This reagent is intended for research use only. The kit is designed to quantify thyroxine (T4) levels in zebrafish serum, plasma, and other related liquid samples. The method employed is a double-antibody sandwich immunoassay. In this procedure, a microtiter plate is pre-coated with purified anti-T4 antibodies, creating a solid-phase antibody. The sample containing T4 is then added, allowing the antigen to bind to the immobilized antibodies. Subsequently, HRP-conjugated anti-T4 antibodies are introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. After thorough washing to remove unbound components, the substrate TMB is added. Under the catalytic action of HRP, TMB turns blue, and the addition of an acidic stop solution changes the color to yellow. The intensity of the yellow color is directly proportional to the concentration of T4 in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the T4 concentration is determined by comparing the OD values against a standard curve generated from known concentrations. This assay provides a reliable and sensitive method for quantifying T4 in zebrafish biological samples, making it suitable for various research applications such as developmental biology, endocrinology, and toxicology studies.
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