Use of Heavy Water (D2O) in Developing Thermostable Recombinant p26 Protein Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Equine Infectious Anemia Virus Infection
نویسندگان
چکیده
Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4 °C, 37 °C, 42 °C, and 45 °C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37 °C < 42 °C < 45 °C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45 °C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.
منابع مشابه
Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.
We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.
متن کاملSerological method using recombinant S2 protein to differentiate equine infectious anemia virus (EIAV)-infected and EIAV-vaccinated horses.
We recently reported a highly protective attenuated live virus vaccine for equine infectious anemia virus (EIAV) based on a proviral construct (EIAVUKDeltaS2) with a genetically engineered mutation in the viral S2 gene that eliminates expression of this accessory protein. While the EIAVUKDeltaS2 vaccine provides protection from detectable infection by experimental challenge with highly virulent...
متن کاملDevelopment of a recombinant protein-based dot-blot hybridization assay for the detection of antibody to chicken infectious bronchitis virus
Nucleocapsid (N) protein of infectious bronchitis virus (IBV), one of the viral structural proteins, inducesstrong antibody response in natural infection. In this study, a simple, recombinant N protein-based dot-blottest was developed to serologically examine chicken serum samples for the presence of IBV antibody.Initially, 72 serum samples were tested for the presence of IBV antibody using a c...
متن کاملStandardization of an Enzyme-Linked Immunosorbent Assay for Detection of Infectious Bronchitis Virus Antibody.
An indirect enzyme–linked immunosorbent assay (ELISA) was developed for screening of antibody to avian infectious bronchitis virus (IBV). Antigen was prepared from whole-purified IBV Massachusetts serotype (BR 801 strain). Optimum dilution with minimum background for antigen concentration, rabbit anti-chicken conjugate and sera in developed ELISA was determined 0.1μg/ml, 1:3000 and 1:100, respe...
متن کاملAn investigation of equine infectious anaemia infection in the central Anatolia region of Turkey.
In this study, 162 horses, 80 donkeys and 51 mule serum samples were collected in Konya city. Additionally, 64 horse serum samples from Ankara and 49 samples from Kayseri city were included in the study. A total of 406 serum samples were examined by agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for antibody to equine infectious anaemia virus (EIAV) and no positiv...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
دوره 2014 شماره
صفحات -
تاریخ انتشار 2014