Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection.
Abstract: 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.
Publication Date: 2014-01-09 PubMed ID: 24523642PubMed Central: PMC3913021DOI: 10.1155/2014/620906Google Scholar: Lookup
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- Journal Article
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- Non-U.S. Gov't
Summary
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The research article discusses a study on the use of heavy water (D2O) to increase the thermal stability of a recombinant p26 protein-based enzyme-linked immunosorbent assay (ELISA) for diagnosing equine infectious anemia virus infection.
Overview of Research
- The study focuses on the effects of heavy water (Deuterium Oxide or D2O) on the thermal stability of the p26 protein in Enzyme-Linked Immunosorbent Assay (ELISA). ELISA is a commonly used assay technique to detect the presence of proteins, such as antibodies, in a sample.
- The researchers primarily aimed to increase the thermal and temporal stability of the in-situ p26 protein. This protein is used in ELISA to test for equine infectious anemia virus (EIAV) infection.
Methodology
- In order to test the thermostabilization of p26 protein, the carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O. This buffer preparation was used to coat the ELISA plates.
- Thermal stability of the p26 protein was thoroughly tested at different temperatures, namely at 4°C, 37°C, 42°C, and 45°C over a range of time from two weeks to ten months.
- A total of 42 serum samples (12 positive and 30 negative) were evaluated in ELISA throughout the study. Moreover, the titer strength of the positive serum samples varied, with four samples each being weak, medium, and strong.
Findings
- The research revealed that there was a gradual escalation in the stabilizing effect when 80% D2O was used, particularly at elevated temperatures (37 °C < 42 °C < 45 °C).
- This study found that using 80% D2O allowed for the thermal protection of the recombinant p26 protein in the ELISA plates for up to two months, even at an incubation temperature of 45°C.
Conclusion
- Findings from this research could potentially contribute to the development of more cost-effective ELISA reagents. These reagents would remain stable for longer durations and across a wider temperature range, thereby extending their shelf life and making them more heat tolerant.
Cite This Article
APA
Singha H, Goyal SK, Malik P, Singh RK.
(2014).
Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection.
ScientificWorldJournal, 2014, 620906.
https://doi.org/10.1155/2014/620906 Publication
Researcher Affiliations
- Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
- Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
- Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
- Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
MeSH Terms
- Animals
- Antibodies, Viral / immunology
- Deuterium Oxide
- Enzyme-Linked Immunosorbent Assay / methods
- Equine Infectious Anemia / diagnosis
- Horses
- Infectious Anemia Virus, Equine / immunology
- Protein Stability
- Recombinant Proteins
- Sensitivity and Specificity
- Temperature
- Viral Proteins / immunology
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