Development and application of an indirect ELISA for detecting equine IgG antibodies against Getah virus with recombinant E2 domain protein

Abstract

Getah virus (GETV) disease is a mosquito-borne infectious disease that causes fever, aseptic meningitis, and abortion in a variety of animals. Currently, the epidemic trend of GETV disease increases seriously worldwide, especially in China, posing a potential threat to animal safety and public health. However, there are few reports about the epidemiological investigation of GETV disease in China as well as a lack of commercial diagnostic kit for GETV antibody. Therefore, the establishment of a rapid, sensitive and suitable GETV antibody detection method for large-scale samples is an urgent request to fully understand the prevalence of GETV disease. Here, a recombinant plasmid pET22b-GETV-E2d that contained the domain of GETV-E2 (E2d) fused to His-tag was constructed to express recombinant protein E2d (rE2d) in Escherichia coli. The rE2d was mainly expressed in inclusion bodies. And it was purified successfully by nickel affinity column so that it could be used to develop an indirect ELISA (rE2d-ELISA). After optimizing reaction conditions of rE2d-ELISA, the cut-off value was determined as 0.396 with 100 equine sera tested by virus neutralization test (VNT). Furthermore, rE2d-ELISA method showed the positive rate of IgG antibodies against GETV was 54.3% based on testing 646 clinical serum samples obtained in Xinjiang whereas the overall coincidence rate between rE2d-ELISA and VNT was 94.0%, with 98.2% sensitivity and 92.6% specificity. The findings suggest that the developed IgG ELISA employing recombinant E2d promises was an efficient and low-cost type of antibody detection method for horse, which will benefit for prevention of GETV outbreaks in horses.

Development of a Blocking ELISA Method for Detection of Getah Virus Antibodies in Horse Sera

“…The sensitivity, specificity, and accuracy of B-ELISA were compared with those of the VN test using 175 horse serum samples. The sensitivity (91.4%) of the B-ELISA for horse serum compared with the VN test was slightly lower than those of the I-ELISA using the recombinant E2 domain protein or a synthetic peptide (93.3%-98.2%) (10,12). The specificity (94.0%) of the B-ELISA was similar to that of the I-ELISA using recombinant or synthetic proteins (92.6-95.0%) (10,12).…”

“…Therefore, we developed a new B-ELISA using a purified antigen and mAb to measure GETV-specific antibodies in horse sera. Several I-ELISAs have been developed to measure anti-GETV antibodies in animals (10,12,14). An I-ELISA developed using a purified recombinant GETV E2 domain protein expressed in E. coli cannot be applied to several animals bitten by mosquitoes carrying GETV.…”

“…The number of GETV outbreaks has increased from horses to pigs, cattle, blue foxes, and red pandas (11). The seropositivity rates of GETV in horses vary by country and season, ranging between 17 and 39% (12). The incidence and risk of GETV infection in horse populations are increasing; however, serological studies of GETV in horses are still lacking in South Korea.…”

“…The virus-neutralization (VN) test is the classical and general method for measuring GETV-specific antibodies in animals (13). Although the VN test is accurate and useful for measuring anti-GETV antibodies, it requires skilled technicians, cell culture equipment and reagents (12). Therefore, it is necessary to develop a relatively simple assay, such as an enzyme-linked immunosorbent assay (ELISA) to measure GETV-specific antibodies in the sera of several animals.…”

“…It is easy to handle, and requires only small amounts of serum. Therefore, an indirect ELISA (I-ELISA) was developed using recombinant E2 protein expressed in E. coli for swine and horse sera (12,14). An ELISA using a 20-mer synthetic peptide for the E2 glycoprotein has been reported to detect GETV infection in horses (15).…”