The Preparation Of AKR1B10 Detection Method Based On Quantum Dot Marker

Objective:To develop a sensitive and specific sandwich fluorescence immunoassay employing quantum dots-modified streptavidin with biotin labeled rabbit anti-AKR1B10 polyclonal antibody and custome made AKR1B10 recombinant protein and the goat anti-AKR1B10 polyclonal antibody.Methods:1.With the CCGKEK polypeptide as a template,water soluble Au quantum dots are to be synthesized.The Au quantum dots and streptavidin are coupled through EDC/NHSS method to obtain the QDs-labeled streptavidin QDs-SA.2.To construct a PET-15b/AKR1B10 plasmid and to transform it into Escherichia coli DH5a to express the protein of AKR1B10 with a his-tag.The Ni-NTA resin column is to be used to purify the his-tag-AKR1B10 protein.3.The polyclonal antibody is obtained by immunizing rabbits with AKR1B10 recombinant protein.An ELISA assay is used to quantitatively analyze the antibody titer,and then purify the antibody.The rabbit anti-AKR1B10 polyclonal antibody is to be coupled with N-hydroxysuccinimide biotin(BNHS)to obtain biotin-labeled rabbit anti-AKR1B10 polyclonal antibody.4.A fluorescence immunoassay kit is to be developed using biotin-avidin amplification system and gold quantum dot-labeled technique based on the Sandwich ELISA.The goat anti-AKR1B10 polyclonal antibody used as the solid-phase antibody,and the biotin-labeled rabbit anti-AKR1B10 polyclonal antibody as the detection antibody,with the signal amplification by Au quantum dot-labeled streptavidin,the content of AKR1B10 protein can be finally measured through the relative fluorescence intensity.Results:1.The QDs and the SA-coupled QDs shows similar fluorescence spectrum curves,with a excitation peak at 332nm and emission peaks between 401 and 405 nm.The dispersibility and spectral properties of the QDs were pertained after SA coupling.About 184 ?g of streptavidin can conjucated with per mg of Au quantum dots.2.The plasmid of PET-15b/AKR1B10 was successfully constructed,confirmed by colony PCR and sequencing analysis.The recombinant plasmid expressed a secretory protein with a molecular weight about 36kDa in E.coli DH5?,which were then purified by a Ni-NTA resin column.The purified protein as antigen successfully immunized rabbit to produce antiserum.3.The titer of rabbit-anti AKR1B10 polyclonal antibody is 1:80000.The specificity of the purified antibody is confirmed by a weak cross reaction with the AKR1B1 protein.The biotin and rabbit anti-AKR1B10 polyclonal antibodies are successfully conjugated.4.The optimal concentration for goat anti-AKR1B10 polyclonal antibody is 6?g/mL when used for coating,0.2?g/mL for biotinlated detecting antibody,and the quantum dots labeled sreptovidin should be diluted to 1:100.Conclusion:A sandwich immunoassay based ELISA with quantum dots for AKR1B10 detection has been established.