The kit employs a double-antibody sandwich immunoassay to quantify the level of osteocalcin (OC) in the sample. A microwell plate is first coated with a purified rat-specific anti-osteocalcin antibody, creating a solid-phase capture reagent. The sample, containing OC, is then added to the wells, allowing the OC molecules to bind to the immobilized antibodies. After washing to remove unbound components, a horseradish peroxidase (HRP)-labeled anti-osteocalcin antibody is introduced, forming a complex consisting of the captured OC, the primary antibody, and the HRP-conjugated secondary antibody.
Following another thorough wash to eliminate any non-specific binding, the substrate TMB (3,3',5,5'-tetramethylbenzidine) is added. In the presence of HRP, TMB undergoes a color change from clear to blue. When an acidic stop solution is added, the color shifts to yellow. The intensity of this yellow color is directly proportional to the concentration of osteocalcin in the original sample.
The optical density (OD) at 450 nm is measured using a microplate reader. By comparing the OD values to a pre-established standard curve, the exact concentration of rat osteocalcin in each sample can be accurately determined. This method ensures high specificity and sensitivity, making it ideal for quantitative analysis of OC in biological specimens such as serum or plasma.
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