This kit is designed to measure the concentration of thyroxine in zebrafish serum, plasma, and other related liquid samples. The experimental method is based on the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique. In this procedure, a microtiter plate is pre-coated with purified antibodies specific to zebrafish thyroxine, forming a solid-phase antibody. After adding the sample containing thyroxine, the antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then introduced, which binds to the captured thyroxine, forming a stable antibody-antigen-enzyme complex. Following a thorough washing step to remove unbound components, a TMB (3,3',5,5'-tetramethylbenzidine) substrate is added. Under the catalytic action of HRP, TMB turns blue, and when an acidic stop solution is added, it changes to a yellow color. The intensity of the yellow color is directly proportional to the thyroxine concentration in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the thyroxine levels are determined by comparing the OD values against a pre-established standard curve. This method ensures high specificity and sensitivity for accurate quantification of thyroxine in zebrafish biological samples.
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