Urinary microalbumin (mALB) enzyme-linked immunosorbent assay

Urinary microalbumin (mALB) enzyme-linked immunosorbent assay (ELISA) procedure: 1. **Preparation of standard curve**: Place 10 standard wells on the ELISA plate. Add 100 μl of standard solution to the first and second wells, followed by 50 μl of standard diluent into each. Mix thoroughly. Then transfer 100 μl from the first and second wells into the third and fourth wells, respectively. Add 4 μl of standard diluent to the fourth well and mix again. Discard 50 μl from the third and fourth wells, then add 50 μl of standard diluent into the fifth and sixth wells. Add 50 μl of standard diluent to the sixth well and mix. Transfer 50 μl from the fifth and sixth wells into the seventh and eighth wells, then add 50 μl of standard diluent to each. Finally, transfer 50 μl from the seventh and eighth wells into the ninth and tenth wells, and add 50 μl of standard diluent to each. Discard 50 μl from the ninth and tenth wells. After dilution, the final concentrations in each well are 600 μg/L, 400 μg/L, 200 μg/L, 100 μg/L, and 50 μg/L, respectively. 2. **Sample addition**: Set up blank control wells (no sample or enzyme reagent added, but follow all other steps). For the test samples, add 40 μl of sample diluent to each sample well, followed by 10 μl of the sample (final dilution factor of 5). Carefully pipette the sample into the bottom of the well, avoiding contact with the walls, and gently mix. 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 buffer (30× for 20T or 20× for 48T) with distilled water as instructed. 5. **Washing**: Remove the sealing film, discard the liquid, and blot dry. Fill each well with washing solution, let it sit for 30 seconds, then discard. Repeat this process five times, and pat the plate dry. 6. **Enzyme reagent addition**: Add 50 μl of enzyme-labeled reagent to each well except the blank controls. 7. **Second incubation**: Seal the plate again and incubate at 37°C for 30 minutes. 8. **Second washing**: Follow the same washing procedure as step 5. 9. **Color development**: Add 50 μl of color reagent A, followed by 50 μl of color reagent B. Gently mix and incubate at 37°C for 15 minutes, protecting the plate from light. 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. This detailed procedure ensures accurate detection of urinary microalbumin levels, which is critical for early diagnosis of kidney damage in patients with diabetes or hypertension. Always follow proper laboratory safety protocols and ensure all reagents are properly stored and handled.

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