Development and Application of an Indirect ELISA for the Detection of gp45 Antibodies to Equine Infectious Anemia Virus

Equine infectious anemia is an important viral infection affecting horses worldwide. Although an effective equine infectious anemia virus (EIAV) vaccine has been developed in China, the control of the disease is mainly based on identification of the horses infected by EIAV through laboratory diagnostic tests. The agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA) based on detecting p26 antibodies are widely used, but there is an increasing need for developing a more sensitive diagnostic test utilizing other EIAV proteins. Equine infectious anemia virus glycoprotein gp45 plays a similar role to gp41 in human immunodeficiency virus by mediating virus-host membrane fusion. The gp45 ectodomain was constructed by removal of the disulfide-bond loop region, and the recombinant protein (rgp45) was expressed in Escherichia coli followed by purification. An indirect rgp45 ELISA has been established and validated. There was an excellent agreement between the ELISA results using rgp45 and AGID test results. After 546 serum samples were tested, the relative sensitivity and specificity of the ELISA were 90.0% and 99.3%, respectively. Moreover, this assay showed sufficient reproducibility after detecting intrarun and interrun coefficients of variation. Therefore, this indirect ELISA represents a sensitive and specific quantitative tool for the serologic detection of EIAV to facilitate the prevention and control of this disease. In addition, this method could be applied to evaluate the effective protection of vaccines by detecting neutralizing antibodies to the ectodomain of gp45, which has strong immunity and biological function.