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

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

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

Noncompetitive Chromogenic Lateral-Flow Immunoassay for Simultaneous Detection of Microcystins and Nodularin

Abstract: Cyanobacterial blooms cause local and global health issues by contaminating surface waters. Microcystins and nodularins are cyclic cyanobacterial peptide toxins comprising numerous natural variants. Most of them are potent hepatotoxins, tumor promoters, and at least microcystin-LR is possibly carcinogenic. In drinking water, the World Health Organization (WHO) recommended the provisional guideline value of 1 µg/L for microcystin-LR. For water used for recreational activity, the guidance values for microcystin concentration varies mostly between 4–25 µg/L in different countries. Current immunoassays or lateral flow strips for microcystin/nodularin are based on indirect competitive method, which are generally more prone to sample interference and sometimes hard to interpret compared to two-site immunoassays. Simple, sensitive, and easy to interpret user-friendly methods for first line screening of microcystin/nodularin near water sources are needed for assessment of water quality and safety. We describe the development of a two-site sandwich format lateral-flow assay for the rapid detection of microcystins and nodularin-R. A unique antibody fragment capable of broadly recognizing immunocomplexes consisting of a capture antibody bound to microcystins/nodularin-R was used to develop the simple lateral flow immunoassay. The assay can visually detect the major hepatotoxins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R) at and below the concentration of 4 µg/L. The signal is directly proportional to the concentration of the respective toxin, and the use of alkaline phosphatase activity offers a cost efficient alternative by eliminating the need of toxin conjugates or other labeling system. The easy to interpret assay has the potential to serve as a microcystins/nodularin screening tool for those involved in water quality monitoring such as municipal authorities, researchers, as well as general public concerned of bathing water quality. Keywords: noncompetitive immunoassay; cyanotoxin; microcystin; nodularin; lateral flow</div

Oppilaitoksen ja yritysten yhteistyön merkitys henkilöstön osaamisen kehittämisessä näyttötutkinnoilla

Kehittämishankkeen tavoitteena oli selvittää koulutuksen vaikutuksia työelämässä ja aikuiskoulutuksen roolia henkilöstön kehittämisessä. Toteutuneiden koulutusten positiiviset palautteet antoivat aiheen tarkastella koulutuksen vaikutuksia ja konkreettisia hyötyjä. Työ toteutettiin haastattelemalla sellaisia yrityksiä, joissa on järjestetty koulutusta usean vuoden ajan. Taustatietoa koulutuksien merkitykselle etsittiin laajasta LeoSpanin tutkimuksesta ”Support of Persons in the Process of Accreditation of Non-formal learning”, hakevan toiminnan ”Vaikuttava hakeva toiminta nostehankkeissa” –tutkimusraportista sekä kirjallisuudesta. Työ osoitti, että koulutuksella on varsin laaja merkitys työelämässä. Koulutuksen vaikutuksia ovatkin mm. työmotivaation, työturvallisuuden, moniosaamisen sekä työssäjaksamisen lisääntyminen sekä tiedon ja osaamisen laaja-alaistuminen. Työn tulosten perusteella voidaan todeta, että koulutus nähdään tärkeänä henkilöstön kehittämisessä ja on investointi yritykseltä. Kehittämishankkeen esille nostamat asiat toimivat myös vuoropuheluna oppilaitoksen ja yritysten välillä ja antavat näkökulmia koulutuksen kehittämiseen yhä paremmaksi.Aim of this development project was to find out results of the education in working life and role of adult education in staff training. Positive feedback from realised trainings gave the reason to analyse effects and concrete benefits of training. This project was made by interviewing the companies were this kind of education was arranged a long time. Background for the meaning of education was searched from Leo Spans research “Support of Persons in the Process of Accreditation of Non-formal learning”, research report of Noste project and literatures. This project showed that education has a quite wide meaning in working life. Effects of the education are increasing in motivation at work, occupational safety, multi handing and also comprehensiveness of knowledge and pragmatics. From results of this project we can see that education is very important in staff training and this training is also investment from the company. Subjects which this development project shows up can also be the conversation between educational establishment and companies and gives standpoints for developing the education still better

Uusi ammatti- ja erikoisammattitutkintojen toteutusmalli

Opinnäytetyönä tehtiin uusi toteutusmalli Jyväskylän aikuisopiston Tekniikan ja liikenteen –yksikössä toteutettaville ammatti- ja erikoisammattitutkinnoille. Uuteen toteutusmalliin koottiin keskeiset asiat tutkintojen rakentumisesta, valmistavasta koulutuksesta, työelämälähtöisyydestä ja –vastaavuudesta sekä elinikäisestä oppimisesta, opetusmenetelmistä ja –teknologiasta sekä alakohtaisista erityishuomioista. Opinnäytetyön toimeksiantajana oli Jyväskylän aikuisopisto. Opinnäytetyö toteutettiin benchmarkaamalla tekniikan erikoisammattitutkintoa sekä teemahaastattelemalla kymmentä kouluttajaa ammattialoilla, joiden tutkintoihin Jyväskylän aikuisopisto järjestää koulutusta. Sekä benchmarkauksen että teemahaastattelujen tulosten pohjalta kehitettiin uusi toteutusmalli, jota eri alat voivat vapaasti soveltaa koulutuksissa. Benchmarkattavaksi valittiin ammattialoista riippumattomana tekniikan erikoisammattitutkinto, koska sen toteutus oli mahdollisimman hyvin yleistettävissä ja verrattavissa erilaisiin tutkintoihin. Teemahaastattelut puolestaan kohdennettiin alasidonnaisiin tutkintoihin, jotta saatiin kattava ja monipuolinen näkemys toiminnasta. Uusi toteutusmalli tehtiin ammatti- ja erikoisammattitutkinnoille, joissa oletuksena oli opiskelijalla tietyllä tasolla oleva aikaisempi osaaminen. Uuden ammatti- ja erikoisammattitutkintojen toteutusmallin tuloksena avattiin erilaiset menetelmät ja tavat koulutusten toteuttamisessa eri alojen yhteiseen käyttöön. Usein alojen toimintatavat olivat vakiintuneita ja koulutuksia toteutettiin samoilla menetelmillä. Uuden mallin myötä eri alat voivat vertaisoppia toisilta ja kehittää toimintaansa. Uuden yleisen mallin pohjalta alakohtaisten mallien tekeminen jalkautettiin ammattialoille niissä olevan substanssiosaamisen vuoksi. Ammattialat voivat ottaa uusia menetelmiä käyttöön tarpeen mukaan ja viimeistään tutkinnon perusteiden uudistuessa. Koulutussuunnitteluun ja esimiestyöhön saatiin opinnäytetyön kautta uusia näkökulmia tapojen ja menetelmien monipuolisuudesta ja koulutustoiminnan kehittämisestä. Käyttäjäkokemusten lisääntyessä toteutusmallia voidaan jatkossa kehittää lisää muuttuvassa yhteiskunnassa

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&ndash;115 ng/mL

Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin

Nodularin (NOD) is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) monoclonal antibody (Mab). One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R) over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR). It was expressed in Escherichia coli as a single chain antibody fragment (scFv) fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD) of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R) water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain