Near real-time immuno-optical sensor for diagnosing single point mutation: a model system: sensor for factor V Leiden diagnosis.
Near real-time immuno-optical sensor for diagnosing single point mutation: a model system: sensor for factor V Leiden diagnosis.
复制标题
DOI：
10.1016/j.bios.2009.02.004
复制
发表时间：
2009-05-15
影响因子：
12.6
通讯作者：
Peiper SC
中科院分区：
文献类型：
--
作者：
Kang KA;Ren Y;Sharma VR;Peiper SC
凝血因子V莱顿(FVL)是凝血因子V(FV)的异常。它是一种遗传性凝血障碍，发病率高(占普通人群的3-7%)。最常见的FVL类型是由单一氨基酸突变引起的，因此，目前只能通过DNA分析进行诊断，这需要很长时间和昂贵的费用。我们开发了一种快速、准确、经济的夹心免疫光学传感方法。为了制备抗Fv或FvL的单抗，与另一分子有最小的交叉反应，利用了Fv或FvL突变位点附近的20个氨基酸序列(20-mer)。抗体首先用20-MERS筛选，然后用天然FV或FVL分子筛选，它们表现出一定的交叉反应。利用FV或FVL分子亲和力最强的两种抗体，制备了FV和FVL首选传感器。在验证了抗体对这两个不同分子的亲和力水平随着分析物浓度的变化保持不变后，开发了一个双传感器系统来定量血浆样品中的Fv和FvL。该系统在0-12μg/ml血浆的生理浓度范围内，以0.5μg/ml血浆的最大误差定量Fv和Fvl水平。这两种分子的水平不仅可以提供患者是否患有FVL，还可以提供疾病的严重程度(纯合子和不同程度的杂合子)。
Factor V leiden (FVL) is an abnormality of factor V (FV), a blood coagulation factor. It is a hereditary blood coagulation disorder with a high frequency (3–7% of general population). The most common type of FVL is caused by a single amino acid mutation and, therefore, its diagnosis is currently done only by DNA analysis, which takes a long time and is expensive. We have developed a rapid, accurate, and cost-effective, sandwich immuno-optical sensing method. To produce monoclonal antibodies against FV or FVL, having minimal cross-reactivity with the other molecule, a 20 amino acid sequence (20-mer) of FV or FVL at around the mutation site was utilized. The antibodies were screened first with the 20-mers and then with native FV or FVL molecules and they showed some cross reactivity. Using two antibodies having strongest affinity to either FV or FVL molecule, a FV and a FVL preferred sensors, were produced. After verifying that the levels of the antibody affinity to the two different molecules remained constant with changes in analyte concentration, a two-sensor system is developed to quantify FV and FVL in plasma samples. The system quantified the levels of FV and FVL at the maximum error of 0.5 μg/ml-plasma, in their physiological concentration range of 0–12 μg/ml-plasma. The levels of both molecules may provide us whether the patient has FVL or not but also the seriousness level of the disease (homozygous and different level of heterozygous).
登录
查看更多内容
DOI：
10.1073/pnas.84.14.4846
发表时间：
1987-07-01
影响因子：
11.1
作者：
JENNY, RJ;PITTMAN, DD;MANN, KG
通讯作者：
MANN, KG
影响因子：
4.8
作者：
KALAFATIS, M;BERTINA, RM;MANN, KG
通讯作者：
MANN, KG
影响因子：
11.1
作者：
Makris, M;Rosendaal, FR;Preston, FE
通讯作者：
Preston, FE
影响因子：
2.9
作者：
Hockin, MF;Cawthern, KM;Mann, KG
通讯作者：
Mann, KG
影响因子：
3.8
作者：
Tang, L;Kwon, HJ;Kang, KA
通讯作者：
Kang, KA