Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood

Lu, Shiying; Zhou, Yu; Lin, Clement; Meng, Xian-Mei; Yan, Dong-Ming; Li, Zhaohui; Yu, Shiyu; Liu, Zengshan; Ren, Hong-Lin

doi:10.1007/s11356-012-0819-y

Cited by 29 publications

(15 citation statements)

References 19 publications

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“…Although the developed SPR-based assay described is not as sensitive as ELISA-based assays commercially available and previously described in the literature, such as DSP ELISA Kit (L35000420-096, Biosense Laboratories AS, Norway) and the indirect competitive ELISA developed by Lu et al (2011), it has proven to be a robust (% CV < 7.3%) and highly reproducible assay, detecting OA at nanogram concentrations and around the mandated cut-off point. No extensive sample clean-up procedure is required, by comparison to HPLC and MS-based methods, which makes the assay more convenient with a comprehensive extraction protocol.…”

Section: Application Of the Assay To Marine Sample Analysismentioning

confidence: 95%

Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish

Berre

Kilcoyne

Kane

2015

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Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes.

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Section: Application Of the Assay To Marine Sample Analysismentioning

confidence: 95%

Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish

Berre

Kilcoyne

Kane

2015

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“…To synthesize the hapten-OVA conjugates used as coating detective antigens, the mixed anhydride method was adopted using isobutyl chloroformate as the coupling reagent. Conjugates of OA-BCP and OA-BSA respectively serving as the immunogen and the coating antigen were prepared according to a previous research method (Lu et al, 2012). Purification was performed by dialyzing extensively with PBS, followed by treatment with a protein concentrator.…”

Section: Preparation Of Hapten-protein Conjugatesmentioning

confidence: 99%

Evaluation of a water-soluble adjuvant for the development of monoclonal antibodies against small-molecule compounds

Liu

Lan

et al. 2016

J. Zhejiang Univ. Sci. B

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A water-soluble adjuvant named QuickAntibody (QA) was introduced into the procedure of mouse immunization for the development of hapten-specific monoclonal antibodies (mAbs), using four kinds of pesticides as model compounds. Compared with conventional Freund's adjuvants, QA treatments offered relatively low but acceptable antiserum titers after three inoculations, gave little adverse effects to the experimental animals, and were preferable in harvesting splenocytes during the steps of cell fusion. Afterwards, hybridomas from the QA group were prepared and screened by both non-competitive and competitive indirect enzyme-linked immunosorbent assays (ELISAs). The efficiency of gaining immune-positive hybridomas was satisfactory, and the resultant mAbs showed sensitivities (half maximal inhibitory concentration (IC 50)) of 0.91, 2.46, 3.72, and 6.22 ng/ml to triazophos, parathion, chlorpyrifos, and fenpropathrin, respectively. Additionally, the performance of QA adjuvant was further confirmed by acquiring a high-affinity mAb against okadaic acid (IC 50 of 0.36 ng/ml) after three immunizations. These newly developed mAbs showed similar or even better sensitivities compared with previously reported mAbs specific to the corresponding analytes. This study suggested that the easy-to-use adjuvant could be applicable to the efficient generation of highly sensitive mAbs against small compounds.

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“…The goat anti-mouse IgG antibody was purchased from Beijing Boshide Biotechnology Development Center (Beijing, China). An IgG1 monoclonal antibody against OA was previously generated in our laboratory (Lu et al 2011). The limits of detection (LOD) by ELISA and LFIC were 0.45 and 10 ng/ml, respectively.…”

Section: Reagents and Materialsmentioning

confidence: 99%

“…Sample analysis using ELISA (Lu et al 2011) The OA-BSA solid phase antigen was diluted with 0.05 mol/l bicarbonate buffer (pH 9.6) and immobilised on a micro-well plate followed by incubation at 4°C overnight. The McAb against OA was diluted 1:20,000 for detection.…”

Section: Sample Preparationmentioning

confidence: 99%

Contamination of commercially available seafood by key diarrhetic shellfish poisons along the coast of China

Lin

Liu

Tan³

et al. 2014

Environ Sci Pollut Res

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With the increasing number of outbreaks of food-borne diseases caused by okadaic acid (OA) and its analogue dinophysistoxin-1 (DTX-1), two key diarrhetic shellfish poison (DSP) toxins, OA and DTX-1, have become a serious threat to public health and have attracted significant public attention in China. The aim of our study was to monitor OA and DTX-1 contamination in commercially available seafood and to provide references for tracking these toxins and preventing disease outbreaks. From 2010 to 2012, 40 species were collected from six coastal cities of four inland seas in China. An enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic (LFIC) test strip were used to analyse the samples, and the results were further confirmed using a commercially available ELISA kit. The monitoring results indicated that 23 of 40 species were positive for contamination. In addition, 14 of the positive species were determined to be inedible because the content of OA and DTX-1 was above the regulatory limit. Simultaneously, we verified that the digestive glands of shellfish tended to accumulate toxin, in contrast to the flesh. The highest concentrations of OA and DTX-1 were recorded in Scapharca broughtonii, which was collected from Qing Dao, in relation to the other analysed species. Moreover, the Arca family as well as Mytilus galloprovincialis were severely contaminated by OA and its analogue. The above results indicate that some of the commercially available seafood from the coastal cities in China may be inedible due to serious marine toxin contamination. The results of this study might play an important role in protecting consumer health and safety screening of marine products.

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Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood

Abstract: The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.

Cited by 29 publications

References 19 publications

Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish

Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish

Evaluation of a water-soluble adjuvant for the development of monoclonal antibodies against small-molecule compounds

Contamination of commercially available seafood by key diarrhetic shellfish poisons along the coast of China

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