Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth.

Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1 prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes monoamines.

4-Aminobutyrate (GABA) accumulates in apple fruit during controlled atmosphere storage. A potential source of GABA is the polyamine putrescine, which can be oxidized via copper-containing amine oxidase (CuAO), resulting in the production 4-aminobutanal/Δ(1)-pyrroline, with the consumption of O2 and release of H2O2 and ammonia. Five putative CuAO genes (MdAO genes) were cloned from apple (Malus domestica Borkh. cv. Empire) fruit, and the deduced amino acid sequences found to contain the active sites typically conserved in CuAOs. Genes encoding two of these enzymes, MdAO1 and MdAO2, were highly expressed in apple fruit and selected for further analysis. Amino acid sequence analysis predicted the presence of a C-terminal peroxisomal targeting signal 1 tripeptide in MdAO1 and an N-terminal signal peptide and N-glycosylation site in MdAO2. Transient expression of green fluorescent fusion proteins in Arabidopsis protoplasts or onion epidermal cells revealed a peroxisomal localization for MdAO1 and an extracellular localization for MdAO2. The enzymatic activities of purified recombinant MdAO1 and MdAO2 were measured continuously as H2O2 production using a coupled reaction. MdAO1 did not use monoamines or polyamines and displayed high catalytic efficiency for 1,3-diaminopropane, putrescine and cadaverine, whereas MdAO2 exclusively utilized aliphatic and aromatic monoamines, including 2-phenylethylamine and tyramine. Together, these results indicate that MdAO1 may contribute to GABA production via putrescine oxidation in the peroxisome of apple fruit under controlled atmosphere conditions. MdAO2 seems to be involved in deamination of 2-phenylethylamine, which is a step in the biosynthesis of 2-phenylethanol, a contributor to fruit flavor and flower fragrance.

Plant-based solutions for veterinary immunotherapeutics and prophylactics.

An alarming increase in emergence of antibiotic resistance among pathogens worldwide has become a serious threat to our ability to treat infectious diseases according to the World Health Organization. Extensive use of antibiotics by livestock producers promotes the spread of new resistant strains, some of zoonotic concern, which increases food-borne illness in humans and causes significant economic burden on healthcare systems. Furthermore, consumer preferences for meat/poultry/fish produced without the use of antibiotics shape today's market demand. So, it is viewed as inevitable by the One Health Initiative that humans need to reduce the use of antibiotics and turn to alternative, improved means to control disease: vaccination and prophylactics. Besides the intense research focused on novel therapeutic molecules, both these strategies rely heavily on the availability of cost-effective, efficient and scalable production platforms which will allow large-volume manufacturing for vaccines, antibodies and other biopharmaceuticals. Within this context, plant-based platforms for production of recombinant therapeutic proteins offer significant advantages over conventional expression systems, including lack of animal pathogens, low production costs, fast turnaround and response times and rapid, nearly-unlimited scalability. Also, because dried leaves and seeds can be stored at room temperature for lengthy periods without loss of recombinant proteins, plant expression systems have the potential to offer lucrative benefits from the development of edible vaccines and prophylactics, as these would not require "cold chain" storage and transportation, and could be administered in mass volumes with minimal processing. Several biotechnology companies currently have developed and adopted plant-based platforms for commercial production of recombinant protein therapeutics. In this manuscript, we outline the challenges in the process of livestock immunization as well as the current plant biotechnology developments aimed to address these challenges.

Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts.

To study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAi-based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5' and 3' UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5'UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

Antibody-dependent cell-mediated cytotoxicity- and complement-dependent cytotoxicity-independent bactericidal activity of an IgG against Pseudomonas aeruginosa O6ad.

In addition to Ag recognition, some Abs are capable of killing target organisms in the absence of phagocytes and complement. In this study, we report that an anti-Pseudomonas aeruginosa O6ad LPS IgG(1), tobacco-expressed human S20 IgG(1) (te-hS20), as well as its recombinant Fab and single-chain variable fragment (scFv) fragments have cellular- and complement-independent bactericidal activity. te-hS20 and its Fab and scFv significantly reduced viability of P. aeruginosa O6ad in dose- and time-dependent manners in vitro and also showed lower levels of bactericidal activity against P. aeruginosa PAO1, but had no activity against P. aeruginosa O10, Escherichia coli TG1, and Streptococcus agalactiae. The H chain and its Fd fragment both had significant Ag-binding and bactericidal activities against P. aeruginosa O6ad. Bactericidal activity was completely inhibited with specific LPS Ag, suggesting that Ag binding is involved in the bactericidal mechanism. Live/dead cell staining and electron microscopic observations indicate that the bactericidal effect was due to disruption of the cell wall and suggest inhibition of cell division. In addition to te-hS20, the Fab and scFv were also protective in vivo, as leukopenic mice had prolonged and improved survival after administration of these Ab fragments followed by challenge with P. aeruginosa O6ad cells at 80-90% lethal dose, supporting a bactericidal mechanism independent of phagocytes and complement. Understanding of the bactericidal mechanism will allow assessment of the potential for therapeutic application of these Abs.

