CAJ | 학술논문

目的：基于多克隆抗体免疫分析，构建并优化一种超灵敏、均相的光激化学发光法（AlphaLISA），用以检测水体中微囊藻毒素（MC）-LR含量。方法采用竞争原理，MC-LR人工抗原偶联的发光微球与标准品或样品MC-LR共同竞争限量一抗，再被二抗捕获，然后再通过生物素-亲和素与感光微球形成成免疫复合物，此时感光微球与发光微球发生接近，则产生并传递能量，发出特殊荧光。选择了合适的一抗和二抗工作稀释度，比较缓冲体系和反应时间的作用，从而优化了反应条件。结果通过优化反应条件，即筛选合适的一抗和二抗反应浓度、缓冲体系和反应时间，从而构建了MC-LR AlphaLISA检测法，其总反应时间40 min，灵敏度0．006μg·L-1，可测线性范围0．006～5μg·L-1，变异系数小于10％，平均添加回收率107．7％，与MC-RR和MC-RY的交叉反应率分别为13．2％和0．91％。结论本研究方法灵敏度高，特异性好，免疫反应迅速，适用于多样本量水体的MC-LR检测。
목적：기우다극륭항체면역분석，구건병우화일충초령민、균상적광격화학발광법（AlphaLISA），용이검측수체중미낭조독소（MC）-LR함량。방법채용경쟁원리，MC-LR인공항원우련적발광미구여표준품혹양품MC-LR공동경쟁한량일항，재피이항포획，연후재통과생물소-친화소여감광미구형성성면역복합물，차시감광미구여발광미구발생접근，칙산생병전체능량，발출특수형광。선택료합괄적일항화이항공작희석도，비교완충체계화반응시간적작용，종이우화료반응조건。결과통과우화반응조건，즉사선합괄적일항화이항반응농도、완충체계화반응시간，종이구건료MC-LR AlphaLISA검측법，기총반응시간40 min，령민도0．006μg·L-1，가측선성범위0．006～5μg·L-1，변이계수소우10％，평균첨가회수솔107．7％，여MC-RR화MC-RY적교차반응솔분별위13．2％화0．91％。결론본연구방법령민도고，특이성호，면역반응신속，괄용우다양본량수체적MC-LR검측。
OBJECTIVE Apolyclonalantibody-basedhomogeneouschemiluminescenceimmunoas-say was developed and optimized using AlphaLISA technology for the quantitative detection of microcys-tin-LR(MC-LR)inwatersamples.METHODS Thismethodwasbasedonacompetitivemodelin which an immune complex was formed from the ingegral binding of artificial MC-LR antigen-coated lumi-nescene beads,free MC-LR standards or sa mples,antibody and biotinylated second antibody.Next sensor bead were added that approached the i mmune co mplex through biotin-streptavidin interaction. With the exciting light,the energy was passed from the sensor luminescer before a special emission light could be observed.To opti mize the reaction conditions,working dilutions of polyclonal antibody and bioti-nylated second antibody were assayed while the effect of buffer syste ms and ti me of each reaction were evaluated.RESULTS Maininfluencingfactorsoftheassaywerediscussedasworkingdilutionsofpoly-clonal antibody and biotinylated goat anti rabbit IgG,assay buffer and reacting ti me.After opti mization of reaction conditions,MC-LR AlphaLISA could be finished in 40 min,with a sensitivity of 0.006 μg·L-1 and a dynamic range of 0.006 -5 μg·L-1 .The coefficient of variation was below 10% and average recovery was 1 07.7%.Moreover,the cross reactivity rates of MC-RR and MC-RY to MC-LR were 13.2%and0.91%,respectively.CONCLUSION Thismethodishighlysensitiveandspecific,time-saving and quite suitable for high throughput determination of MC-LR water samples.