Vaccination is the method of choice to control AIV in poultry industry in many countries. Fast, efficient, and inexpensive DIVA test has a vital role in the success of this strategy. To address this issue, we expressed the M2e peptide as a recombinant M2e-MBP protein in E.coli
. After purification by affinity chromatography, its specificity to detect M2e antibodies was confirmed by both Western blotting and ELISA. Synthetic M2e peptide has been used previously in chickens 
. Application of M2e as recombinant protein, instead of as a peptide, has dual benefits. Recombinant M2e provides a continuous access to an inexpensive reagent for a large scale screening. The second benefit of recombinant M2e protein is the opportunity of demonstrating the M2e-based assay specificity by another method, in this case Western blotting, which is not possible when M2e is used as a peptide.
The expressed recombinant M2e-MBP protein had an expected size of 45.1 kd corresponded to 2.6 kd M2e and 42.5 kd MBP. Another minor protein of 42.5 kd corresponding to truncated M2e-MBP, was also co-purified with M2e-MBP and was not possible to remove from the preparation of M2e-MBP by additional purification.
Western blotting recognised the M2e-MBP protein of 45.1 kd length only by antisera to live AIV whereas antisera to inactivated H5N1 did not react with the M2e-MBP. This reaction in Western blotting was demonstrated to be due to M2e only and not due to MBP.
The comparison of M2e-MBP and peptide ELISA using reference sera indicted that M2e-MBP antigen was equally capable of detecting M2e positive sera without any significant background noise, or non-specific reaction. All sera that were positive by peptide ELISA were also positive in M2e-MBP ELISA, and in full agreement with the results obtained by M2e-MBP based Western blotting, indicated that M2e expressed as fusion M2eMBP protein is antigenically functional. The recombinant M2e-MBP was expressed in large quantities and allowed sera to be tested for M2e antibodies at a relatively high dilution of 1/100. The signal to noise ratio in ELISA was significantly different and allowed clear differentiation of M2e positive reference sera.
UP to now, M2e peptide has been expressed in Salmonella
, Pichia pastoris
, plants and viruses for the purpose of studying the protective immunity induced by the M2e 
. Only in one study the M2e was expressed as GST fusion protein in E. coli
and used to analyse the antibody response to the swine influenza virus M2e 
. Application of efficient and high productive E.coli
platform in this study provides the opportunity of production of a large amount of recombinant M2e protein in a short period of time. Further dilution of the producd protein (1/100 dilution, because of high ratio in this study) as a result of high recombinant protein yield in E.coli
platform significantly decreases the non-specific resulting in more accurate DIVA test.
The comparison of the results of M2e-MBP ELISA and HI (HA-based) tests demonstrated the excellent ability of M2e-MBP antigen to discriminate between antibodies produced against live virus challenge and killed virus vaccination for DIVA serological test. M2e-MBP ELISA was highly sensitive to live virus infection as the ELISA OD of vaccinated chickens greatly increased by more than 8 time when vaccinated chickens were infected with the live AIV virus ().
Furthermore, the robustness of the method reinforced by large scale M2e-MBP ELISA in differentiation of infected chickens from non-infected and vaccinated chickens (). With negative field sera the background noises in M2e MBP ELISA were however higher. In order to reduce the of biases of non-specific binding, each serum sample was tested in duplicate in the presence and absence of M2e MBP antigen and background noise subtracted from OD obtained on M2e MBP. This non-specific binding of a percentage of field sera is largely independent of the antigen used for coating as shown by others 
and also was occurred in M2e peptide ELISA.
The newly designed M2e-MBP ELISA system using as a DIVA tool has some limitations in its application in old chicken and the serum samples that were haemolysed or lipemic. Some of the field serum samples from old flocks (older than 44 weeks) had different degrees of non-specific reactions with M2e-MBP antigen. Western blot analysis using MBP purified fusion protein revealed that these samples had different reactivity just with MBP fusion protein but not with M2e.
Also some of the serum samples from layer or free range chicks had non-specific reactions to MBP fusion protein and/or just the ELISA plates alone. To overcome these problems using fresh, non-haemolysed and preferably non-lipemic serum samples would be an advantageous. Where it is necessary the results can be confirmed by Western blotting or subtracting the MBP value from the whole antigen. Second critical item in reliability of the M2e-MBP ELISA is purity of the antigen, in some cases during purification procedure of the antigen some of E.coli proteins could contaminate purified protein, and these residues will react with chicken antibodies, increasing the background of the tested samples in ELISA. Regarding some non-specific binding encountered in indirect M2e-MBP ELISA, being either due to the nature of the antigen or the properties of the chicken serum, we suggest that the use of a competitive or blocking ELISA for the detection of M2e antibodies might be an approach to reduce non-specific reactions with chicken sera.
H5N1-originated recombinant M2e-MBP protein or synthetic M2e peptide reacts with a wide range of other AIV strains antibodies such as H5N2, H9N2, H7N7, H11N6, etc. While the major surface influenza glycoproteins, HA and NA undergo major antigenic changes resulting in short-time effectiveness of available vaccines, the extracellular domain of matrix protein 2 (M2e) is strongly invariable and conserved. Therefore, vaccines based on extracellular domain of M2e are capable in inducing broad-spectrum immunity and inhibit virus replication up to 90–100% protection in mice and heterosubtypic immunity in pigs 
. In fact, the recent concept of a “universal influenza vaccine” relies on the conserved ectodomain of the influenza A protein 
In addition to the above-mentioned advantages, extracellular domain of M2e opens a new vista in avian influenza management via DIVA test. Hydrophilic structure of M2e as well as its invariability and broad-spectrum reactivity show that M2e is an appropriate candidate for both vaccine production and DIVA test. The high performance of M2e subunit for DIVA test can be explained by considerable lower number of M2 molecules per virion (20–60) comparing to HA and NA. Due to larger amount of HA molecule per virion. HA can induce a very strong immune response in both vaccinate (killed virus) and infected chickens. In contrast, the amount of M2e subunit in vaccine is very low, and immune response can be detected after live virus infection as presented in .