2026-05-17T05:46:40Zhttps://repisalud.isciii.es/rest/oai/requestoai:repisalud.isciii.es:20.500.12105/74432024-09-27T22:04:19Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609
Repisalud
author
Orbegozo-Medina, Ricardo A
author
Martínez-Sernández, Victoria
author
Perteguer-Prieto, Maria Jesus
author
Hernandez-Gonzalez, Ana
author
Mezo, Mercedes
author
González-Warleta, Marta
author
Romarís, Fernanda
author
Paniagua, Esperanza
author
Garate, Teresa
author
Ubeira, Florencio M
funder
Ministerio de Economía y Competitividad (España)
funder
Ministerio de Economía, Industria y Competitividad (España)
funder
Xunta de Galicia (España)
2019-04-10T11:27:23Z
2019-04-10T11:27:23Z
2019-02-01
PLoS One. 2019 Feb 1;14(2):e0211035.
10.1371/journal.pone.0211035
1932-6203
PloS one
30707711
http://hdl.handle.net/20.500.12105/7443
Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.
eng
In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA
journal article
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URL
https://repisalud.isciii.es/bitstreams/f416761d-a4bc-490e-964e-bd7fcc988efd/download
File
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application/pdf
InPlateRecapturingOf_2019.pdf
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https://repisalud.isciii.es/bitstreams/b84bc3b3-c9df-4e07-9c1d-e3b619b81ffd/download
File
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text/plain
InPlateRecapturingOf_2019.pdf.txt