Multiplex Assay Kit for Autophagy Related Protein 5 (ATG5), etc. by FLIA (Flow Luminescence Immunoassay)

Analyte-specific antibodies are pre-coated onto color-coded microparticles. Microparticles, standards, and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest. After washing away any unbound substances, a biotinylated antibody cocktail specific to the analytes of interest is added to each well. Following a wash to remove any unbound biotinylated antibody, Streptavidin-Phycoerythrin conjugate (Streptavidin-PE), which binds to the biotinylated detection antibodies, is added to each well. A final wash removes unbound Streptavidin-PE and the microparticles are resuspended in buffer and read using the Luminex or Bio-Plex analyzer.The MFI developed is proportional to the concentration of analytes of interest in the sample.

1. Preparation of standards, reagents and samples before the experiment; <br/> 2. Add 100μL standard or sample to each well, <br/> &nbsp; &nbsp; &nbsp; &nbsp; add 10μL magnetic beads, and incubate 90min at 37&deg; C on shaker; <br/> 3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37&deg; C on shaker; <br/> 4. Wash plate on magnetic frame for three times; <br/> 5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37&deg; C on shaker; <br/> 6. Wash plate on magnetic frame for three times; <br/> 7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.<br/>

<p> Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Autophagy Related Protein 5 (ATG5), etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively. <br/> Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Autophagy Related Protein 5 (ATG5), etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate. <br/> CV (%) = SD/meanX100 <br/> Intra-Assay: CV<10% <br/> Inter-Assay: CV<12% <br/> </p>

<p> The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. <br/> To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end. </p>

<p> This assay has high sensitivity and excellent specificity for detection of Autophagy Related Protein 5 (ATG5), etc. by FLIA (Flow Luminescence Immunoassay) .<br/> No significant cross-reactivity or interference between Autophagy Related Protein 5 (ATG5), etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed. </p>