**Rabies Ag Quantitative Detection Kit Instructions (Operating Procedure)**
Before starting, ensure all reagents are at room temperature. Remove the kit from the refrigerator and allow it to equilibrate for 30 minutes.
Next, prepare the washing solution by diluting the 20x concentrated wash buffer with distilled water to the original concentration.
Now, set up the microtiter plate. Place the coated plates on a rack and label the wells: standard, sample, and blank control. Record the positions for clarity. Add 50 µL of standard solution into each standard well. For the sample wells, first add 10 µL of the test sample, then 40 µL of sample diluent (diluting the sample 5-fold). The blank control well should remain empty.
Incubate the plate for 30 minutes at 37°C in a water bath or incubator. After incubation, wash the plate thoroughly. Discard the liquid, blot dry on absorbent paper, then fill each well with washing solution. Let sit for 1 minute, discard again, blot dry, and repeat this process four times. A washer can be used for efficiency.
Add 50 µL of the enzyme-labeled working solution to each well, except for the blank control. Incubate again for 30 minutes under the same conditions.
Wash the plate following the same procedure as step 5.
Proceed with 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.
Afterward, terminate the reaction by adding 50 µL of stop solution to each well. The color will change from blue to yellow.
Measure the absorbance at 450 nm within 15 minutes using a microplate reader. First, zero the Instrument using the blank well.
Finally, calculate the results. Plot the standard curve using the known concentrations and their corresponding OD values. Use the regression equation to determine the sample concentration based on its OD reading. You may also use software tools for this calculation. Multiply the calculated value by the dilution factor to obtain the actual concentration. Always check for consistency and accuracy throughout the procedure.
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