(The FASEB Journal. 2002;16:1855-1860.)
© 2002 FASEB
Transgenic plants expressing antibodies: a model for phytoremediation
PASCAL M. W. DRAKE,
DANIEL CHARGELEGUE,
NICHOLAS D. VINE,
CRAIG J. VAN DOLLEWEERD,
PATRICIA OBREGON and
JULIAN K.-C. MA1
Department of Oral Medicine and Pathology, Unit of Immunology, Guy’s Tower, Guy’s Hospital, GKT Dental Institute, London Bridge, London SE1 9RT, UK
1Correspondence: Department of Oral Medicine and Pathology, Unit of Immunology, 28th Floor, Guy’s Tower, Guy’s Hospital, GKT Dental Institute, London Bridge, London SE1 9RT, UK. E-mail: julian.ma{at}kcl.ac.uk/.
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ABSTRACT
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The feasibility of using antibody expressing transgenic plants either to neutralize bioactive molecules in the rhizosphere, or to accumulate and concentrate the molecules in leaves has been demonstrated in a model system consisting of hydroponic Nicotiana plant cultures expressing a murine monoclonal IgG1. Two transgenic plant types were used; in the first, functional antibody was rhizosecreted and shown to bind with antigen in the surrounding medium to form an immune complex. In the second, a transmembrane sequence retained monoclonal antibody in the plants, on the plasma membrane. Antigen added to the nutrient medium around the roots of mIgG plants was transported within 24 h to the topmost leaves of the plant where it was sequestered as an immune complex by binding to antibody on the cell membrane. Concentration of immune complex in the leaf tissue remained constant over a 72 h period after removal of antigen from nutrient medium. Free antigen was not detected in the leaves of wild-type plants. The two strategies of rhizosecretion-mediated binding and sequestration in leaf tissue could potentially be used in the phytoremediation of any pollutant for which it is possible to generate a monoclonal antibody.—Drake, P. M. W., Chargelegue, D., Vine, N. D., van Dolleweerd, C. J., Obregon, P., Ma, J. K.-C. Transgenic plants expressing antibodies: a model for phytoremediation.
Key Words: hydroponic medium • recombinant antibody • transgenic plants
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INTRODUCTION
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PHYTOREMEDIATION IS THE use of plants to extract, sequester, or detoxify pollutants in soils and surface waters. This process is an environmentally responsible alternative to the physical processes for restoration of contaminated soil (1)
. Phytoremediation strategies for radionuclide and heavy metal pollutants (e.g., arsenic, cadmium, chromium, lead) focus on hyperaccumulation in above-ground tissues, followed by removal of plant biomass from the site with subsequent recycling or disposal of contaminated material (2)
. Several plant species, notably in the Brassicaceae, are natural hyperaccumulators of heavy metals. For example, Alyssum lesbiacum can be grown in Ni (II)-rich soil; nickel is rapidly transported into the plant and accumulates to >3% of the dry weight of above-ground tissues (3)
.
Genetic modification has the potential to enhance the natural remediation capacities of plants, and the principle of binding and sequestration of heavy metal pollutants has been demonstrated in transgenic plants engineered to express mammalian metallothionein proteins (4
, 5)
. The majority of environmental pollutants, however, are not heavy metals and therefore not amenable to this phytoremediation strategy. Examples include a range of pesticides and several suspected endocrine-disrupting chemical families (such as phthalates, alkylphenols, polychlorinated biphenyls, bisphenol A, dioxins, and both natural and synthetic steroid estrogens) that are implicated in reproductive system disorders in exposed animal species (6)
.
Theoretically, the phytoremediation capabilities of plants could be extended to these types of chemicals by generating transgenic plants expressing an antibody specific to a particular pollutant. Transgenic plants have been used to produce full-length immunoglobulin G (IgG) antibody (7)
, multimeric secretory antibody (8)
, and a wide range of functional antibody fragments (9
10
11)
. In the present study, we have used a model system—hydroponic tobacco plant cultures expressing the murine monoclonal antibody Guy’s 13—to demonstrate the feasibility of using antibody-expressing plants for phytoremediation. Guy’s 13, a neutralizing antibody, recognizes a 185 kDa cell surface protein (SA I/II) of Streptococcus mutans (12)
. Two transgenic plant types were studied: in the first (IgG plants), functional full-length antibody was produced and secreted from the roots into the surrounding nutrient medium. The aim was to determine whether secreted antibody would bind SA I/II in the aqueous environment surrounding the roots. In the second plant type (mIgG plants), antibody was expressed and retained in the plant cell plasma membrane due to the presence of a transmembrane sequence (13)
. Experiments were undertaken to measure the ability of these plants to bind SA I/II via membrane-associated antibody expressed in the leaves after uptake of antigen in the roots and transport through the plant.
