Recombinant M2e protein-based ELISA: a novel and inexpensive approach for differentiating avian influenza infected chickens from vaccinated ones
Authors: Risa Indriani
Jagoda Igniatovic
N L P Indi Dharmayanti
Farhid Hemmatzadeh
Esmaeil Ebrahimie
Sumarningsih Sumarningsih
Simson Tarigan
Journal: PloS one (2013)
Discipline: Multidisciplinary
Keywords: avian influenza; serological diagnostic tests; vaccinated; naturally infected; DIVA
Affiliations: School of Animal and Veterinary Sciences, The University of Adelaide, Adelaide, Australia. 

Abstract:  Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test. 

Retracted: No

PubPeer comments:
 April 2021 Expression of Concern, found here: https: journals.plos.org plosone article?id=10.1371 journal.pone.0250485 From the statement:  After this article [1] was published, concerns were raised about results reported in Figure 1. When levels are adjusted to visualize the background in this figure: * Figure 1B, lanes 2, 6, and 7 appear similar. * Figure 1C, lanes 1, 2, 3, and 4 appear similar. * Figure 1C, lanes 5 and 7 appear similar. The authors noted that duplications may have been due to errors made when negative data were being prepared for the manuscript. They provided the available raw image data for Figure 1 from the original experiments; these images were not of sufficient quality to provide clear information as to the issues or results. However, lab records and image data for replication experiments (S1u2013S3 Files) lend support for the overall results that none of the Figure 1B or 1C samples yielded positive MBP blotting results, and that the Scotland, Vietnam, Turkey, and Denmark sera, but no other samples tested, reacted with M2e-MBP. Here is Figure 1 as marked and reported to the Editors in September 2014. ![file] ,