Using clenbuterol kit for clenbuterol residues in pork tissue, were detected and analyzed after extraction, competition with a microplate reader. This method is more suitable for on-site inspection , with fast detection speed and high sensitivity , which is a good monitoring method to ensure the health and safety of meat.
Enzyme-linked immunosorbent assay is currently the best method of detection. ELISA methods include double antibody sandwich assay for antigen, indirect assay for antibodies, and competitive assay for antibodies. In this paper, the antibody is tested by the competition method in the enzyme-linked immunosorbent assay, and the principle is that the polyclonal antibody can be combined with the clenbuterol hydrochloride or the coated antigen. These coating antigens are immobilized on the wall of the ELISA plate, and when the sample contains clenbuterol, it competes with the coated antigen for binding a limited amount of binding sites in the antibody. Since the number of binding sites of antibodies in each well is the same, when the concentration of clenbuterol in the sample is low, more antibody sites are bound to the coated antigen, and more antibodies are immobilized on the wall of the microplate. Combined with more enzyme-labeled secondary antibodies, the result is dark blue. In contrast, the sample had a high concentration of clenbuterol and the result was a light blue color. After the addition of the stop solution, the blue color turned yellow and then measured with a microplate reader. The use of competitive enzyme-linked immunosorbent assay for the detection of clenbuterol has the characteristics of high speed and high sensitivity. It is suitable for on-site detection and has a guiding role in the development of clenbuterol detection.
1 Experimental materials and methods
1.1 Preparation of raw materials
A representative skinless pork with a certain batch is taken to remove impurities and fat. Mix the fine meat with a shear homogenizer and place it in the freezer for free. Take 5 g of the mashed sample, add 25 mL, and shake 50 HCl for 1.5 h to achieve homogenization. Weigh 5g of homogenate (equivalent to 1g liver or muscle), add to the centrifuge tube, and centrifuge at 10,000r/min for 15min. The supernatant was taken to another centrifuge tube, and 1 mol of NaOH 300 ul was added and mixed for 15 min. Add 4 mL, 500 mmol KH2PO4 (pH 3.0), and mix quickly and store in a refrigerator at 4 ° C for at least 1.5 h. Centrifuge for 15 min at 10,000 r/min and separate the supernatant.
1.2 Reagents Clenbuterol-Enzyme-Linked Immunosorbent
1.3 MK3 FC microplate reader, Wellwash 4mk2 washer, OMNI series multi-sample prep, high-energy hybrid Mixer, Macro, hand-held TH shear homogenizer Finnpipette single- channel 8, 12 micro-sampler (by mainland agents) Pingliyang company friendship hotline 13581963618) domestic centrifuge domestic electric thermostat water bath.
1.4 Method
1.4.1 Wash all reagents back to room temperature. The concentrated washing solution was diluted 10 times with distilled water. The enzyme-linked immunoplate was taken out and allowed to equilibrate for 5 min at room temperature. Add 300 uL of the washing solution to each well, place it for 1 min, then remove the washing solution, repeat 3 times, and dry the residual washing liquid on the plate on the absorbent paper.
1.4.2 The antibody in the competition kit was diluted 1:1000. Add the standard in the order of 1 to 3 on the plate during the loading, repeat 100 times per well, add the other samples to the sample to be tested, 100uL antibody per well. Be careful not to let the tip of the sample contaminate the sample with the antibody. Standards, then place the plate in a wet box and compete for 30 min at 37 degrees Celsius.
1.4.3 The secondary antibody-labeled enzyme in the secondary antibody kit is diluted 1:1000. Add 200 μL of the prepared secondary antibody-labeled enzyme to each well on the enzyme-linked plate and place it in a wet box at 37 ° C for 30 min.
1.4.4 Add substrate to the substrate. Substrate A and substrate B. Mix in equal volume. Add 200 μL of the substrate on each plate to develop color. After color development, add 50 μL to each well. The stop solution terminates the reaction. The optical density (OD) value at 450 nm of each standard sample and each sample was measured on a microplate reader, and a 200 ng/mL well of a clenbuterol standard solution was used as a 0-well.
2 Discussion
2.1 Storage of the kit The kit is stored at 4 degrees Celsius. The antibody and the enzyme-labeled secondary antibody (IGg-HRP) are easily denatured at normal temperature, and must be stored frozen, and directly diluted when used.
2.2 Loading There are 3 loading steps in the experiment, ie adding the specimen, enzyme conjugate and substrate. When adding the sample, add the sample to the bottom of the ELISA plate hole with a micro-sampler. Hold the middle of the micro-sampler with your left hand, avoid adding it to the upper part of the hole wall, and prevent splashing and bubble generation, resulting in experimental error. . When adding the enzyme conjugate application liquid and the substrate application liquid, a quantitative multi-channel dosing device can be used to complete the dosing process quickly.
2.3 Insulation There were two antigen-antibody reactions in the experiment, ie, after adding the specimen and adding the enzyme combination. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation or incubation. Because ELISA is a solid-phase immunoassay, the binding of antigens and antibodies occurs only on the surface of the solid phase. The specimens added to the wells in the wells do not have the same chance of binding to the solid phase, only the closest to the pore walls. The antigen in a layer of solution is directly in contact with the antibody. This is a process of gradual equilibrium, so it needs to be diffused to reach the end of the reaction. The same applies to the binding of the enzyme-labeled antibody added thereafter to the solid phase antigen. The temperature of the incubation is usually 37 degrees Celsius, which is also the suitable temperature for most antigen-antibody binding. The two antigen-antibody reactions are generally carried out at 37 ° C for 1 to 2 h, and the product formation reaches a peak. To speed up the reaction, increase the temperature of the reaction, but not more than 43 degrees Celsius. The method of heat preservation is to use a water bath, and the plate is placed in a stainless steel electric heating constant temperature water bath. Note that the ELISA plate can be placed in a water bath, and the bottom of the ELISA plate should be attached to the water surface to quickly balance the temperature. In order to avoid evaporation, the plate should not be measured at the same time more than two plates at a time.
2.4 Washing Washing is a critical step in the ELISA process. ELISA relies on washing to achieve the goal of separating free and bound enzyme labels. The substance remaining in the pores of the plate which is not capable of binding to the solid phase antigen or antibody, and the interfering substance which is non-specifically adsorbed to the solid phase carrier during the reaction are removed by washing. The adsorption of proteins by plastics such as polystyrene is universal, and such non-specifically adsorbed interfering substances should be washed away during washing. If the washing plate is contaminated or the washing water is contaminated by the free enzyme label, the washing frequency is insufficient or the water injection amount is insufficient, and the interval after washing the plate is too long, the plate hole is dried, etc., which directly affects the final result of the test, and the experiment does not produce color. The experiment failed.
2.5 Color development and colorimetric color development is the last step of the incubation reaction in the ELISA, where the enzyme catalyzes the colorless substrate to produce a colored product. The temperature and time of the reaction are still factors that affect color development. The negative holes remain colorless for a certain period of time, while the positive holes are colored for a prolonged period of time. Properly increasing the temperature helps accelerate color development. In the quantitative determination, the reaction temperature and time after the addition of the substrate should be as accurate as specified. The acidic stop solution H2SO4 converts blue to yellow, at which point the absorbance can be read at a specific wavelength (450 nm). Before colorimetry, dry the liquid attached to the bottom of the plate with a clean absorbent paper, and then place the plate correctly in the colorimetric frame of the enzyme-based colorimeter. In the case of a soft-board test, the plate must be placed in a standard 96-well frame for colorimetric analysis. And cut the edge of the soft board before adding the substrate liquid to make the soft board
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