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MATERIALS AND METHODS
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Plant material
Assessments were performed on Nicotiana tabacum (var. Xanthii) using two transgenic plant types, both expressing Guy’s 13 monoclonal antibody. IgG plants were homozygous double transgenic plants coexpressing light and heavy chains of the murine IgG1. The assembly of functional IgG by these plants and extracellular secretion into the apoplasm have been described (14
, 15)
. In previous studies, IgG plants were shown to rhizosecrete full-length functional immunoglobulins into surrounding hydroponic medium (unpublished data). mIgG plants were double transgenic plants produced in the same way, expressing the same immunoglobulin light chain along with the membrane heavy chain of Guy’s 13 monoclonal antibody. The latter is identical to the secreted heavy chain except for the addition of an extra 71 amino acid residues at the COOH terminus that comprise a 17 residue acidic extracellular portion, a 26 residue hydrophobic intramembrane portion, and a 28 residue hydrophilic intracellular portion in mammalian cells (16)
. In plants, this sequence is also sufficient to retain recombinant IgG in the plasmalemma (13)
. We have previously shown that antibody is not rhizosecreted from mIgG plants (unpublished data), nor is it released in soluble form from macerated leaves unless the cell membranes are treated with detergent. Wild-type nontransformed plants were used as negative controls in experimental assessments.
Establishment of hydroponic cultures
Seeds of IgG, mIgG, and WT plants were surface sterilized by immersion in 20% v/v bleach (Domestos, Lever Brothers, London, UK), washed in sterile distilled water, and sown onto MS medium (17)
solidified with 0.7% w/v agar. Seedlings were allowed to reach a length of 
1 cm, then transferred to liquid culture under sterile conditions. Plants were placed through a perforation in a plastic platform in a 150 mL glass jar containing 25 mL of liquid MS medium (15 g/L sucrose) so that the root was immersed in liquid medium and the shoot was above the platform. IgG, mIgG, and WT hydroponic cultures were established and medium was replaced every 28 days. Plants were maintained at 25°C with a 16 h photoperiod in an environmental incubator (Percival, Perry, IA) and grown to a height of 7 cm before experimental assessments.
Formation and harvesting of immune complex
Experiments were designed to test for immune complex formation in the medium surrounding roots of IgG plants and in leaf tissue of mIgG plant cultures. Before the experimental assay, MS medium was removed from the hydroponic cultures and replaced with 25 mL of fresh MS medium (15 g/L sucrose) containing 8 g/L gelatin. Gelatin was included in the culture medium as it had been shown to stabilize antibody in MS medium (18
, 19)
. After 30 min, an Escherichia coli expressed recombinant fragment of SA I/II (comprising amino acids 39–983 of native SA I/II) was carefully pipetted directly into the medium surrounding the roots at a dilution of either 0.1% v/v (for IgG plants) or 1% v/v (for mIgG plants), giving final antigen concentrations of 0.05 and 0.5 µg/mL respectively.
For IgG plants, 200 µL of medium surrounding the roots was taken from each plant just before addition of recombinant SA I/II and 24 h after inoculation. These samples were frozen at -20°C, before assaying for immune complex formation by ELISA and Western blot. Wild-type plants subjected to identical treatment were used as controls.
In assessments using mIgG plants, leaf samples (100 mg) were removed from leaves toward the apex of the plant 24 h after addition of antigen to the medium. Care was taken to prevent medium (containing antigen) coming into contact with the leaf material. The leaf material was ground in phosphate-buffered saline (PBS) containing 2.5% bovine serum albumin (BSA), 10 µg/mL leupeptin, and 1% v/v dilution Triton X-100. Samples were frozen at -20°C, then assayed for immune complex formation by ELISA and Western blot. Control assessments consisted of WT plants given an identical treatment and mIgG plants maintained in medium lacking antigen.
In the second experiment, mIgG plants were placed in fresh medium and antigen was added as before. Leaf samples were immediately removed (time=0). Additional leaf samples were removed at intervals of 4 h, 16 h, 36 h, and 8 days. Leaf samples were ground and frozen as described previously, then assayed for immune complex formation by ELISA.
