Investigation of MCPA (4-Chloro-2-ethylphenoxyacetate) resistance in wild radish (Raphanus raphanistrum L.).

The phenoxy herbicides (e.g., 2,4-D and MCPA) are used widely in agriculture for the selective control of broadleaf weeds. In Western Australia, the reliance on phenoxy herbicides has resulted in the widespread evolution of phenoxy resistance in wild radish (Raphanus raphanistrum) populations. In this research the inheritance and mechanism of MCPA resistance in wild radish were determined. Following classical breeding procedures, F1, F2, and backcross progeny were generated. The F1 progeny showed an intermediate response to MCPA, compared to parents, suggesting that MCPA resistance in wild radish is inherited as an incompletely dominant trait. Segregation ratios observed in F2 (3:1; resistant:susceptible) and backcross progeny (1:1; resistant to susceptible) indicated that the MCPA resistance is controlled by a single gene in wild radish. Radiolabeled MCPA studies suggested no difference in MCPA uptake or metabolism between resistant and susceptible wild radish; however, resistant plants rapidly translocated more (14)C-MCPA to roots than susceptible plants, which may have been exuded from the plant. Understanding the genetic basis and mechanism of phenoxy resistance in wild radish will help formulate prudent weed management strategies to reduce the incidence of phenoxy resistance.

Plant-produced trastuzumab inhibits the growth of HER2 positive cancer cells.

To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.

Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

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.

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.

Comparison of a direct ELISA and an HPLC method for glyphosate determinations in water.

A competitive direct enzyme-linked immunosorbent assay (ELISA) and high-pressure liquid chromatographic (HPLC) methods were compared in terms of accuracy and precision for the detection and quantification of glyphosate-spiked Nanopure, tap, and river waters. The ELISA had a detection limit of 0.6 ng mL(-)(1) and a linear working range of 1-25 ng mL(-)(1), whereas the HPLC method had a detection limit of 50 ng mL(-)(1) and a linear working range of 100-10000 ng mL(-)(l). No statistically significant differences (95% confidence interval) were found between the ELISA and HPLC analysis of the three water matrixes. The coefficients of variation obtained with the ELISA in tap water were between 10 and 19%, whereas the coefficients of variation for the HPLC analysis were between 7 and 15%. The use of ELISA for the analysis of glyphosate in water is a cost-effective and reliable method capable of meeting water quality guidelines established for Europe and North America.