Dataset Information

Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

ABSTRACT: Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full-size version in leaves of Nicotiana tabacum and Nicotiana benthamiana to facilitate inexpensive and scalable production. The specific antigen-binding activity of the purified antibody was verified by surface plasmon resonance spectroscopy and ELISA, confirming the same binding specificity as its hybridoma-derived counterpart. The plant-derived antibody was used to design a lateral flow immunoassay (dipstick) for the sensitive detection of [Arg4]-microcystins at concentrations of 100-300 ng/L in freshwater samples collected at different sites. Plant-based production will likely reduce the cost of the antibody, currently the most expensive component of the dipstick immunoassay, and will allow the development of further antibody-based analytical devices and water purification adsorbents for the efficient removal of toxic contaminants.

SUBMITTER: Melnik S

PROVIDER: S-EPMC5785354 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

Melnik Stanislav S Neumann Anna-Cathrine AC Karongo Ryan R Dirndorfer Sebastian S Stübler Martin M Ibl Verena V Niessner Reinhard R Knopp Dietmar D Stoger Eva E

Plant biotechnology journal 20170605 1

Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence o ...[more]

PMID: 28421663

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Project description:BackgroundCulture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples.Methodology/principal findingsFirst, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 ?L of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei.Conclusions/significanceThe LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.

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Dataset Information

Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

Publications

Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

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