In the third experiment (designed to test the persistence of immune complex in mIgG leaf tissue), plants were maintained in medium containing antigen for 24 h. The medium was then removed and roots were washed in 10 changes of sterile distilled water. Fresh medium lacking antigen was then given to the plant cultures and leaf samples were removed at 24 h intervals for a 72 h period. After removal, samples were ground and frozen as described previously before assessment for immune complex in by ELISA.
Detection of free antigen in WT plants
Wild-type plant cultures were maintained in medium containing 1% v/v dilution of recombinant SAI/II or medium in which antigen had been omitted. After 24 h, leaf samples were removed, ground, and frozen as described, and subsequently tested by ELISA for the presence of free antigen.
ELISA
All incubations were performed at 37°C.
To detect the immune complex, ELISA plates (Nunc Immobilon, Watford, UK) were coated with a 7.5 µg/mL solution (in PBS) of affinity-purified sheep anti-rabbit immunoglobulins (The Binding Site, Birmingham, UK). Plates were incubated for 2 h, washed, then blocked by addition of 2.5% w/v BSA in PBS with another 2 h incubation. Plates were washed three times with distilled water containing 0.1% Tween 20 (H2OT), dried, then stored at -20°C for use in subsequent assays. For the assay, a rabbit polyclonal anti-SA I/II antiserum diluted 1/500 v/v (in PBS) was added to the plates, followed by 2 h incubation. Plates were then washed three times with H2OT. Leaf (mIgG plants) or medium (IgG plants) samples (and corresponding WT controls) were defrosted and centrifuged (20,000 g, 10 min 4°C), and aliquots were added to the ELISA plate wells. Plates were incubated an additional 2 h. Secondary antibody was an affinity-purified, HRPO-conjugated anti-mouse IgG1 (The Binding Site, UK) and incubation was for 2 h. Plates were washed and developed by addition of tetramethylbenzidine dihydrochloride peroxidase substrate (Sigma, Poole, Dorset, UK). Color development was allowed to proceed for 10 min, then 25 µL of 2M H2SO4 was added to each well and optical density was read at 450 nm on an ELISA plate reader (Anthos, East Sussex, UK).
To detect free antigen, the coating of plates with sheep anti-rabbit immunoglobulins, blocking with BSA, addition of rabbit anti-SA I/II antiserum, incubation of leaf samples in ELISA wells, and subsequent washing were as described earlier. A 10% v/v dilution (in PBS) of Guy’s 13 hybridoma culture supernatant (containing 1 µg/mL antibody) was added to the wells and incubated for 2 h, followed by washing. Addition of secondary antibody, color development, and measurement of optical density were again as described previously.
Western blot
Pooled liquid root medium (250 µL; IgG and WT plants) or 700 µL of crude leaf extracts (mIgG and WT plants) were mixed with a 100 µL aliquot (1:1 v/v) of protein G-Sepharose beads (Sigma) for 3 h at 4°C. The Sepharose was washed three times in PBS containing 0.1% Tween 20 and bound antibodies were eluted by boiling with SDS-PAGE sample buffer [75 mM Tris-HCl (pH 6.8), 2% SDS, 5% ß-mercaptoethanol]. Eluted samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose. The blot was incubated for 2 h at 37°C in PBS with 0.05% Tween 20 and 1% nonfat dry milk. Detection of proteins bound to the nitrocellulose was with a rabbit anti-SA I/II antiserum, followed by an alkaline phosphatase-labeled goat anti-rabbit IgG antiserum (Sigma). Visualization was with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad, Hemel Hempstead, UK).
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RESULTS
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Immune complex formation in the rhizosphere of IgG plants
The capture ELISA for detection of immune complexes involves capture of antigen (by antisera on the solid phase), followed by detection of cocaptured mouse immunoglobulin heavy chain. A positive control consisting of Guy’s 13 IgG hybridoma cell culture supernatant (0.1 µg/mL IgG) mixed in vitro with a crude streptococcal antigen preparation (0.5 µg/mL) in plant nutrient medium demonstrated this ELISA to be functional and specific. No reactivity was detected in medium containing only antibody or only antigen or lacking both.
Using the plant material, Fig. 1
shows that immune complex formation between rhizosecreted Guy’s 13 antibody and exogenously applied SA I/II occurred in the plant nutrient medium surrounding the roots. Assay of medium aliquots harvested before and after inoculation of SA I/II demonstrated that an antigen/antibody complex had occurred over a 24 h period, but not in samples from WT plants. No difference was observed in the OD 450 readings of medium samples taken from IgG and nontransformed WT plants before addition of antigen.
