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**ELISA Kit Operating Instructions – Rat Cholesterol (CH)**
1. **Standard Dilution and Loading:**
Place 10 standard wells on the microplate. Add 100 μL of standard solution to the first and second wells. Mix 50 μL of the diluted standard and mix well. Then transfer 100 μL from the first and second wells into the third and fourth wells, followed by adding 50 μL of standard diluent to each. Discard 50 μL from the third and fourth wells, then add 50 μL to the fifth and sixth wells. Repeat this step for the seventh and eighth wells, and finally add 50 μL to the ninth and tenth wells. After each step, mix thoroughly. The final concentrations in the wells will be 480 ng/L, 320 ng/L, 160 ng/L, 80 ng/L, and 40 ng/L, respectively.
2. **Sample Addition:**
Set up blank control wells (no sample or enzyme reagent added, but follow all other steps). Add 40 μL of sample diluent to each test well, then add 10 μL of the sample (final dilution: 5x). Carefully pipette the sample to the bottom of the well without touching the walls. Gently mix the solution.
3. **Incubation:**
Seal the plate with an adhesive film and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation:**
Dilute the concentrated washing solution (30 times for 48T) with distilled water and set aside.
5. **Washing:**
Remove the sealing film, discard the liquid, and blot dry. Fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process five times. Gently pat the plate dry.
6. **Enzyme Reagent Addition:**
Add 50 μL of enzyme-labeled reagent to each well, except for the blank control wells.
7. **Second Incubation:**
Seal the plate again and incubate at 37°C for another 30 minutes.
8. **Second Washing:**
Follow the same washing procedure as step 5.
9. **Color Development:**
Add 50 μL of color developer A, followed by 50 μL of color developer B. Gently mix the solution and incubate at 37°C for 15 minutes, avoiding light exposure.
10. **Stop Reaction:**
Add 50 μL of stop solution to each well to terminate the reaction. The color will change from blue to yellow.
11. **Measurement:**
Measure the absorbance (OD value) at 450 nm using a microplate reader. Ensure measurements are taken within 15 minutes after adding the stop solution.
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