme that produces an insoluble, colored reaction product. Use of alkaline phosphatase conjugated antibodies will allow for detection sensitivity in the picogram range. With this technique, it was determined that there were quantitative differences, as determined by densitometry, in some chloroplast proteins between triazine-resistant and -susceptible lines of canola while other proteins were unchanged (20). Dot blotting/slot blotting. Dot and slot blots are variations on the immunoblot that allow for rapid screening of large numbers of samples for antigen quantitation. In these methods, extracts are loaded into circular (dot) or rectangular (slot) wells. The bottom of these wells is formed by a membrane identical to that used for immunoblotting. The sample is drawn through this membrane by mild vacuum rather than by electrical potential. The blocking, antibody, and wash steps are as described above for immunoblotting. Because electrophoresis and electroblotting steps have been eliminated, dot and slot blotting are much faster than

-200 kDa

-97 kDa

- 68 kDa

-43 kDa

-26kDa

-18 kDa

A~ ~~~~~~~~ I

Figure 4. Immunoprecipitation of in vitro-translated mRNA from Chlorella vulgaris Beij. under different nitrogen regimes. Ammonia-grown cells, which have very low levels of nitrate reductase activity, were transferred to nitrate- containing medium. Total mRNA isolated from amn-gon(A) or -

induced (I) cells was translated in vitro in the presence of 35S-methionine. Immulnoreactive translation products were isolated usin antinitrate reductase followed by Protein A-sepharose. The precipitates were electrophoresed and autoradiographed.

immunoblotting. Commercially available dot and slot blotters allow for processing 48 to 96 samples per membrane.

MICROSCOPIC PROTOCOLS

Plant cells have inherent problems in immunocytochemical analysis because antibodies cannot penetrate the cell wall. This lack of penetration has been overcome by digesting the cell wall away with cellulytic enzymes, cutting frozen thin or semithin sections, or embedding the material in resins and then cutting thin or semithin sections. Indeed, progress in immunocytochemical analysis of plant tissues has gone through considerable change in recent years with improvement in techniques related to specimen preparation. Because of the variety and complexity of these techniques, we will address only those with direct relevance to weed science. Immunofluorescence. Immunofluorescence has been used most widely in plant science in investigating the plant "4cytoskeleton." Microtubules and microfilaments of plant



Figure 5. Immunofluorescence micrographs of control (A) and terbutol-treated (B) onion root tip cells stained for tubulin with a monoclonal antibody to tabulin. In the control cell, a normal spindle microtubule array is noted, with the microtubules originating from a broad region at each of the poles. In the terbutol- treated cell, the spindle microtubules are oriented in a starburst pattern at both poles. x440.

cells are not well fixed utilizing standard electron microscopic protocols. Even when well fixed, plant microtubules and microfilaments are difficult to analyze in thin-section transmission electron microscopy because of their small size. These cytoskeletal elements often form arrays that require tedious reconstruction of serial sections to establish their three-dimensional orientation. Wick et al. (25) described a procedure that alleviates many of these problems. Root tips are briefly fixed in a buffer that stabilizes microtubule arrays. Cell walls are then softened by cellulytic enzymes to allow passage of antibodies into the cell. Initial fixation maintains cell shape even though most of the cell wall is digested. Samples are then incubated in a primary antiserum that recognizes tubulin and then in a secondary antiserum that has been raised against the whole IgG molecule of the type found in the primary antiserum. This secondary antibody is coupled to a fluorescent tracer molecule such as fluorescein (FITC) or rhodamine. Samples are then mounted in a medium which prevents fading of fluorescence and examined with a fluorescence microscope. The pattem of microtubule arrays can be readily determined by focusing up and down through the cell (Figure 5a).

Many of the herbicides that disrupt mitosis do so by disrupting formation and organization of microtubules (9). Immunofluorescence provides a quick way of determining the effect of the herbicide on the microtubule arrays. Terbutol (2,6-di-tert-butyl-p-totyl methylcarbamate)-induced star anaphases (Figure Sb), abnormal phragmoplast microtubules

after DCPA (dimethyl 2,3,5,6-tetrachloro-1,4-benzenedicar- boxylate) treatment (11), and small kinetochore tufts of microtubules after pronamide [3,5-dichloro(N-1,1-dimethyl- 2-propynyl)benzamide] and dithiopyr (S,S-dimethyl 2- (difluoremethyl)4-(2-methylpropyl)-6-trifluoromethyl)-3,5- pyridinedicarbothioate) treatment (10) are readily observable in the immunofluorescence preparation but would be difficult to decipher using traditional thin-section analysis. Thus, immunofluorescence has substantially enhanced analysis of mitotic disrupter herbicides. Immunogold. For detection of antibodies at the electron microscopic level, a secondary label must be electron opaque. Earlier techniques used antibodies tagged with the electron opaque protein ferritin (6) or utilized peroxidase with subsequent conversion of the enzyme product to an electron opaque substance by osmium (6). Both of these protocols have drawbacks, however. Ferritin tends to adhere nonspecifi- cally to some membranes. Peroxidase methods give a diffuse reaction product, and certain cellular components that are very osmiophilic also appear as spurious reactive sites. Thus, providing a convincing inmunocytochemical localization with these two protocols is difficult.

