Development of a Bead-based 96-well Filtration Plate Competitive Immunoassay for the Detection of Gentamycin

• Main Authors: Tien Yu Jessica, 何天瑜
• Other Authors: Chien-Sheng Chen
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/17978667331153687283

碩士 === 國立中央大學 === 系統生物與生物資訊研究所 === 101 === We developed a sensitive, simple, inexpensive and rapid immunoassay platform for the detection of Gentamycin in milk. This platform was composed of liposomal nanovesicle amplification system, Gentamycin sulfate beads and 96-well filtration plates. Gentamycin beads were constructed as a competitor of analyte for the recognition by antibody. The incubation for this competitive assay was conducted in a bottom-sealed 96-well filtration plate. By running washing buffers through the unsealed 96-well filtration plate with gravity but without any spin or vacuum force, the antibody-Gentamycin bead complexes were separated from the rest of the components in the solution. Fluorescent dye-loaded protein G-liposomal nanovesicles were added to specifically bind to antibodies on the beads retained in the sealed 96-well filtration plate. After washing unbound nanovesicles, millions of fluorescent dye molecules were released by adding a detergent solution to lyse liposomal nanovesicles. Results showed that the limit of detection (LOD) of this novel detection platform in TBS and in skim milk were 52.65 ng/mL and 14.16 ng/mL, which are both sufficient for detecting the 200 ng/mL Codex maximum residual level (MRL). The dynamic ranges were both from each of their LODs to 100μg/mL. The LOQs were 257.06 ng/mL and 236.85 ng/mL for detection in TBS and skim milk respectively. The 50% inhibition concentrations (IC50) in TBS and skim milk were 199.66 ng/mL and 360.81 ng/mL, respectively. We also demonstrated the good specificity of this platform by comparing detection results between pure Gentamycin solution and a mixture solution of Gentamycin with other 5 different antibiotics. The entire assay with 60 samples was conducted within 2 h. These results demonstrated that this novel biosensing platform not only fulfilled most benefits of magnetic bead-based assays, but also was inexpensive and convenient by replacing the magnetic separation with a 96-well filtration plate separation.