Abstract：
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：蓖麻毒素是一种潜在的生物战剂。它是从蓖麻种子中分离出的植物毒素。目前尚无蓖麻毒素中毒的解毒剂，患者只能根据症状接受支持治疗。这突出了早期发现的重要性，以避免事故的严重性和减少危险因素。考虑到这一点,我们的研究旨在开发一种高灵敏度和特异性的夹心ELISA来检测蓖麻毒素。
方法:从蓖麻种子中纯化蓖麻毒素。使用蛋白A/G柱从兔抗血清和杂交瘤细胞（1H6F1）上清液中产生抗蓖麻毒素多克隆和单克隆抗体。使用间接ELISA进行抗体滴度估计。开发了基于链霉亲和素-生物素的夹心ELISA，线性范围,测定内和测定间变异系数(CV)，并确定与其他类似毒素的交叉反应性。还检查了掺入蓖麻毒素的人血浆样品的干扰。
结果：发现ELISA的LOD为0.45ng/ml，线性范围为0.90-62ng/ml，测定内和测定间CV分别为3.34%至5%和5.17%至10.80%。该测定不与其他类似的核糖体失活蛋白(RIP)毒素交叉反应。在加标血浆样品中检测到蓖麻毒素。
结论：开发的检测方法对检测蓖麻毒素具有高度敏感性和特异性，并且与其他类似类型的毒素没有交叉反应。该测定法可以检测加标血浆样品中的蓖麻毒素，因此它有可能用于蓖麻毒素中毒后的临床样本分析。
方法:从蓖麻种子中纯化蓖麻毒素。使用蛋白A/G柱从兔抗血清和杂交瘤细胞（1H6F1）上清液中产生抗蓖麻毒素多克隆和单克隆抗体。使用间接ELISA进行抗体滴度估计。开发了基于链霉亲和素-生物素的夹心ELISA，线性范围,测定内和测定间变异系数(CV)，并确定与其他类似毒素的交叉反应性。还检查了掺入蓖麻毒素的人血浆样品的干扰。
结果：发现ELISA的LOD为0.45ng/ml，线性范围为0.90-62ng/ml，测定内和测定间CV分别为3.34%至5%和5.17%至10.80%。该测定不与其他类似的核糖体失活蛋白(RIP)毒素交叉反应。在加标血浆样品中检测到蓖麻毒素。
结论：开发的检测方法对检测蓖麻毒素具有高度敏感性和特异性，并且与其他类似类型的毒素没有交叉反应。该测定法可以检测加标血浆样品中的蓖麻毒素，因此它有可能用于蓖麻毒素中毒后的临床样本分析。
