**Rabies Ag Quantitative Detection Kit Operating Instructions**
Before starting, make sure all reagents and materials are ready. First, take the kit out of the refrigerator and allow it to reach room temperature by leaving it for 30 minutes. This ensures optimal performance of the reagents.
Next, prepare the washing solution. The provided washing solution is 20 times concentrated. Dilute it with distilled water to the original concentration as instructed.
Now, set up the microtiter plate. Place the enzyme-labeled plates on the frame and label each well: standard wells, sample wells, and blank control wells. Record the positions to avoid confusion. Add 50 μL of the standard solution to each standard well. For the sample, first add 10 μL of the sample, then 40 μL of the diluent (making a 5-fold dilution). Do not add anything to the blank control.
Incubate the plate at 37°C for 30 minutes. After incubation, wash the plate thoroughly. Remove the liquid, gently pat dry on absorbent paper, then fill each well with washing solution. Let it stand for 1 minute, discard the liquid, and repeat this process four times. A washer can also be used if available.
Add 50 μL of the enzyme-labeled working solution to each well, except the blank control. Incubate again for 30 minutes under the same conditions.
Wash the plate again following the same washing procedure as step 5.
For color development, add 50 μL of developer A to each well, followed by 50 μL of developer B. Incubate at 37°C for 15 minutes. The reaction will turn the solution from blue to yellow.
Afterward, stop the reaction by adding 50 μL of the stop solution to each well. This halts the color change.
Measure the absorbance immediately after stopping the reaction. Use the blank well to zero the Instrument, then measure the OD value at 450 nm within 15 minutes.
Finally, calculate the results. Plot a standard curve using the known concentrations and their corresponding OD values. Use the regression equation to determine the concentration of the sample based on its OD reading. You may also use software tools for more accurate calculations. Remember to multiply the calculated concentration by the dilution factor to obtain the actual sample concentration.
Always follow safety guidelines and handle all reagents with care. Ensure proper storage of unused components and dispose of waste according to local regulations.
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