Casein Detection Kit (ELISA) Instruction Manual

Casein Quantitative Detection Kit (ELISA) User's Manual Kit Name Casein Quantitative Detection Kit (ELISA) Quantitative detection of casein in bovine serum, plasma and related fluid samples ( Casein) content. [Detection Principle] This kit uses a double antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). The standard product and the sample to be tested are added to a pre-coated casein antibody clear enzyme-labeled plate, incubated for a sufficient period of time, washed to remove unbound components, and then the enzyme-labeled working solution is added for sufficient time to incubate. Afterwards, the unbound components are washed away. Substances A and B were sequentially added, and the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), which became yellow under the action of acid. The depth of color and the casein in the sample (Casein) ) The concentration was positively correlated, and the OD value was measured at a wavelength of 450 nm. According to the OD values ​​of the standard product and the sample, the content of the cow casein (Casein) in the sample was calculated. [kit composition] 1 Enzyme labeling plate 12 wells × 8 strips 7 Chromogenic reagent B liquid 6 mL2 Standard product 0.3 mL*6 tube 8 Stop solution 6 mL3 20-fold concentrated washing solution 25 mL 9 Manual 1 copy 4 Biotin 1 mL 10 Plate 2 sheets Enzyme Labeling Reagent 10mL 11 Sealed Bag 1 6 Reagent A Liquid 6mL Remarks: The standard product (S5→S0) concentration is: 400, 200, 100, 50, 25, 0 μg/mL. Needed but not provided reagents and equipment] 1, 37 °C incubator 2, standard spectrograph 3, precision pipettes and disposable tips 4, distilled water 5, disposable test tubes 6, absorbent paper [Procedure] 1 Preparation: Remove the kit from the refrigerator and re-equilibrate at room temperature for 30 minutes. 2. Dosing solution: Dilute the 20-fold concentrated washing solution with distilled water to an original multiple of the washing solution. 3, set the sample hole: take a sufficient number of enzyme standard coated plate, fixed on the frame, set the standard product hole, sample hole to be tested and blank control hole, record the location of each hole. 4. Add the standard: Add 50 μL of standard substance to the standard well; blank control wells are not added. 5. Add the sample to be tested: Add 10 μL of biotin to the hole of the sample to be tested, and then add 50 μL of the sample to be tested. Blank wells should not be added. 6. Add Enzymatic Labeling Working Solution: Add 100 μL Enzyme Labeling Working Solution to each well; blank control wells are not added. 7. Incubation: Incubate in 37°C water bath or incubator for 60 min. 8. Wash plate: discard the liquid, pat dry on the absorbent paper, fill the washing liquid in each hole, let it stand for 1 min, shake off the washing solution, and pat dry on the blotting paper, so repeatedly wash the plate 4 times (can also be used to press the plate washer Manual operation wash plate). 9. Color development: Add 50 μL of color developer solution A to each well, add 50 μL of color developer solution B, and develop color at 37°C for 15 min. 10. Termination: Remove the microtiter plate and add 50 μL of stop solution per well to stop the reaction (color changes from blue to yellow). 11. Assay: Zero the blank well and measure the absorbance (OD) of each well with a wavelength of 450 nm within 15 minutes after termination. 12. Calculation: According to the concentration of the standard product and the corresponding OD value, calculate the linear regression equation of the standard curve. Then according to the OD value of the sample, calculate the corresponding sample concentration in the regression equation, and also use various application software. Calculations. The final concentration is the actual measured concentration multiplied by the dilution factor. Sample Requirements 1. The sample should not contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP). 2. As soon as possible after specimen collection, the extraction is carried out and the extraction is carried out according to the relevant literature. After the extraction, the experiment should be carried out as soon as possible. If it cannot be tested immediately, the specimen can be stored at -20°C, but repeated freezing and thawing should be avoided. 3, the sample should be fully centrifuged, without hemolysis and particles. 【Precautions】 1. The experiment shall be conducted strictly in accordance with the instructions. The determination of the experimental results must be based on the reading of the microplate reader. 2. If the enzyme-label coated plate is not used up after it has been opened, it should be immediately put into a sealed bag and stored with desiccant. 3. It is recommended that all standard products, samples, and blank controls be duplicated and averaged to reduce experimental error. 4, if the color is too light, the substrate incubation time can be appropriately extended. 5. In order to avoid cross-contamination, a standard tip, a sample, and a blank control must each be replaced with a pipette tip; common components such as enzyme-labeled working solution, sample diluent, and substrate must be cantilevered and must not touch the microwells. Do not reuse the sealing film. 6, the use of the kit shelf life, different batch number of reagents can not be mixed. 7, the substrate B is sensitive to light, to avoid prolonged exposure to light. 【Summary of Operation Procedure】Prepare reagents, samples and standards Add the prepared samples, standards and ELISA reagents, wash the plate at 37°C for 60 minutes, add the color solutions A and B, and develop at 37°C for 15 minutes. Stop solution read OD value calculation within 15 minutes 【Scope of detection】 12.5μg/mL - 400μg/mL [Specifications] 96T / box [Storage] 2-8 °C, protected from light and moisture. 【Valid period】6 months