Development of single-chain variable fragment (scFv) antibodies against hapten benzo[a]pyrene: a binding study.

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V(L)) and heavy (V(H)) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V(L) and V(H) chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V(L) and V(H) domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V(L) and V(H) combinations. On the basis of our data, it was shown that the V(H) plays the major role for specific binding of B[a]P, whilst the V(L) can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V(L) in providing cross-reactivity.

Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.

Multivalent anchoring and oriented display of single-domain antibodies on cellulose.

Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 °C for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.

A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes.

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

Isolation and affinity maturation of hapten-specific antibodies.

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.

Isolation, characterization and pentamerization of alpha-cobrotoxin specific single-domain antibodies from a naïve phage display library: preliminary findings for antivenom development.

Conventional antivenoms to snakebite generated from the serum of immunized animals, often elicit adverse reactions and have mismatched pharmacokinetic profiles with their target toxins due to antibody/toxin size discrepancies which results in poor neutralization. Furthermore, animal immunization protocols are often lengthy and have batch to batch variability. Recombinant V(H)H-based antivenoms may help overcome these problems. Three V(H)H fragments with specificity to alpha-cobrotoxin, a snake neurotoxin from Naja kaouthia venom, were isolated from a naïve llama V(H)H phage-display library. Alpha-cobrotoxin-binding specificity was determined using a phage-displayed V(H)H ELISA format. Sequence analysis shows two of the three clones differ by only two amino acid substitutions, while the third is unique. Surface plasmon resonance analysis determined the K(D) values of the interactions to be 2, 3 and 3 microM. These affinities are too low for alpha-cobrotoxin detection in a standard ELISA format, or for practical use as therapeutic agents. However, improved functional affinity was obtained via antibody pentamerization and alpha-cobrotoxin detection was possible using a pentabody-based ELISA. Development of antivenoms composed of a mixture of antibody fragments, such as V(H)Hs and V(H)H multimers, may help match the pharmacokinetic profiles of complex venoms, improving antivenom biodistribution, and toxin neutralization while reducing adverse effects in humans.

Picloram resistance in transgenic tobacco expressing an anti-picloram scFv antibody is due to reduced translocation.

Picloram resistance exhibited by transgenic tobacco (Nicotiana tabacum) plants expressing an anti-picloram single-chain variable fragment (scFv) antibody was investigated through the study of homozygous lines expressing the antibody. Dose-response bioassays, using foliar application of picloram, showed that these homozygous transgenic plants were resistant to at least 5 g of ai ha-1 picloram and grew normally to produce seed, whereas wild-type plants did not survive. Although these lines had improved resistance compared with those previously reported, significant improvements are still required to achieve field-level resistance. Uptake and translocation studies demonstrated that [14C]picloram translocation from treated leaves to the apical meristem was reduced in transgenic versus wild-type plants. The presence of [14C]picloram visualized by autoradiography and quantified by liquid scintillation spectrometry, demonstrated the distribution of more picloram in the treated leaf and less in the apical meristem of transgenic plants when compared to wild-type plants. No differences between transgenic and wild-type plants were found in the distribution of [14C]clopyralid, a herbicide with structural similarity to picloram as well as the same mechanism of action. No differences were found in the metabolism of [14C]picloram. Taken together, these results suggest that reduced translocation to the site of action is a major mechanism responsible for picloram resistance in tobacco plants expressing this anti-picloram antibody.

Basis for antagonism by sodium bentazon of tritosulfuron toxicity to white bean (Phaseolus vulgaris L.).

