Development and optimization of ultrasensitive homogenous immunoassay for microcystin-LR
10.3867/j.issn.1000-3002.2014.03.021
- VernacularTitle:微囊藻毒素-LR 超灵敏均相免疫分析方法的建立与优化
- Author:
Yi ZHANG
;
Biao HUANG
;
Weimin NIU
;
Canpei ZHAO
;
Jian JIN
- Publication Type:Journal Article
- Keywords:
microcystin;
microcystin-LR;
che milu minescent measurments;
ho mogeneous i mmuno-assay
- From:
Chinese Journal of Pharmacology and Toxicology
2014;(3):432-437
- CountryChina
- Language:Chinese
-
Abstract:
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.