Colloidal gold reagents have greatly advanced the use of inmunocytochemistry in higher plants. Colloidal gold particles of different sizes are prepared by perfonming the particle nucleations under different buffer/pH conditions. With these protocols, particles from 2 to 30 nm may be produced with relatively little variation in size. These are then coupled either


WEED SCIENCE

w

' ~ ~~~~~~~~~~~~~~~~~ :4t: '

4 , Y r.- '-

Figure 6. Electron micrographis of a bundle sheath cell of Jolinsoigrass [Sorghum halepense (L.) Pers.] embedded in L. R. White resin and labeled using antiribulose bisphosphate carboxylase and 15-nm protein A-gold. Note that all of the inmmunogold labeling (some noted with arrows) is associated with the stroma of the bundle sheath chloroplast (C) and is relatively randomly distributed in the stroma. w = wall, bar = 1.0 pm.

to secondary antibodies or Protein A (a protein that recognizes the IgG from number of species by a pseudoim- mime reaction) to form a complex that can detect the location of the primary antibody.

The inmmunogold technique may be preformed directly on thin or semithin plastic sections. The sample is fixed and processed almost identically to normal transmission electron microscopic procedures except that the tissue is embedded in a nonepoxy resin (22). The tissue sections are cut with an ultramicrotome and mounted on gold or nickel grids. If the tissue is osmicated, the sections are "deosmicated" with a treatment of meta-periodate. Immunological processing of the grids includes: blocking to prevent nonspecific binding, primary antibody treatment, washing steps, secondary antibody or Protein A-gold treatment, and washing steps. The grids are then lightly post stained (relative to "normal" thin- section staining) and observed with a transmission electron microscope. The same reactions may be performed on light microscopic sections except that the colloidal gold product is intensified by reaction with silver. This not only increases the size of the gold particle but also enhances it for photography by converting the pinkish colloidal gold to dark-grey silver deposits (19).

A successful localization of ribulose bisphosphate carboxylase using immunogold is shown in Figure 6 (21). Electron opaque spheres are found in the stroma of the chioroplast but other cellular structures are unlabeled. Because the immunogold process reacts only with the surface of the section, one can actually compare densities of labeling between tissues in the same section and determine relative concentrations of the antigen in the different cell types. Immunogold is a very powerful technique, offering high resolution and consistency, and is now the method of choice for subcellar (and suborganellar) izmmunolocalization.

PROSPECTUS AND CONCLUSIONS

Imnmunological protocols vary greatly in the type and amount of complexity of information that they generate

Table 1. Comparison of nonmicroscopic immunological techniques.

Technique Advantages Disadvantages

Ouchterlony Simple, quick, informativeNot quantitative Radial diffusion Simple, quick Wastes antisera Rocket electrophoresis Excellent quantitation Wastes antisera Immunoprecipitation Simple, quick Semiquantitative ELISA Quantitative Expensive setup Dot/slot blotting Quantitative Expensive setup Immunoblotting Extensive information Expensive setup

(Table 1). Generally, procedures that are classified as nonprecipitating reactions are most popular because they require very little primary antiserum for analysis and have a high information yield. However, these protocols generally require more costly equipment and technical expertise than those involving precipitating reactions. Among immunocytochemical protocols, there has been a tremendous growth of the technology involving immunogold reagents. Immunochemistry has been introduced into many labs with the advent of new plastic resins that preserve antigenicity and the availability of collodial gold-labeled secondary antibodies and Protein A. Immunogold-silver gradually is replacing immunofluorescence because it is a permanent record, not subject to fading.

In weed science, immunological protocols probably will be heavily used in the areas of weed biology, herbicide mode-of- action, and soil science. In weed biology, immunological methods have already been utilized to monitor differences in chloroplast proteins of triazine-resistant and -susceptible biotypes (20). In weed taxonomy, immunology may be useful in distinguishing morphologically variable species or in chemosystematic studies of genera. Huber and Sautter (3) recently have performed the first immunolocalization of a herbicide. With this protocol, it should be possible to detect sites of herbicide binding and accumulation. The use of antibodies against herbicides may be useful in detection and measurement of herbicides in ground water or soil by the ELISA protocol. Clearly, we have seen only the beginning of the many applications of immunological techniques likely to emerge in weed science research.

