Dataset Information

Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

ABSTRACT: BACKGROUND: So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology. RESULTS: An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC50 of striatal B was 36.0 ng mL-1 with an inter-assay coefficient of variation (CV) of 13.2% (n = 5). Recoveries from striatal B spiked samples in the assay were in the range of 97.3 - 125.9%. A good correlation between the striatal B concentration measured by the ELISA and by HPLC-DAD (y = 1.1122× - 0.1585, R2?=?0.9942) was obtained from linear regression analysis. The suitability of the ELISA for detection of cyathane type diterpenoids in submerged cultures and fruiting bodies of H. erinaceus was studied. It showed cross reactivity with supernatants from submerged cultures and extracts thereof, but did not show cross reactivity with extracts from fruiting bodies. CONCLUSIONS: The developed method is appropriate for qualitative and quantitative detection of cyathane diterpenoids in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening method for discovering new cyathane diterpenoids and new potential producers of them.

SUBMITTER: Shen T

PROVIDER: S-EPMC4239385 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

Shen Tian T Hof Lena M LM Hausmann Heike H Stadler Marc M Zorn Holger H

BMC biotechnology 20141118

<h4>Background</h4>So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology.<h4>Results</h4>An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to ...[more]

PMID: 25404227

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Project description:Enterovirus A71 (EVA71) is a medically important virus that is commonly associated with hand, foot, and mouth disease (HFMD). It is responsible for periodic outbreaks, resulting in significant economic impact and loss of life. Vaccination offers the potential to control future outbreaks, and vaccine development has been increasingly the focus of global research efforts. However, antigenic characterization of vaccine candidates is challenging because there are few tools to characterize the different antigenic forms of the virus. As with other picornaviruses, EVA71 virions exist in two antigenic states, native (NAg) and expanded (HAg). It is likely that the composition of vaccines, in terms of the proportions of NAg and HAg, will be important for vaccine efficacy and batch-to-batch consistency. This paper describes the development of a single-chain fused variable (scFv) domain fragment and the optimization of a sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of the NAg conformation of EVA71. NAg specificity of the scFv was demonstrated using purified EVA71, and conversion of NAg to HAg by heating resulted in a loss of binding. We have thus developed an effective tool for characterization of the specific antigenic state of EVA71. IMPORTANCE EVA71 is a medically important virus that is commonly associated with HFMD, resulting in periodic outbreaks, significant economic impact, and loss of life. Vaccination offers the potential to curtail future outbreaks, and vaccine development has been increasingly the focus of global research efforts. However, antigenic characterization of vaccine candidates is challenging because there are very limited effective tools to characterize the different antigenic forms of EV71. As with other picornaviruses, EVA71 virions exist in two antigenic states, native and expanded. This paper describes the development of an scFv and the optimization of a sandwich ELISA for the specific detection of the native conformation of EVA71 as an effective tool for characterization of the specific antigenic state of EVA71.

Project description:ObjectiveOrganophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment.MethodsA broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively.ResultsUnder the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides.ConclusionsThe established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

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Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

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Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

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