Highly sensitive fluorescence quantification of picloram using immunorecognition liposome.

Picloram is a widely used chlorinated herbicide, which is quite persistent and mobile in soil and water with adverse health and environmental risks. A simple and efficient method with high sensitivity and good selectivity was developed in this work to analyze picloram. The aldehyde group functionalized quartz glass plate was used to catch picloram by Schiff base reaction, and reacted with the liposomes-labeled antibody. The fluorescein isothiocyanate (FITC) solution was encapsulated in the liposomes. After being released from the liposomes, the fluorescence of FITC was measured by a fluorimeter. It was found that the fluorescence intensity is linearly correlated to the logarithm of picloram concentration, ranging from 1.0 × 10(-4) to 100 ng mL(-1), with a detection limit of 1.0 × 10(-5) ng mL(-1). Picloram concentration in real wastewater samples were accurately measured by the proposed method and HPLC, the results of the two methods were approximately the same. The proposed method showed high sensitivity and good selectivity, and could be an efficient tool for picloram quantitative analysis.

[Liposome immunosensor for picloram in water based on electrochemical polymerization].

A "sandwich-type" immunosensor for the determination of picloram in water environment was developed based on the immunoliposomes prepared by crosslinking rabbit antibody against picloram (anti-picloram) and potassium ferrocyanide-encapsulated liposomes with glutaraldehyde. The working conditions including modification components on the electrode were optimized. The best performance is obtained using 0.5% of Nafion, 10 mg x mL(-1) of multiwalled carbon nanotubes (MWCNTs) and 50 microg x mL(-1) of anti-picloram. The preparation and detection process of immunosensor was as follows. Cyclic voltammetry was applied to urge the electrochemical polymerization of 3,4-ethylenedioxythiophene (EDOT) and the anti-picloram was immobilized on the modified glassy carbon electrode (GCE). Then the electrode was incubated with the analytes and immunoliposomes sequentially. The bound liposomes were lysed with TritonX-100, and square-wave voltammetry was applied to determine current response of picloram concentration. The whole process was able to be completed in 70 min. The immunosensor has good reproductivity after being soaked in 0.1 mol x L(-1) of H3PO4 in 5 min. The result shows that lower detection limit for picloram is 10(-10) mol x L(-1) with linear range of 10(-10)-10(-4) mol x L(-1), which meets the detection requirement of picloram for the safety of drinking water.

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.

Increasing expression of an anti-picloram single-chain variable fragment (ScFv) antibody and resistance to picloram in transgenic tobacco (Nicotiana tabacum).

Systematic research involving four chimeric gene constructions designed to express the same anti-picloram single-chain variable fragment (scFv) antibody is described. Agrobacterium-mediated transformation produced at least 25 transgenic tobacco plants with each of these, and the number of T-DNA loci in each plant was determined using kanamycin-resistance segregation assays. The relative amounts of active and total scFv in each plant were evaluated using quantitative enzyme-linked immunosorbent assay and immunoblot technologies, respectively. No significant differences in scFv activity were found among the four groups of single-locus plants, although the 35S/M construct was found to produce significantly more total anti-picloram scFv than the other three constructs. A dose-response bioassay involving T(1) seedlings from several of the highest expressers of active scFv demonstrated resistance to a constant exposure of picloram at 5 x 10(-)(8) M. Other approaches for increasing antibody-based herbicide resistance are discussed, as further improvements are needed before practical application of this technology.

Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.

Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.

Synthesis of ligand-specific phage-display ScFv against the herbicide picloram by direct cloning from hyperimmunized mouse.

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and heavy domain DNA were joined to produce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein gIIIp. The phage-display scFv library (10(4) clones) was selected against picloram conjugated to ovalbumin. After five rounds of panning, individual clones were analyzed. ScFv with different affinities to picloram (IC(50) values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. The increased avidity of the phage vector enhanced the selection (i.e., panning) of multiple picloram-specific recombinant antibodies. Stringent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of the V(L) belonged to the V kappa 9A family joined to J kappa 2 segments. All of the V(H) belonged to the V(H)()7183 family and joined to two different J segments (i.e., J(H)()2 or J(H)()4). Different from the immune response to large molecular weight molecules (MW > 10,000 Da), which requires both VDJ segment rearrangement and somatic hypermutations, production of high-affinity antibodies to picloram, a small ligand having a formula weight of 241.5 Da, predominantly requires somatic hypermutations.