DETECTION OF RICIN BY COLORIMETRIC AND CHEMILUMINESCENCE ELISA
DETECTION OF RICIN BY COLORIMETRIC AND CHEMILUMINESCENCE ELISA
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DOI：
10.1016/0041-0101(94)90409-x
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发表时间：
1994-11-01
期刊：
影响因子：
2.8
通讯作者：
MERRILL, GA
中科院分区：
文献类型：
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作者：
POLI, MA;RIVERA, VR;MERRILL, GA
开发了一种高度灵敏和特异性的 ELISA 来检测生物体液中的蓖麻毒素。该测定利用亲和纯化的山羊多克隆抗体从溶液中吸附蓖麻毒素。然后使用相同的抗体（生物素化的）形成夹心，并且亲和素连接的碱性磷酸酶允许显色并测量 405 nm 处的光密度。我们的常规检测使用 0-10 ng/ml 蓖麻毒素范围内的标准曲线，在检测缓冲液以及 1:10 稀释的人尿或 1:50 稀释的掺有蓖麻毒素的人血清中精确定量低于 1 ng/ml（100 pg/孔）。在所有基质中，加标样品中测量的蓖麻毒素的准确度通常在预期值的 5% 以内。变异系数范围为 10 ng/ml 时的 3-10% 至 2.5 ng/ml 时的 8-25%。还研究了常规测定的两种变化。首先，延长的孵育时间和额外的显色时间可以在低至 1:2 的血清稀释度中进行准确定量。其次，生物素化抗体和亲和素连接酶的浓度从 1:250 增加到 1:70，使测定的灵敏度提高了 10 倍，实现了至少 100 pg/ml（10 pg/孔）的检测限。该测定还配置为基于化学发光的格式，允许在 0.1-1 ng/ml 范围内进行定量，但其变异性比比色测定稍大。
A highly sensitive and specific ELISA was developed to detect ricin in biological fluids. The assay utilizes an affinity-purified goat polyclonal antibody to adsorb ricin from solution. The same antibody (biotinylated) is then used to form a sandwich, and avidin-linked alkaline phosphatase allows color development and measurement of optical density at 405 nm. Our routine assay uses a standard curve over the range of 0-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in assay buffer as well as in a 1:10 dilution of human urine or 1:50 dilution of human serum spiked with ricin. Ricin measured in spiked samples demonstrated accuracy typically within 5% of the expected value in all matrices. The coefficient of variation ranged from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml. Two variations on the routine assay were also investigated. First, lengthened incubation times and additional time for color development allowed accurate quantitation in serum dilutions as low as 1:2. Second, increased concentrations of biotinylated antibody and avidin-linked enzyme from 1:250 to 1:70 enhanced the sensitivity of the assay 10-fold, achieving a detection limit of at least 100 pg/ml (10 pg/well). The assay was also configured to a format based upon chemiluminescence, which allowed quantitation in the 0.1-1 ng/ml range, but was subject to slightly greater variability than the colorimetric assay.