Gillian Strachan

Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat

Single-chain antibody fragments (scAbs), which have a human C-kappa constant domain and a hexa-histidine tail attached to the carboxy terminus of the single-chain Fv (ScFv) fragments to facilitate purification, have been raised against the herbicides paraquat and atrazine and expressed in transgenic Nicotiana tabacum cv. Samsun NN. Prior to purification, the anti-atrazine scAb is expressed as up to 0.014% of soluble leaf protein and has a binding profile in ELISA, against an atrazine-bovine serum albumin (BSA) conjugate, similar to that of the scAb produced in Escherichia coli. Competition ELISA has shown that the plant-derived scAb also recognises free atrazine. Following antibody affinity purification to isolate dimers, the affinity for immobilised antigen approaches that of the parental monoclonal antibody. This was confirmed by surface plasmon resonance analysis. The purified scAb also recognises related triazine herbicides. When isolated from cell-suspension cultures, the anti-paraquat scAb binds to a paraquat conjugate in a concentration-dependent manner, with a profile similar to the parental monoclonal antibody. This is the first demonstration that functional scAbs against organic pollutants can be produced in transgenic plants and that the scAbs may be appropriate for the development of immunoassay-based detection systems.

Use of bacterial biosensors to interpret the toxicity and mixture toxicity of herbicides in freshwater.

The dose response relationship between seven commonly used herbicides and four luminescence-based bacterial biosensors was characterised. As herbicide concentration increased the light emitted by the test organism declined in a concentration dependent manner. These dose responses were used to compare the predicted vs. observed response of a biosensor in the presence of multiple contaminants. For the majority of herbicide interactions, the relationship was not additive but primarily antagonistic and sometimes synergistic. These biosensors provide a sensitive test and are able to screen a large volume and wide range of samples with relative rapidity and ease of interpretation. In this study biosensor technology has been successfully applied to interpret the interactive effects of herbicides in freshwater environments.

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

Production of soluble single-chain T-cell receptor fragments in Escherichia coli trxB mutants.

Antibodies and T cell receptors (TCR) both belong to the immunoglobulin superfamily whose members are characterised by the possession of one or more immunoglobulin domains. The production of soluble single chain antibody fragments in Escherichia coli has, in recent years, become a routine laboratory procedure. In contrast, the production of T cell receptors in bacteria has remained problematic as the majority of the recombinant protein is insoluble. In this paper we show that single chain TCR produced in E. coli BL21 (DE3) and directed to the periplasm was also insoluble and that this was in part due to the failure of the cell protein processing machinery to cleave the pelB leader sequence. This problem was overcome by expressing the single chain TCR in the cytoplasm of E. coli which carry an inactive thioredoxin reductase gene. This strain allows the formation of disulphide bonds in the cell cytoplasm which we believe encourages the correct folding of the recombinant protein. We have constructed both a human and mouse single chain TCR in these bacteria and demonstrated using BIAcore technology that these molecules have folded in a conformation which allows their recognition by conformational specific ligands. In addition, we have used one of our soluble single chain TCR preparations to isolate a TCR specific Fab molecule from a phage antibody library.

Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules

Single‐chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co‐expression with the colE1‐compatible, arabinose‐inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats.

Neutralisation and binding of VHS virus by monovalent antibody fragments.

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.

Anti-mesotrione single chain antibody fragments derived using either phage display or via hybridoma technology: Selection, characterization and stabilization

Single-chain antibody fragments (scAb), specific for the triketone herbicide mesotrione, have been expressed and purified from the bacterium Escherichia coli. ScAbs were identified following the cloning of variable heavy and light chains from anti-mesotrione monoclonal antibodies (mAbs) and from panning an immunized phage display library. Both the monoclonal and phage derived scAbs were produced from the same immunized mouse spleen. In competition ELISA, the mAb derived scAbs (35 and 36) showed similar specificity for mesotrione compared with the parental mAbs. MAb 35 was the most sensitive with a limit of detection (IC20 value) within EC legislative limits for drinking water at 0.04 μg l−1. The best phage scAb (H6) had sensitivities approximately 20× less than scAb 35. In competition ELISA, scAb 35 showed reduced sensitivity to sulcotrione, a structurally related triketone, with IC50 values in the μM range. The introduction of an interdomain disulphide bond into scAb 35 resulted in greater stability in non-physiological environments. Immunoassay may provide a simple, cheap and rapid test for detection of mesotrione in a range of environments including soil and food.

Immunomethods for Detecting a Broad Range of Polychlorinated Biphenyls

Aroclor 1254 (consisting of a mixture of over 100 different congeners of polychlorinated biphenyls (PCBs) was adsorbed on to the surface of polystyrene microbeads and used to immunize mice and rabbits. High titre antisera were produced. PCBs were also adsorbed on to antimony pentoxide microbeads which were then used effectively as immobilized antigen in microbead‐based ELISA assays. Antisera raised with Aroclor 1254‐coated beads contained antibodies with high affinity (as determined by BIAcore analysis) against tetracholoro‐biphenyls even though these congeners represent only a minor fraction in Aroclors.