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Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis
Shivani Dixit1, Jagrati Parashar1, Ram Kumar Dhaked2
1Division of Pharmacology & Toxicology, Defence Research Development & Establishment, Defence Research Development Organization, Gwalior, India.
Background:
Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin.
Methods:
Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked.
Results:
The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples.
Conclusion:
The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.
