A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET)

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR).</p

A homogeneous assay for rapid detection of cyanobacterial peptide hepatotoxins: Microcystins and nodularins

Background-aimToxic cyanobacterial blooms creates local and global problems by contaminating surface water resources with their potent toxins having adverse health effect for both humans and animals. Two structurally related families of cyclic peptides, microcystins (MC) and nodularins (Nod), are the most commonly reported and troublesome cyanobacterial hepatotoxin. For the assessment of water quality and safety, simple and rapid screening methods are required for analysis of water samples to detect the possible presence of MC/Nod. We report a mix-and-measure type simple and rapid non-competitive homogenous screening assay for MC/Nod based on time resolved fluorescence resonance energy transfer (TR-FRET).MethodsTo demonstrate the homogenous assay a generic anti-immunocomplex (anti-IC) scFv (single-chain variable fragment) isolated from our in house synthetic antibody library was crucial together with a generic anti-adda specific antibody recognizing the common adda (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) part of the microcystins and nodularins. The anti-IC scFv labeled with alexa 680 and the anti-adda antibody labeled with europium enabled the FRET assay to occur in the presence of MC or Nod. In the presence of toxin in sample, FRET occurs only at the close proximity of the two fluorophores when anti-IC scFv binds specifically to the anti-adda-antibody:MC/Nod immunocomplex and sensitized emission of fluoresce signal was detected at 730 nm in time resolved mode.ResultsUsing only 20 μl of water sample, the rapid (15 min or less) wash-free assay was capable of detecting all the tested nine major hepatotoxins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R) with sensitivities well below the World Health Organization guideline limit of 1 μg/L.ConclusionsThe mix and measure type assay without requiring any washing step has a great potential as a quick screening tool for MC/Nod detection from a large number of water samples.</p

Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL. View Full-TextKeywords: antibody library; phage display; mycotoxin; deoxynivalenol; immunoassay</div

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotransla-tional translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44-and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency

Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman&#39;s rank correlation, &rho;&nbsp;&gt;&nbsp;0.66, P&nbsp;&lt;&nbsp;0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC&ndash;MS). Positive correlations were observed also for single lakes.</p

Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years. However, in the microtiter plate, it has been challenging to attain cell densities similar to well-aerated shake-flask culture, due to the poor mixing resulting in oxygen limitation. To solve this problem, we investigated the influence of multiple cultivation parameters on P. pastoris cell growth, including the architecture of 96-deepwell plate (96-DWP), shaking throw diameter, shaking frequency, culture volume/well, and media composition. In the optimised conditions, a cell density of OD600 ~50 (dry cell weight ~13 g/L) with >99% cell viability was achieved in the casamino acids supplemented buffered-minimal-media in 300 to 1000 μl culture volume/well. We have devised a simplified method for coating of the culture supernatant on the polystyrene surface for immunoassay. Clones for secretory expression of envelope domain III of dengue virus serotype-1 under the control of inducible and constitutive promoter were screened using the developed method. Described microscale cultivation strategy can be used for rapid high-throughput screening of P. pastoris clones, media optimization, and high-throughput recombinant protein production. The knowledge gained through this work may also be applied, to other suspension cultures, with some modifications.</p

Single-step noncompetitive immunocomplex immunoassay for rapid aflatoxin detection

Owing to the high carcinogenicity of aflatoxins, these toxic secondary metabolites pose a severe risk to human and animal health and can have major economic implications. Herein, we report the development of a noncompetitive immunoassay for aflatoxins based on a monoclonal capture antibody and a unique anti-immunocomplex (anti-IC) antibody fragment (scFv) isolated from a synthetic antibody repertoire. The anti-IC scFv recognizes the immunocomplex and enables the development of noncompetitive sandwich-type assays despite the small size of the analyte. The single-step assay developed in this work, with a detection limit of 70 pg mL−1, could detect aflatoxins within 15 min. The assay was applied to the analysis of spiked food samples, and the results showed that the method could provide a rapid and simple tool for aflatoxin detection. Moreover, the work demonstrates the potential of anti-IC antibodies and non-competitive immunoassays for the analysis of small molecule contaminants. </p

Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris

Background: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of Dengue Viruses (DENV) that places &#8764;40% of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope Domain-III (EDIII) antigen of Dengue Virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose. Results: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino Acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20°C than 30°C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20°C in the presence of 1% CA. The overall increase in EDIII titer was &#8764;9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV. Conclusions: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement

Non-competitive ELISA with broad specificity for microcystins and nodularins

Simple and cost-effective methods with sufficient sensitivities for preliminary screening of cyanobacterial toxins are in high demand for assessing water quality and safety. We have recently developed a highly sensitive and rapid time-resolved fluorometry based noncompetitive immunoassay for detection of microcystins and nodularins. The assay is based on a synthetic broad-specific anti-immunocomplexantibody SA51D1 capable of recognizing the immunocomplex formed by a generic anti-Adda monoclonal antibody (mAb) bound to either microcystins or nodularins. Using the same antibody pair, here we describe a very simple and cost-efficient non-competitive ELISA test for microcystins and nodularins based on conventional alkaline phosphatase (AP) activity measurement. The recombinant SA51D1 single-chain fragment of antibody variable domain (scFv) was produced as a fusion with bacterial alkaline phosphatase in Escherichia coli. After one step affinity purification through His-tag, the scFv-AP fusion protein could directly be used in the assay. For the assay, toxin standard/sample, biotinylated anti-Adda mAb and the scFv-AP were incubated together for one hour on streptavidin-coated microtiter wells, washed and AP activity was then measured by incubating (1 h at 37°C) with chromogenic substrate para-nitrophenylphosphate (pNPP). The assay was capable of detecting all the eleven tested toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LA -LY, -LF -LW, -WR, and nodularin-R) below WHO guide line value of 1 μg L–1. The detection limit (based on blank+3SD response) for microcystin-LR was 0.2 μg L–1. The assay was verified using spiked (0.25-4 μg L–1 of microcystin-LR) tap, river and lake water samples with recoveries from 64 to 101%. The assay showed good correlation (r2>0.9) with four reference methods for its performance in detecting extracted intracellular microcystin/nodularin from 17 natural surface water samples. The described easy-to-perform assay has a high potential to be used in resource-poor settings as quantitative measurements can be obtained using a simple ELISA reader or easy-to-interpret qualitative results by visual readout. Based on the non-competitive format, the assay does not need any chemical toxin conjugates and offers robustness as compared to the currently available competitive format assays.</p

Detection of bladder cancer with aberrantly fucosylated ITGA3

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay