Determination of progesterone in milk by enzyme-linked immunosorbent assay - Master's thesis - Dissertation

EL-C1600N100013-B

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Fast cow's milk progesterone

Enzyme linked immunosorbent assay

The determination is based on the combination of free progesterone and horseradish peroxidase-labeled progesterone and the progesterone antibody adsorbed on the pore wall of the microtiter reaction plate. The reaction time is 2 hours, the ABTS substrate solution is reacted for 1 hour, and the reaction stop solution is added. The 410 nm absorption value was measured later. When the amount of the specific antibody is constant, and is smaller than the labeled and labeled antigen, the higher the concentration of the unlabeled antigen, the more the binding of the labeled antigen and the antibody is to the sputum, and the lighter the color. The agility was 5.2 pg/well, the intra-assay coefficient of variation was 9.06%, and the inter-assay coefficient of variation was 12.9%. The enzyme bound to the immune complex forms a colored product upon catalytic hydrolysis and redox reaction when the corresponding enzyme substrate is encountered.

Through the determination of progesterone content in milk, the basic principles and detection techniques of enzyme-linked immunosorbent assay (AID) were studied. The depth of color development was positively correlated with the amount of enzyme, but negatively correlated with the antigen content in the sample. Similar to the RIA method, the test antigen is competitively bound to the specific antibody by the enzyme-labeled antigen, and the color reaction produced by the enzyme-catalyzed substrate labeled on the antigen is tested (added). These enzyme markers (complexes) retain their immunological and enzymatic activities and then react with the corresponding antibodies or antigens to form enzyme-labeled immune complexes. Enzyme-linked immunosorbent assay (ELISA kit) is a chemical method that combines an enzyme with an antigen or antibody to form an enzyme label (or immunologically combines an enzyme with an anti-enzyme antibody to form an immune complex).

Prepare the enzyme progesterone dilution curve and select the enzyme progesterone working concentration to coat the enzyme standard plate with the primary antiserum dilution, dilute the enzyme progesterone to different concentrations, draw the enzyme progesterone dilution curve, select the slope The maximum dilution with a certain optical density value is the working concentration. Pretreatment of the enzyme plate The newly prepared plate contains organic components. It is soaked in absolute ethanol for more than two hours before use, washed with distilled water, dried at 37 °C or dried in the shade. Prepare progesterone antiserum dilution curve and select progesterone antiserum working concentration to dilute progesterone antiserum to different concentrations, coat the different pores of the unified enzyme plate, measure the optical density, and select the best anti-serum dilution Working concentration. As the concentration of antiserum decreases, the optical density decreases. Generally, the dilution at the primary optical density of 1.0 is the working concentration of progesterone antiserum. Prepare the enzyme progesterone dilution curve and select the enzyme progesterone working concentration to coat the enzyme standard plate with the primary antiserum dilution, dilute the enzyme progesterone to different concentrations, draw the enzyme progesterone dilution curve, select the slope The maximum dilution with a certain optical density value is the working concentration.