In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA
Autor: | Marta González-Warleta, Victoria Martínez-Sernández, Mercedes Mezo, F. Romarís, Ana Hernández-González, M.J. Perteguer, Teresa Gárate, Florencio M. Ubeira, Esperanza Paniagua, Ricardo A. Orbegozo-Medina |
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Přispěvatelé: | Ministerio de Economía y Competitividad (España), Ministerio de Economía, Industria y Competitividad (España), Gobierno de Galicia |
Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
Serum Proteins Flatworms Antigen Processing and Recognition Helminth genetics Biochemistry Inclusion bodies law.invention Antibodies Monoclonal Murine-Derived law Contaminants Medicine and Health Sciences Enzyme-Linked Immunoassays Materials Mammals Multidisciplinary biology Chemistry Eukaryota Ruminants General Medicine Recombinant Proteins Vertebrates Physical Sciences Monoclonal Recombinant DNA Medicine Target protein Antibody General Agricultural and Biological Sciences Research Article medicine.drug_class Science Immunology Materials Science 030106 microbiology Antibodies Helminth Enzyme-Linked Immunosorbent Assay Research and Analysis Methods Monoclonal antibody Trematodes General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Antigen Bovines Helminths medicine Animals Immunoassays Sheep Organisms Biology and Life Sciences Proteins Invertebrates Fasciola 030104 developmental biology Antigens Helminth Amniotes Immunologic Techniques biology.protein Cattle |
Zdroj: | PLOS ONE PLoS ONE, Vol 14, Iss 2, p e0211035 (2019) Repisalud Instituto de Salud Carlos III (ISCIII) PLoS ONE |
ISSN: | 1932-6203 2011-3056 |
DOI: | 10.1371/journal.pone.0211035 |
Popis: | 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. This work was supported by: Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03 and AGL2014-57125R], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02] and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). VMS is supported by a contract under the grant ED431B 2017/18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí |
Databáze: | OpenAIRE |
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