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Figure 1. ELISA demonstrating immune complex formation in plant culture medium surrounding roots of IgG plants (n=5) compared to WT nontransformed controls (n=5) 24 h after addition of 0.1% v/v recombinant SA I/II (0.05 µg/mL). Time 0 represents samples taken immediately before antigen addition. Results are mean OD450 readings ± SE.
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Immune complexes could also be affinity purified from medium samples by incubation with protein G-labeled Sepharose beads, which is a ligand for immunoglobulin heavy chains. After elution, samples were probed with anti-SA I/II antisera in Western blots (Fig. 2
). No SA I/II was recovered from the medium from WT plants, even though these samples had been inoculated with SA I/II in the same way as the IgG transgenic plants. This demonstrates the specificity of the extraction procedure using protein G as the affinity ligand. Furthermore, Guy’s 13 IgG monoclonal antibody (G13 IgG) shows no cross-reactivity with the antisera used in the Western blot. Two clear bands were detected from the IgG samples, which correspond approximately to the expected electrophoretic migration pattern of the recombinant SA I/II fragment. The higher band probably represents the full-length recombinant antigen fragment, whereas the lower band is likely to be a proteolytic degradation fragment. The original recombinant E. coli extract contains a number of degradation products of the expressed SA I/II fragment, resulting in the presence of multiple bands between 25 and 150 kDa that are recognized by the polyclonal rabbit anti-SA I/II antiserum (rSAI/II lane). In contrast, the antigen captured by plant IgG is represented by two dominant bands (recognized by the same polyclonal antiserum). This is a reflection of the epitope recognized by Guy’s 13, which is conformational, and requires sequence from two distant regions of the molecule (unpublished data). Therefore small fragments (containing only one of the regions, or less) would not be captured.
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Figure 2. Western blot demonstrating copurification of SA I/II with Guy’s 13 IgG from pooled liquid medium surrounding plant roots. The medium was incubated with protein G-Sepharose and affinity-purified eluates were separated by 10% SDS-PAGE. Detection of blotted proteins was with a rabbit anti-SA I/II antiserum, followed by alkaline phosphatase anti-rabbit IgG antiserum. Position of Mr protein markers is indicated. rSA I/II: E. coli extract containing recombinant SA I/II fragment; G13 IgG: Guy’s 13 hybridoma cell culture supernatant.
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Immune complex formation in the leaves of mIgG plants
ELISA analysis of leaf extracts demonstrated fourfold higher optical density readings in mIgG plants exposed to antigen compared with WT plants given antigen or unexposed mIgG plants (Fig. 3
). Western blot confirmed that immune complex was present in leaf tissue with the detection of SA I/II from crude leaf preparations initially incubated with protein G to purify Guy’s 13 IgG (Fig. 4
). Results from two representative mIgG plants and one WT plant are shown. Leaves from the top of the plant were used in these studies to prevent any possible contamination with antigen in the medium (which could lead to a false positive result after grinding of the leaf tissue). However, we have shown that immune complex was also present at similar concentration in leaves toward the base of the plant (data not shown).
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Figure 3. ELISA comparing immune complex formation in leaves of mIgG plants (n=8) and WT controls (n=7) 24 h after addition of 1% v/v recombinant SA I/II (0.5 µg/mL) to hydroponic culture medium. Also included are results for mIgG plants not exposed to antigen (mIgG, n=5). + SA I/II represents addition of antigen to culture medium. Results are mean OD450 readings ± SE.
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Figure 4. Western blot demonstrating copurification of SA I/II with Guy’s 13 IgG from leaf extracts in mIgG plants. The clarified extract was incubated with protein G-Sepharose and affinity-purified eluates were separated by 10% SDS-PAGE. Detection of blotted proteins was with a rabbit anti-SA I/II antiserum, followed by alkaline phosphatase anti-rabbit IgG antiserum. Position of Mr protein markers is indicated. Samples from 2 representative mIgG and 1 WT plants are shown.
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To demonstrate the formation of immune complexes with time, leaf samples were analyzed at different intervals for up to 8 days. The results showed that immune complexes were clearly detected at 16 h and had reached saturation by 36 h in these small plants (Fig. 5
).
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Figure 5. ELISA demonstrating immune complex formation in leaves of mIgG plants (n=4) compared to WT plants (n=4). Recombinant SA I/II was added to hydroponic culture medium around the roots at a concentration of 1% v/v (0.5 µg/mL); leaves were removed at the intervals indicated. Results are mean OD450 readings ± SE.