White bean (Phaseolus vulgaris L.) was used to study the antagonism caused by Na-bentazon on the phytotoxic action of the sulfonylurea (SU) herbicide tritosulfuron. After 168 h, uptake and translocation of [14C]tritosulfuron were reduced by 60 and 89%, respectively, when Na-bentazon was added to the mixture. Addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon completely eliminated the negative effects on [14C]tritosulfuron uptake but not on its translocation. Scanning electron microscopy revealed that a mixture of Na-bentazon plus tritosulfuron plus DASH HC (0.156%) formed a rough layer of grain-like crystals on the leaf surface, whereas the addition of (NH4)2SO4 or replacement of Na-bentazon with NH4-bentazon resulted in amorphous deposits that may be more easily absorbed. The antagonism of tritosulfuron's phytotoxicity by Na-bentazon involves two separate processes, chemical (uptake effect) and biochemical (translocation effect).

Detection of Listeria monocytogenes and the toxin listeriolysin O in food.

Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus. The mortality rate for those contracting listeriosis is approximately 20%. Currently, the United States has a zero tolerance policy regarding the presence of L. monocytogenes in food, while Canada allows only 100 cfu/g of food. As such, it is essential to be able to detect the pathogen in low numbers in food samples. One of the best ways to detect and confirm the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. The LLO-encoding gene (hlyA) is present only in virulent strains of the species and is required for virulence. LLO is a secreted protein toxin that can be detected easily with the use of blood agar or haemolysis assays and it is well characterized and understood. This paper focuses on some of the common methods used to detect the pathogen and the LLO toxin in food products and comments on some of the potential uses and drawbacks for the food industry.

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin.

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.

MCPA (4-Chloro-2-ethylphenoxyacetate) resistance in hemp-nettle (Galeopsis tetrahit L.).

The physiological basis for MCPA resistance in a hemp-nettle (Galeopsis tetrahit L.) biotype, obtained from a MCPA-resistant field population, was investigated. Dose-response studies revealed that the resistance factor for MCPA, based on GR50 comparisons of total dry weight of resistant (R) and susceptible (S) plants, was 3.3. Resistance factors for fluroxypyr, dicamba, 2,4-D, glyphosate, and chlorsulfuron were 8.2, 1.7, 1.6, 0.7, and 0.6, respectively. MCPA resistance was not due to differences in absorption, because both R and S biotypes absorbed 54% of applied [14C]MCPA 72 h after treatment. However, R plants exported less (45 vs 58% S) recovered 14C out of treated leaves to the apical meristem (6 vs 13% S) and root (32 vs 38% S). In both biotypes, approximately 20% of the 14C recovered in planta was detected as MCPA metabolites. However, less of the 14C recovered in the roots of R plants was MCPA. Therefore, two different mechanisms protect R hemp-nettle from MCPA phytotoxicity: a lower rate of MCPA translocation and a higher rate of MCPA metabolism in the roots. In support of these results, genetic studies indicated that the inheritance of MCPA resistance is governed by at least two nuclear genes with additive effects.

Selection, characterization, and CDR shuffling of naive llama single-domain antibodies selected against auxin and their cross-reactivity with auxinic herbicides from four chemical families.

Indoleacetic acid (IAA)-binding single-domain antibodies (sdAbs) were isolated from a naive phage-display library constructed from the heavy chain antibody repertoire of a Ilama. The highest-affinity sdAb isolated (CSF2A) had a K(D) of 5-20 microM for two IAA-protein conjugates and a K(D) of 20 microM for free IAA. This sdAb also bound to a synthetic auxin analogue, 1-naphthaleneacetic acid (NAA), and to six auxinic herbicides (K(D) values of 0.5-2 mM), but not to serotonin and tryptophan, which are structurally similar to IAA but have no auxinic activity. To understand how sdAb CSF2A binds IAA and to determine which complementary-determining region(s) (CDR) participate(s) most in binding IAA, CSF2A was shuffled with four other sdAb clones by staggered extension process (StEP). After panning against IAA, two shuffled sdAbs were found: sdAb CSB1A, which originated from three different parental clones, and sdAb CSE8A, derived from two parental clones. These shuffled sdAbs and CSF2A were each fused to the B subunit of the Escherichia coli verotoxin, resulting in the formation of the pentamerized sdAbs V2NCSB1A, V2NCSE8A, and V2NCSF2A, which were analyzed by surface plasmon resonance (SPR) along with the sdAbs previously isolated. The shuffled clones had affinity for IAA (20 microM) similar to that of the highest affinity parental clone CSF2A, but much lower affinity for the auxinic herbicides. CDR2 was instrumental in binding IAA, whereas hydrophobic CDR3 was important for binding the auxinic herbicides. A novel SPR methodology is also described for specific immobilization of pentamerized sdAbs, allowing determination of K(D) values of Ab interaction with underivatized, low molecular weight haptens.