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
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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