Sample collection from Healthy population: For the determination of the reference interval, blood and urine samples were collected from healthy healthcare professionals (10 males and 10 females). Individuals were informed of the testing to be performed and only the specified testing was performed on the collected reference specimens. Analyses were conducted with the full respect for the individual's right to confidentiality. Information regarding their age, gender and race was also collected with the healthcare professional's consent to calculate the glomerular filtration rate (GFR). Urine creatinine and serum creatinine was assayed for each of these samples. Freshly collected urine samples were allowed to sit at room temperature for 30 minutes to sediment, and the supernatant was aliquoted and stored at -70 oC until analysis.
Construction of KIM-1 sandwich ELISA (R&D Cat# DY1750, Minneapolis, MN): The wells of Nunc-Maxisorp EIA plates were coated by diluting the capture antibody (72 μg/mL) to a working concentration of 0.4 μg/mL in PBS with 100 μL in each well. The plate was sealed and incubated overnight at room temperature. Each well was aspirated and washed using an automated microplate washer (Bio-Tek) with 400 μL of Wash Buffer (0.05% Tween-20 in PBS), repeating the process two times for a total of three washes. The plates were blocked by adding 300 μL of reagent diluent (1% BSA in PBS, 0.2 μm filtered) to each well and incubated at room temperature for 2 hours. After washing as in the previous step, 100 µL of standard recombinant human KIM-1 (0-2000 pg/mL), control, and urine sample was pipetted to the designated well, covered with an adhesive strip, and placed on the orbital shaker at 400 rpm at room temperature for 2 hours. The plate was washed using the same wash protocol as before, and 100 μL of the biotinylated goat anti-human KIM-1 detection antibody diluted in reagent diluent to a working concentration of 400 ng/mL was added to each well. The plate was covered with a new adhesive strip, incubated at room temperature for 2 hours with continued shaking at 400 rpm. Washing step was repeated and 100 μL of streptavidin-HRP diluted to a working dilution was added to each well. The plate was protected from light, covered with a new adhesive strip, shaken at 400 rpm, and incubated at room temperature for 20 minutes. After washing, 100 µL substrate solution was added to all wells, protected from light, covered with an adhesive strip, shaken at 400 rpm, and incubated at room temperature for 7 minutes. The reaction was stopped by adding 50 µL of stop solution to all wells. The absorbance was measured using a plate reader (BioTek Elx800) at 450 nm with an absorbance correction at 540 nm. The urinary KIM-1 concentration was calculated based on the standard curve and expressed in absolute terms (pg/mL).
Evaluation of the KIM-1 ELISA: The validation of the KIM-1 ELISA was evaluated by measuring linearity, intra-run precision, inter-run precision, analytical sensitivity, recovery, dilution verification, reference range, stability and length of run. Details of the criteria for each are described in the result section.
Sample collection and analysis: Urine samples were collected asceptically directly in urine cups and stored at -70 oC within 4 hours. All statistical analysis was carried out using the program EP evaluator 15 (version 8.0, DG Rhoads).
Analytical Testing: Urinary creatinine was measured by the Jaffe rate-blanked creatinine assay using a Roche/ Hitachi 911 system (Roche Diagnostics, Indianapolis , IN, USA). Serum enzymatic creatinine was measured using the Roche/Hitachi P800 analyzer. Glomerular filteration rate (GFR) was calculated using the MDRD (modification of diet in renal disease) formula 186 X (SerumCreatinine)-1.154 X (Age)-0.203 X (0.742 if female and 1.000 if male) X (1.210 if African American and 1.000 if others) (mL/min/1.73m2) 16 .
Construction of KIM-1 sandwich ELISA (R&D Cat# DY1750, Minneapolis, MN): The wells of Nunc-Maxisorp EIA plates were coated by diluting the capture antibody (72 μg/mL) to a working concentration of 0.4 μg/mL in PBS with 100 μL in each well. The plate was sealed and incubated overnight at room temperature. Each well was aspirated and washed using an automated microplate washer (Bio-Tek) with 400 μL of Wash Buffer (0.05% Tween-20 in PBS), repeating the process two times for a total of three washes. The plates were blocked by adding 300 μL of reagent diluent (1% BSA in PBS, 0.2 μm filtered) to each well and incubated at room temperature for 2 hours. After washing as in the previous step, 100 µL of standard recombinant human KIM-1 (0-2000 pg/mL), control, and urine sample was pipetted to the designated well, covered with an adhesive strip, and placed on the orbital shaker at 400 rpm at room temperature for 2 hours. The plate was washed using the same wash protocol as before, and 100 μL of the biotinylated goat anti-human KIM-1 detection antibody diluted in reagent diluent to a working concentration of 400 ng/mL was added to each well. The plate was covered with a new adhesive strip, incubated at room temperature for 2 hours with continued shaking at 400 rpm. Washing step was repeated and 100 μL of streptavidin-HRP diluted to a working dilution was added to each well. The plate was protected from light, covered with a new adhesive strip, shaken at 400 rpm, and incubated at room temperature for 20 minutes. After washing, 100 µL substrate solution was added to all wells, protected from light, covered with an adhesive strip, shaken at 400 rpm, and incubated at room temperature for 7 minutes. The reaction was stopped by adding 50 µL of stop solution to all wells. The absorbance was measured using a plate reader (BioTek Elx800) at 450 nm with an absorbance correction at 540 nm. The urinary KIM-1 concentration was calculated based on the standard curve and expressed in absolute terms (pg/mL).
Evaluation of the KIM-1 ELISA: The validation of the KIM-1 ELISA was evaluated by measuring linearity, intra-run precision, inter-run precision, analytical sensitivity, recovery, dilution verification, reference range, stability and length of run. Details of the criteria for each are described in the result section.
Sample collection and analysis: Urine samples were collected asceptically directly in urine cups and stored at -70 oC within 4 hours. All statistical analysis was carried out using the program EP evaluator 15 (version 8.0, DG Rhoads).
Analytical Testing: Urinary creatinine was measured by the Jaffe rate-blanked creatinine assay using a Roche/ Hitachi 911 system (Roche Diagnostics, Indianapolis , IN, USA). Serum enzymatic creatinine was measured using the Roche/Hitachi P800 analyzer. Glomerular filteration rate (GFR) was calculated using the MDRD (modification of diet in renal disease) formula 186 X (SerumCreatinine)-1.154 X (Age)-0.203 X (0.742 if female and 1.000 if male) X (1.210 if African American and 1.000 if others) (mL/min/1.73m2) 16 .
