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oai:repisalud.isciii.es:20.500.12105/74432024-09-27T22:04:19Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609

00925njm 22002777a 4500 dc 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 author 2019-02-01 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. 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 In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA