A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

Oriented Functionalization of Magnetic Beads with in Vivo Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples.

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 µg/L of MC.

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC50) were 0.87, 1.17 and 1.47µg/L, their detection limits (IC10) were 0.06, 0.08 and 0.12µg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water.

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 µg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

Multihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin.

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 µg/L, well below the WHO's maximum recommendation of 1 µg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

Automated online optical biosensing system for continuous real-time determination of microcystin-LR with high sensitivity and specificity: early warning for cyanotoxin risk in drinking water sources.

The accelerated eutrophication of surface water sources and climate change have led to an annual occurrence of cyanobacterial blooms in many drinking water resources. To minimize the health risks to the public, cyanotoxin detection methods that are rapid, sensitive, real time, and high frequency must be established. In this study, an innovative automated online optical biosensing system (AOBS) was developed for the rapid detection and early warning of microcystin-LR (MC-LR), one of the most toxic cyanotoxins and most frequently detected in environmental water. In this system, the capturing molecular MC-LR-ovalbumin (MC-LR-OVA) was covalently immobilized onto a biochip surface. By an indirect competitive detection mode, samples containing different concentrations of MC-LR were premixed with a certain concentration of fluorescence-labeled anti-MC-LR-mAb, which binds to MC-LR with high specificity. Then, the sample mixture was pumped onto the biochip surface, and a higher concentration of MC-LR led to less fluorescence-labeled antibody bound onto the biochip surface and thus to lower fluorescence signal. The quantification of MC-LR ranges from 0.2 to 4 µg/L, with a detection limit determined as 0.09 µg/L. The high specificity and selectivity of the sensor were evaluated in terms of its response to a number of potentially interfering cyanotoxins. Potential interference of the environmental sample matrix was assessed by spiked samples, and the recovery of MC-LR ranged from 90 to 120% with relative standard deviation values <8%. The immunoassay performance of the AOBS was validated with respect to that of conventional high-performance liquid chromatography, and the correlation between methods agreed well (R(2) = 0.9762). This system has successfully been applied to long-term, continuous determination and early warning for MC-LR in Lake Tai from June 2011 to May 2012. Thus, the AOBS paves the way for a vital routine online analysis that satisfies the high demand for ensuring the safety of drinking water sources. The AOBS can also serve as early warning system for accidental or intentional water pollution.

Magnetic particle-based enzyme assays and immunoassays for microcystins: from colorimetric to electrochemical detection.

In this work, magnetic particles (MPs) are used as supports for the immobilization of biorecognition molecules for the detection of microcystins (MCs). In one approach, a recombinant protein phosphatase 1 (PP1) has been conjugated to MPs via coordination chemistry, and MC-LR detection has been based on the inhibition of the enzyme activity. In the other approach, a monoclonal antibody (mAb) against MC-LR has been conjugated to protein G-coated MPs, and a direct competitive enzyme-linked immunoparticle assay (ELIPA) has been then performed. Conjugation of biomolecules to MPs has been first checked, and after optimization, MC detection has been performed. The colorimetric PPIA with PP1-MP and the best ELIPA strategy have provided limits of detection (LOD) of 7.4 and 3.9 µg/L of MC-LR, respectively. The electrochemical ELIPA has decreased the LOD to 0.4 µg/L, value below the guideline recommended by the World Health Organisation (WHO). The approaches have been applied to the analysis of a cyanobacterial culture and a natural bloom, and MC equivalent contents have been compared to those obtained by conventional assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results have demonstrated the viability of the use of MPs as biomolecule immobilization supports in biotechnological tools for MCs monitoring.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR.

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy's 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 µg g(-1) leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.

Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 µg/L, with a limit of detection of 0.03 µg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

Photoelectronic characterization of IgG antibody molecule-quantum dot hybrid as biosensing probe.

Quantum dot (QD)-based biomolecule hybrids have recently attracted much attention in specifically identifying and labeling target proteins. In this study, QD encapsulated with immunoglobulin antibodies, as a labeling building block in biosensors, was investigated to clarify the most efficient configuration and photoluminescence behavior. Both the biological recognition capacity and photoluminescence emitting signal of the antibody-coupled nanocrystal were validated through a photoelectrical characterization procedure. Derivation of the optimum number of antibody molecules to be packed onto the QD surface yielded the highest binding capacity for the target antigen. During formation of the bioactive layer, the intrinsic photoluminescence response of the QDs significantly decreased due to photoinduced hole transfer according to their rearranged electronic structure. The thorough study of this assembly provides a validation approach for the careful titration of biosensor probes for optimal reaction kinetics. Furthermore, it contributes to the development of an effective tool for the application and interpretation of QD-based labeling techniques.

