Liquid chromatography based enzyme-amplified immunological assays in fused-silica capillaries at the zeptomole level.
Liquid chromatography based enzyme-amplified immunological assays in fused-silica capillaries at the zeptomole level.
复制标题
基于液相色谱的酶放大免疫学测定，在 Zeptomole 水平的熔融二氧化硅毛细管中进行。
DOI：
10.1021/ac00063a040
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发表时间：
1993
影响因子：
7.4
通讯作者：
Regnier,FE
中科院分区：
文献类型：
--
作者：
deFrutos,M;Paliwal,SK;Regnier,FE
通过使用熔融二氧化硅毛细管，表明减少酶扩增免疫测定的液体体积使测定的扩增速率和灵敏度增加几个数量级。用与碱性磷酸酶（ALP）偶联的F（ab）抗hlgG饱和蛋白G包被-μ-id柱上捕获的人免疫球蛋白G（hlgG）。随后在与对硝基苯基磷酸酯的停流孵育中测定由抗原捕获的缀合酶。然后将停流孵育中产生的对硝基苯酚扫到检测器上，并在405 nm处定量。停流模式下的检测限约为3 fmol。在这种流通式毛细管测定形式中确定了三个问题。第一个原因是毛细血管内免疫复合物的形成速度太慢。在应用于柱之前预处理免疫复合物使灵敏度增加了2个数量级。另一个问题是毛细管内的传质速率限制了对预形成的免疫复合物的捕获。该问题通过在柱中对复合物进行逆流孵育来解决。免疫复合物的预形成和柱内停流结合的组合使检测限降低到约3阿莫尔。最后，减少测定中使用的F（ab）-ALP的量使缀合酶的非特异性结合最小化，并将检测限进一步降低至333 zmol。
Through the use of fused-silica capillaries it was shown that reducing the liquid volume of an enzyme-amplified immunological assay increases the rate of amplification and sensitivity of the assay by several orders of magnitude. Human immunoglobulin G (hlgG) captured on protein G-coated-µ-id columns was saturated with F (ab) anti-hlgG conjugated to alkaline phosphatase (ALP). Conjugated enzyme captured by the antigen was subsequently assayed in a stop-flow incubation with p-nitrophenyl phosphate. p-NitrophenoI producedin the stop-flow incubation was then swept to thedetector and quantitated at 405 nm. The detection limit in the stop-flow mode was approximately 3 fmol. Three problems were identified in this flow-through, capillary assay format. The first was that the rate of immunological complex formation within the cap-illary was too slow. Preforming the immunological complex before application to the column in-creased the sensitivity by 2 orders of magnitude. Another problem was that the rate of mass transfer within the capillary limited capture of the pre-formed immunological complex. This problem was solved bystop-flow incubation of the complex in the column. The combination of preformation of the immunological complex and stop-flow binding within the columnreduced the detection limit to approximately 3 amol. Finally, reducing the amount of F (ab)-ALP used in the assay minimized nonspecific binding of the conjugated enzyme and reduced the detection limit further to 333 zmol.