Enzyme-linked immunosorbent assay (ELISA) [Abstract]: The immunoenzyme-linked immunosorbent assay (ELISA) was reported in 1971 by Swedish scholars Engrall and Perlmann, and Dutch scholars Van Weeman and Schuurs. The basic principle is that the antigen or antibody is bound to the surface of a solid phase carrier in advance without damaging its immunological activity; during the measurement, the test sample (including the side antibody or antigen) and the enzyme-labeled antigen or antibody are determined according to certain The program reacts with the antigen or antibody bound to the solid phase carrier to form an antigen or antibody complex; when the reaction is terminated, the enzyme-labeled antigen or antibody on the solid phase carrier is bound Reported by Swedish scholars Engrall and Perlmann, Dutch scholars Van Weeman and Schuurs in 1971. The basic principle is that the antigen or antibody is bound to the surface of a solid phase carrier in advance without damaging its immune activity; during the measurement, the test sample (including the side antibody or antigen) and the enzyme-labeled antigen or antibody are The procedure reacts with the antigen or antibody bound to the solid phase carrier to form an antigen or antibody complex; when the reaction is terminated, the amount of enzyme-labeled antigen or antibody bound on the solid phase carrier (immune complex) is the same as the antibody or antibody to be tested The amount of antigen is a certain proportion; after washing to remove other substances in the reaction solution, after adding the enzyme reaction substrate, the substrate is catalyzed by the enzyme on the solid support to become a colored product, and finally the colored product can be determined by qualitative or quantitative analysis The amount of antigen or antibody in the sample. Indirect immune enzyme-linked absorption is the most commonly used method for determining antibodies. The principle is to connect the antigen to a solid phase carrier, and the antibody to be tested in the sample is combined with it to form a solid phase antigen-tested antibody complex, and then an enzyme-labeled secondary antibody is used. Combine with the antibody in the solid-phase immune complex to form a solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex, measure the degree of color development after adding the substrate, and perform qualitative or quantitative determination of the test antibody ). [Materials] 1. Antigen: suspension of Rhizobium etli bacteria (see experiment 3); primary antibody: rabbit anti-aitryli serum (see experiment 3), secondary antibody: horseradish peroxidase ( Horseradish Peroxidanse (HRP) labeled goat anti-rabbit immunoglobulin (IgG) (purchased from the Chinese Academy of Military Medical Sciences). 2. 0.015M, pH 7.4 PBS, o-phenylenediamine (OPD) effervescent tablet solution, 2mol / LH2SO4. 3. Enzyme plate. [Method] 1. Coating: Put the bacterial suspension in the well of 96-well polyethylene enzyme plate, and add 100 ml of normal saline. The enzyme plate is placed in a wet box at 4 ℃ overnight, and taken out the next day, using PBS ( The recipe is shown in the appendix) After washing 3-4 times, it is sealed with parafilm and kept in the refrigerator at 4 ℃ for future use. Negative control wells were set, and only normal saline was added to the wells. 2. Reaction with the primary antibody: add 1: 4000 primary antibody 100 ml to the sample well and the control well, incubate in a humidity box at 37 ℃ for 1 hour, drain the liquid, and wash with PBS 3-4 times. 3. Reaction with the secondary antibody: add 1: 4000 goat anti-rabbit IgG 100 ml to the sample well and the control well, incubate in a wet box at 37 ℃ for 3 hours, drain the liquid, and wash with PBS 3-4 times. 4. Color reaction: Add 100 ml of o-phenylenediamine (OPD) effervescent tablet solution dissolved in H 2 O 2 to the sample well and the control well. Terminate the reaction. [Result judgment] The negative control wells are light yellow, and the wells added with primary antibody are orange, which is a positive reaction (Figure 7-2). [Notes]? The reported time is not less than 18 hours. ? The quilt and incubation should be carried out in a wet box. ? Washing the plate must strive to be clean to ensure the success of the experiment.
Orlistat is a long-acting and potent specific gastrointestinal lipase inhibitor. It is white or off-white powder at room temperature. It is insoluble in water, soluble in chloroform, and easily soluble in ethanol. It passes through the stomach and small intestine cavity. Intragastric lipase and pancreatic lipase active codon form a covalent bond to inactivate the enzyme. Fat in food cannot be broken down into free fatty acids and monoacylglycerols, so the fat cannot be absorbed and utilized, thereby reducing the body's caloric intake and controlling body weight. This medicine does not need to be absorbed through the body to exert its effects. At the usual dose, fat absorption can be suppressed by 30%. It is rarely absorbed after oral administration and can be metabolized and inactivated in the intestine. The metabolic site is on the wall of the gastrointestinal tract, and the elimination half-life is about 14 to 19 hours. About 97% of this product is excreted with feces, of which 83% is excreted in the original form.
Orlistat can be used clinically for obesity and hyperlipidemia. Under normal circumstances, 120mg can be taken orally once a day, three times a day, and taken within one hour after a meal. After taking the medicine for 2 weeks, the weight may start to decrease. It can be taken continuously for 6 to 12 months. If the dose is increased to more than 400 mg per day, its effect will no longer be enhanced.
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