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Immune complexes appeared to be stable in the leaf tissue and were detectable 72 h after removal of antigen from the nutrient medium (Fig. 6
). The concentration of leaf complex in leaf tissue remained constant over this period, indicating early saturation of antibody with antigen. It also suggests that antigen captured by mIgG in plants is stable and not processed further by plant cells, and that there is little rerelease into the external environment.
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Figure 6. ELISA demonstrating immune complex persistence in leaves of mIgG plants (n=3) compared to a WT plant (n=1). Recombinant SA I/II was initially added to hydroponic culture medium around the roots at a concentration of 1% v/v ( 0.5 µg/mL). After 24 h incubation, medium was removed, plants were washed 10 times in sterile distilled water, and given fresh medium (lacking antigen). Leaf samples were taken at 24 h intervals for 72 h and tested for immune complex formation. Results are mean OD450 readings ± SE.
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Detection of free antigen in WT plants
WT plants were dosed with SA I/II and after 24 h leaf material was tested by ELISA for the presence of free antigen. Despite using a sensitive ELISA assay for SA I/II (sensitive to concentrations <10 ng/mL), we were unable to detect any free antigen, with identical results obtained for WT plants with or without application of antigen.
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DISCUSSION
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In the present study, ELISA of plant medium around the roots of IgG plants indicated that an immune complex was formed between rhizosecreted Guy’s 13 antibody and SA I/II antigen that had been added to the medium. The copurification of SA I/II after specific affinity purification of Guy’s 13 IgG is further evidence that the two molecules had complexed in the medium surrounding the plant roots. Overall, these results suggest that rhizosecretion of appropriate antibodies in the open aquatic environment could reduce the bioavailability and attenuate harmful effects of environmental pollutants by neutralizing biologically active epitopes. Alternatively, the establishment of rhizosecreting hydroponic cultures within a waste-water treatment plant could provide a decontamination strategy, as immune complexes could also be removed from the aqueous environment by simple precipitation strategies.
ELISA and Western blot analysis demonstrate that plants are able to sequester antigen in the leaves by formation of an immune complex with cell membrane-retained antibody. An experiment was performed to determine that immune complex formation occurred in situ on the plasma membrane and to exclude the possibility that free antigen in leaves only complexed with antibody during the grinding process. Wild-type plants were dosed with SA I/II and an ELISA was undertaken in an attempt to detect free antigen in leaf tissue. Our inability to detect free antigen in WT plants suggests that antigen is transported through the plant and degraded in the leaves. Alternatively, the expression of antibody might be a requirement for antigen uptake. In mIgG plants, antigen may be protected from degradation in leaf tissue by binding to antibody and forming immune complex on the extracellular surface of the plasma membrane. The protein antigen used is large (104 kDa), but comparable to other native xylem sap proteins that have been described (20)
.
Using this model system with a large protein antigen, the principle of antibody-mediated sequestration in plants has been demonstrated. With the experimental system we used, however, we were not able to demonstrate antigen depletion from the plant medium, because the antigen concentrations used were greatly in excess of the antibody capacity of each plant. However, at low antigen concentrations (data not shown), we were unable to demonstrate immune complex formation in the plants, which raises the possibility that protein uptake may be concentration dependent. The uptake of proteins into plants overall is likely to be a complex process requiring further investigation. Further studies are required to determine the exact rate of antigen transport within the plant as well as the status of antigen in the roots and vascular tissues of WT and mIgG plants. Factors such as rate of transport or stability will vary with different pollutants; it seems likely that most of the potential targets for this technology will not be proteins, but organic molecules that are less susceptible to degradation.
We believe that the two strategies of rhizosecretion-mediated antibody neutralization and in planta antigen sequestration demonstrated for Guy’s 13 and SA I/II could be extended to virtually any pollutant to which monoclonal antibodies can be generated. The choice of two strategies would increase the number of possible applications of this technology, extending its use to both soil and surface water environments. It is notable that the protein antigen was stabilized by complexing with membrane antibody in plant leaves for up to 72. Thus, this technology may have another application in phytomining: the extraction and concentration of valuable soil components.
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ACKNOWLEDGMENTS
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We are grateful to Dr. Alessandro Vitale for valuable discussions. This work was funded in part by Wellcome Trust grant 062710 and European Union Framework IV.
Received for publication March 12, 2002.
Accepted for publication August 5, 2002.
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ABSTRACT
INTRODUCTION