An SPR biosensor for the detection of microcystins in drinking water.

A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC(50) 0.67 ± 0.09 µg L(-1), a detection limit of 73 ± 8 ng L(-1), and a dynamic range from 0.2 to 2.0 µg L(-1) for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay-regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 µg L(-1) established by the World Health Organization for drinking water.

Electrochemical immunoassay using quantum dot/antibody probe for identification of cyanobacterial hepatotoxin microcystin-LR.

The presence of cyanobacterial hepatotoxins such as microcystin-LR poses health threats to humans due to their potential for causing severe physiological effects when contaminated drinking water is ingested. Here, the electrochemical detection of microcystin-LR is explored using a quantum dot/antibody (QD/Ab) probe for nanoparticle-based amplification and direct electrochemical transduction. The immunological recognition of microcystin-LR using the QD/Ab probe was amplified and converted to an electrochemical signal by measuring the cadmium ions released from QD based on square wave stripping voltammetry under optimized electrochemical factors. Whereas a qualitative analysis for microcystin-LR was achieved using the specific peak potential of the anodic voltammogram at -0.6 +/- 0.05 V, concentration of the toxin was quantified based on the charge density of the anodic peak; a dynamic range of 0.227 to 50 microg/L and limit of detection of 0.099 microg/L were obtained with high sensitivity. The extracted microcystin-LR from Microcystis aeruginosa was estimated as 1,944 microg/g of dried weight of the microorganism.

Understanding microcystin-LR antibody binding interactions using in silico docking and in vitro mutagenesis.

Microcystins (MCs) are a group of highly potent cyanotoxins that are becoming more widely distributed due to increased global temperatures and climate change. Microcystin-leucine-arginine (MC-LR) is the most potent and most common variant, with a guideline limit of 1 µg/l in drinking water. We previously developed a novel avian single-chain fragment variable (scFv), designated 2G1, for use in an optical-planar waveguide detection system for microcystin determination. This current work investigates interactions between 2G1 and MC-LR at the molecular level through modelling with an avian antibody template and molecular docking by AutoDock Vina to identify key amino acid (AA) residues involved. These potential AA interactions were investigated in vitro by targeted mutagenesis, specifically, by alanine scanning mutations. Glutamic acid (E) was found to play a critical role in the 2G1-MC-LR binding interaction, with the heavy chain glutamic acid (E) 102 (H-E102) forming direct bonds with the arginine (R) residue of MC-LR. In addition, alanine mutation of light chain residue aspartic acid 57 (L-D57) led to an improvement in antigen-binding observed using enzyme-linked immunosorbent assay (ELISA), and was confirmed by surface plasmon resonance (SPR). This work will contribute to improving the binding of recombinant anti-MC-LR to its antigen and aid in the development of a higher sensitivity harmful algal toxin diagnostic.

Microcystin-LR Enrichment from Freshwater by a Recombinant Plant-derived Antibody Using Sol-Gel-Glass Immunoextraction.

Eutrophication of water bodies can promote cyanobacterial (blue-green algae) blooms, which has become a source of increasing concern for both recreational and drinking water use. Many bacterial species can produce toxins that pose threats to wildlife, domestic animals and humans. Microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. For the first time, lab-scale investigations were performed to test the application of a recombinant plant-derived anti-MC-LR antibody immobilized on an immunoaffinity support material to selectively extract the toxin from spiked freshwater samples. As a comparison, its hybridoma-derived counterpart (murine monoclonal antibody) was evaluated. The antibody-doped material was prepared via an optimized sol-gel process; its stability and binding efficiency of MC-LR in spiked freshwater samples were thoroughly tested using the ELISA and orthogonal LC-MS methods. For removal, two column-based procedures with sequential or continuous cyclic sample addition and a suspension mode (moving adsorbent) were tested. Noteworthy the results obtained with a crude antibody fraction were fully compatible with the highly purified preparation. This study paves the way for further investigation being focused on novel applications of plant-derived anti-MC-LR antibodies in bioremediation to selectively deplete the toxin from freshwater: a green and promising technology without secondary pollution.

High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay.

Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 107 CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli (E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 µg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 µg/L and a limit of detection (LOD) of 0.044 µg/L, which is below the maximum residue limit standard (MRLs) of 1.0 µg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2-106.3% with CVs of 2.62-10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.

