Time-resolved Fluoroimmunoassay Based On Nanoparticles For Detecting Pathogens Of Aquaculture

The aquaculture of china has been developing rapidly.However,the frequent occurrence of disease from bacteria leads to great financial loss in aquaculture.The commonly used immunassys,such as enzyme-linked immunosorbent assay and fluorescent antibody method,suffer from low sensitivity and high background fluorescence.Time-resolved fluorescence immunoassay(TRFIA)is a sensitive and efficient means of detection because the background fluorescence of TRFIA can be effectively reduced.TRFIA has been widely used in the immunoassay for pathogens,toxic compounds.The fluorescent nanoparticles combine with a variety of biological macromolecules such as antibodies and their biological activities aren't affected.In this study,TRFIA based on nanoparticles for detecting pathogens of aquaculture was established.The contents are as follows:(1)A direct nanoparticles TRFIA for Aeromonas hydrophila B15 was established.The bacteria were coated on the plate,and the antibody labeled by the fluorescent nanoparticles was used as detection antibody.The coating time,the temperature of coating and the concentration of nanoparticles–antibody were optimized by orthogonal experiment.The dectiton limit was 1.0×10~5cfu/mL.(2)In order to improve the detection sensitivity,the antibody for Aeromonas hydrophila B15 werecoated on the plate,and a double-antibody sandwich nanoparticles TRFIA was established.The concentration of coated antibody,the coating time,immune reaction time and the concentration and concentration of nanoparticles–antibody were optimized by orthogonal experiment.The dectiton limit was 1.0×10~3cfu/mL.(3)The sensitivity of detection for Aeromonas hydrophila B15 can be further improved in biotin-streptavidin systems.The antibody was labeled by biotin,and streptavidin was combined with fluorescent nanoparticles.A avidin-biotin(Biotin-Avidin,BA)-nanoparticles TRFIA for Aeromonas hydrophila B15 was established.The concentration of biotinylated antibody,the combining time of biotinylated antibody and BA-fluorescent nanoparticles and the concentration of BA-fluorescent nanoparticle concentration were optimized by orthogonal experiment.The dectiton limit was 8.0×10~2cfu/mL.