Fast cow's milk progesterone detection
Enzyme-linked immunosorbent assay (ELISA) is a widely used technique in biological and medical research. This method relies on the specific binding between antigens and antibodies, with the aid of an enzyme to produce a detectable signal. In this procedure, free progesterone in the sample competes with horseradish peroxidase-labeled progesterone for binding to progesterone-specific antibodies coated on the microtiter plate. After a 2-hour incubation, the ABTS substrate is added, and the reaction is stopped after one hour. The absorbance at 410 nm is then measured.
The color intensity is inversely proportional to the concentration of the unlabeled antigen in the sample. When the amount of labeled antigen is constant and lower than that of the unlabeled antigen, more unlabeled antigen leads to less binding of the labeled antigen to the antibody, resulting in a lighter color. The sensitivity of this method is 5.2 pg/well, with an intra-assay coefficient of variation of 9.06% and an inter-assay coefficient of variation of 12.9%. The enzyme-bound immune complex generates a colored product when it reacts with its corresponding substrate, enabling quantification through optical density measurements.
By analyzing progesterone levels in milk, this method helps understand the basic principles and techniques of ELISA. Similar to radioimmunoassay (RIA), the test involves competitive binding between the unlabeled antigen and the enzyme-labeled antigen for specific antibodies. The enzyme catalyzes the substrate, producing a measurable color change. These enzyme-labeled complexes retain both their immunological and enzymatic activity, allowing them to bind with specific antibodies or antigens to form detectable immune complexes.
ELISA kits are chemical methods that combine enzymes with antigens or antibodies to create enzyme labels. Alternatively, they may involve the combination of an enzyme with anti-enzyme antibodies to form immune complexes. This technique provides a sensitive and reliable way to detect and quantify various substances, including hormones like progesterone.
To prepare the enzyme progesterone dilution curve, different concentrations of the enzyme-labeled progesterone are tested. The working concentration is determined by selecting the maximum dilution that still produces a consistent optical density. Before use, the enzyme plate should be soaked in absolute ethanol for over two hours, then washed with distilled water and dried at 37°C or in a shaded area.
A similar process is followed for preparing the progesterone antiserum dilution curve. The antiserum is diluted to various concentrations, and the optical density is measured to determine the optimal working concentration. Typically, a dilution that yields an optical density of around 1.0 is selected as the working concentration for the antiserum.
This detailed preparation ensures accurate and reproducible results, making ELISA a powerful tool in diagnostic and research settings.
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