Immunomodulatory effects of cyanobacterial toxin cylindrospermopsin on innate immune cells.

Cylindrospermopsin (CYN), a cyanobacterial toxin, is an important water pollutant with broad biological activity. It has been known mainly from tropical areas, but the area of occurrence of its producers is spreading to temperate climates. It can be found in high concentrations in the environment as well as in purified drinking waters. The aim of the study is to bring a basic information on the ability of CYN to interfere with mammalian innate immunity cells and thus increase the understanding of the immunomodulatory potency of CYN. This study investigated whether immune cells can be a target of CYN either alone or in combination with a model immunomodulatory agent, lipopolysaccharide (LPS). We examined the effects on cellular viability and inflammation signaling of CYN on murine macrophage-like RAW 264.7 cells. Macrophages were treated either with pure toxin (1 µM) or together with a known stimulator of immunologically active cells, bacterial or cyanobacterial LPS. CYN has had a significant effect on production on pro-inflammatory mediator tumor necrosis factor α (TNF-α) which correlates with its effect on reactive oxygen species (ROS) production. We found that CYN potentiated the effect of bacterial and cyanobacterial LPS that was documented by activation of inflammatory signaling pathways including mitogen-activated protein kinase p38 as well as consequent expression of inducible nitric oxide synthase (iNOS) and increased production of pro-inflammatory mediators such as nitric oxide (NO), TNF-α, interleukin-6 (IL-6). Our study brings one of the first information that contributes to the elucidation of immunomodulatory role of CYN in macrophages under normal and pro-inflammatory conditions.

Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody.

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7µg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05µg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation <6.8% for scFv-based IC-ELISA to detect MC-LR spiked in water samples, which met the requirement of indoor testing. The present results indicate that we have obtained a high sensitive anti-MC-LR scFv by the MC-LR-immunized phage library construction and screening, and the scFv-based IC-ELISA can be used for monitoring MC-LR in water samples.

Degradation of cyanobacterial toxin, microcystin LR, using chemical oxidants.

Cyanobacterial toxins, microcystins, are very potent hepatotoxins and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, the effects of several environmental factors on production and degradation of toxins produced by cyanobacteria in Lake Soyang have been studied. A new rapid quantification method of microcystins, using fluorescence for a detection signal and a lateral-flow-type immunochromatography as a separation system, was used. Chlorine, potassium permanganate, and hydrogen peroxide were used as chemical oxidants for the degradation of microcystin LR. When chlorine was used, the efficiency of degradation was the highest. The degradation reaction took 40 minutes.

Enhancing recombinant antibody performance by optimally engineering its format.

Antibody-based sensors are now widely used in therapeutics, diagnostics, and in environmental monitoring. Recombinant antibodies are becoming integral parts of such devices due to their reported high affinities, their capacity for engineering to achieve highly defined performance characteristics and the fact that their production can be optimized to a significant degree. To aid as a model for the identification of important analyte binding residues within the antibody sub-structure and elucidate the docking characteristics of small molecules such as metabolites, illicit drugs, biotherapeutics (proteins, peptides and nucleic acids) or toxins towards the antibody, herein we report the binding of the harmful cyanobacterial-toxin, microcystin-leucine-arginine (MC-LR) to a single chain fragment variable (scFv) antibody fragment. Analysis of the binding of MC-LR to this scFv was used to identify key residues of interest and to show how 'freely-available' and 'easily-accessible' computer-based webservers can be utilized to initiate an investigation into the binding characteristics of interacting molecules. In this study, a detailed investigation of the sub-structure of the anti-MC-LR (scFv) was carried out and antibody/small-molecule binding interactions were analyzed. The profile elucidated using computational analysis revealed amino acids of importance in the complementarity determining region light chain region 3 (CDRL3) and framework region 3 (FR3) of the heavy chain. Important amino acid residues within CDRL3 and FR3 were mutated in vitro and sensitivity and binding profiles were examined. It was identified that phenylalanine (F) at position 91 and aspartate (D) at position 92 of the light chain region, and arginine (R) at position 66 in framework region 3 (FR3) of the heavy chain were nvolved in binding. The introduction of an auxiliary antibody domain to the variable heavy and variable light (scFv) to ascertain its influence on stability and binding was also examined. The strategy adopted provided a deeper knowledge of scFv sub-structure and identified the regions and amino acids essential to antibody/small-molecule binding.