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Fluorescence immunoassay of α-1-fetoprotein with hemin as a mimetic enzyme labelling reagent |
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| Authors: | Qing-Zhi Zhu Feng-Hua Liu J-G Xu Wen-Jin Su Jian-Wei Huang |
| Institution: | The Research Laboratory of SEDC of Analytical Science for Material and Life Chemistry, Department of Chemistry, Xiamen University, Xiamen 361005, P.R. China e-mail: jgxu@xmu.edu.cn, CN Department of Biology, Xiamen University, Xiamen 361005, P.R. China, CN Xiamen Municipal Health and Anti-Epidemic Station, Xiamen 361005, P.R. China, CN |
| Abstract: | A novel mimetic enzyme immunoassay to determine α-1-fetoprotein (AFP) in solution was developed. Hemin, a horseradish peroxidase substitute, was used as a labelling reagent to catalyze the reaction of p-hydroxyphenylacetic (HPA) and hydrogen peroxide in alkaline media. In the competitive immunoassay, monoclonal anti-AFP antibody was coated on a 96-well plate (polystyrene) and a constant amount of hemin-labelled AFP and a known volume of test solution were added. Non-labelled and hemin-labelled AFP compete for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed hemin-AFP conjugate moiety was determined by measuring the fluorescence produced in a solution containing HPA and hydrogen peroxide. The calibration graph for AFP was linear over the range 0 ～ 380 ng/ well with a detection limit of 1.0 ng/well. The method has been applied to determine the AFP in human blood serum with satisfactory results. |
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