ACKNOWLEDGMENTS

We would like to acknowledge the work of Steven J. Stegink and Larry P. Lehnen in the laboratory of Kevin C. Vaughn. Thanks are extended to Lynn Libous-Bailey for excellent technical assistance. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

LITERATURE CITED

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2. Hall, J. C., R.J.A. Deschamps, and M. R. McDermott. 1990. Immnunoassays to detect and quantitate herbicides in the environment. Weed Technol. 4:226-234.

3. Huber, S. J. and C. Sautter. 1986. Inmunofluorescence localization of conjugated atrazine in leaf pieces of corn (Zea mays). J. Plant Dis. and Prot. 93:608-613.

4. Huse, W. D., L. Sastry, S. A. Iverson, A. S. Kang, M. Alting-Mees, D. R. Burton, S. J. Benkovic, and R. A. Lemer. 1989. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246:1275-1281.

5. Knox, R. B., J. Heslop-Harrison, and C. E. Reed. 1970. Immunolocali- zation of antigen associated with the pollen grain wall by immunofluorescence. Nature 229:1066-1068.

6. Knox, R. B., H.I.M.V. Vithanage, and B. J. Howlett. 1980. Botanical immunocytochemistry: a review with special reference to pollen antigens and allergens. Histochem. J. 12:247-272.

7. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

8. Laine, A. -C. and L. Faye. 1988. Significant immunological cross- reactivity of plant glycoproteins. Electrophoresis 9:844-845.

9. Lehnen, L. P., Jr., M. A. Vaughan, and K. C. Vaughn. 1990. Terbutol affects spindle microtubule organizing centres. J. Exp. Bot. 41: 537-546.

10. Lehnen, L. P., Jr. and K. C. Vaughn. 1991. Immunofluorescence and electron microscopic investigations of the effects of dithiopyr on onion root tips. Pestic. Biochem. Physiol. 40:58-67.

11. Lehnen, L. P., Jr. and K. C. Vaughn. 1991. Immunofluorescence and electron microscopic of DCPA-treated oat roots. Pestic. Biochem. Physiol. 40:47-57.

12. Linskens, H. F. and J. F. Jackson, eds. 1986. Immunology in Plant Sciences. Springer-Verlag, Berlin. 263 pp.

13. Ouchterlony, 0. 1968. Pages 33-48 in Handbook of immunodiffusion and Trnmunoelectrophoresis. Ann Arbor Science Publisher, Inc., Ann Arbor. MI.

14. Robins, R. J. 1986. The measurement of low-molecular-weight, non- immunogenic compounds by immunoassay. Pages 86-141 in H. F. Linskens and J. F. Jackson, eds. Tmmunology in Plant Sciences. Springer-Verlag, Berlin.

15. Sherman, T. D. and E. A. Funkhouser. 1989. Induction and synthesis of nitrate reductase in Chlorella vulgaris. Arch. Biochem. Biophys. 274: 525-531.

16. Stegink, S. J. and K. C. Vaughn. 1990. lImunotaxonomy of nodule- specific proteins. Cytobios 61:7-19.

17. Stegink, S. J., K. C. Vaughn, and D. P. Verma. 1987. Antigenic similarity in urate oxidases of major ureide producing legumes and its cofrelation with the type of peroxisome in uninfected cells of nodules. Plant Cell Physiol. 28:387-396.

18. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354.

19. Vaughan, M. A. and K. C. Vaughn. 1987. Pronamide disrupts mitosis in a unique manner. Pestic. Biochem. Physiol. 28:182-193.

20. Vaughn, K. C. 1986. Characterization of triazine-resistant and -susceptible isolines of canola (Brassica napus L.). Plant Physiol. 82: 859-863.

21. Vaughn, K. C. 1987. Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts. Plant Physiol. 84:188-196.

22. Vaughn, K. C. 1989. Subperoxisomal localization of glycolate oxidase. Histochemistry 91:99-105.

23. Wang, M. 1990. Anti-idiotypes used in immunohistochemistry. Histochem. J. 22:519-525.

24. Wang, T. L. 1986. Tmmunology in Plant Science. Cambridge University Press, Cambridge. 228 pp.

25. Wick, S. M., R. W. Seagull, M. Osbor, K. Weber, and BE.S. Gunning. 1981. Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J. Cell Biol. 89:605-609.


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Immunology in Weed Science