In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

Ricardo A Orbegozo-Medina,Mercedes Mezo,Marta González-Warleta,Teresa Gárate,Fernanda Romarís,Florencio M Ubeira,María J Perteguer,Ana Hernández-González,Esperanza Paniagua,Victoria Martínez-Sernández,Vikas Nanda

doi:10.1371/journal.pone.0211035

Abstract

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.

Highlights

The detection of serum antibodies induced by parasitic infections using ELISA methods depends on the availability of enough amounts of specific antigens, either natural or recombinant, to be used in the immunoassays
Epitope tagging is a genetic engineering technique in which a protein encoded by a cloned gene is fused to an epitope recognized by a known antibody [37,38]
This strategy allows rapid affinity-purification of recombinant proteins, but it can be used for protein detection in WB, flow cytometry and ELISA [39]

Summary

Introduction

The detection of serum antibodies induced by parasitic infections using ELISA methods depends on the availability of enough amounts of specific antigens, either natural or recombinant, to be used in the immunoassays. For this purpose, it would be expected that recombinant. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economıa y Competitividad (Programa de Formacion de Personal Investigador). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion