TITLE: Creatinine Companion Protocol Assay (Exocell)
AUTHORS: Kathi Burke (Frank Brosius lab), Peter Reifsnyder (Ed Leiter Lab)

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This assay is designed for companion use with Exocell’s immunospecific ELISAs for urine albumin excretion, allowing expression of results as µg albumin per mg creatinine in the urine. Adaptation of creatinine measurement to microtiter wells enables convenient, simultaneous determination of albumin and creatinine concentrations in the same specimen, using analogous microtiter plate formats. Normalization of albumin excretion in relation to creatinine facilitates classification according to defined ranges of microalbuminuria, the established marker of the early diabetic renal dysfunction. The urine albumin-to-creatinine ration in a random urine specimen is an accepted alternative to cumbersome 24 urine collections in the detection and monitoring of microalbuminuria.</div><div class = "text-block"><span style = "font-weight:bold;">The procedure is an adaptation of the alkaline picrate method and entails determination of the differential absorbance in a sample before and after the additions of acid to correct for color generation due to interfering substances.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bicd4kcn.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bice4kcn.jpg" /></div></div>

[STEPS]
?. [Urine Collection Protocols]
Spot UrineBetween 8:00 and 10:00 a.m., the mouse is picked up and induced to urinate into a 1.5 ml microfuge tube. If this is not successful, or not enough is collected, attempts on successive days can be combined (urine should be stored at 4ºC between attempts). Another way to collect spot urine is to place the mouse in a special cage (no food or water) the size of a 96-well plate. I have a custom made apparatus with compartments for eight mice. The mice stand on a 96-well plate for 2 hours. Urine is collected from the various wells the mouse urinates in, taking care to avoid any urine contaminated by feces.24-hour Mice are weighed before placement into Techniplast 24-hr urine collection cages. Mice remain in the cages for 72 hours. The urine is collected and mice are weighed after each 24 hour period. Urine is stored at 4ºC until analysis. Food and water are replenished as necessary. The sample from the third 24-hour period is the preferred sample for albumin and creatinine measurement. In general, if the sample is stored at 4ºC, then analysis will occur within 7 days. If longer stored is required, the sample can be frozen at -60ºC for two months.Kit Contents: ♦ 96 well microtiter plate (2) ♦ Picrate Reagent (2X 10ml) ♦ 1 N NaOH ♦ Acid Reagent (2X 12ml) ♦ Standards (1,3, and 10 mg/dL)
?. [Urine Collection Protocols]
Procedure:1. Reagent Preparation: Add 2.0 ml of 1 N NaOH to a bottle of 10 ml Picrate Reagent. This is a working solution of alkaline picrate (picrate working solution), and must be used immediately after preparation. 2. Sample Dilution: For human samples, prepare 1:20 dilution in distilled water in disposable microfuge tubes. For animal samples, appropriate dilution will depend on method of collection. If washing of collection apparatus is employed, further dilution may not be required. Otherwise, a 1:20 dilution is acceptable. 3. Add 20 µl of water to wells A1 and A2. These are control blanks. 4. Add 20 µl of Creatinine Standard, 10 mg/dl to wells A3 and A4. 5. Add 20 µl of Creatinine Standard 3 mg/dl to wells A5 and A6. 6. Add 20 µl of Creatinine Standard 1 mg/dl to wells A7 and A8. 7. Add a 20 aliquot of diluted sample to wells A9 and A10. 8. Continue the addition of sample aliquots to the rest of the plate. 9. Add 100 µl picrate working solution to each of the wells. 10. Let stand for 10 minutes on the bench top. 11. Determine the absorbance of the wells on a plate reader set at approximately 500 nm. Well A1 serves as “Blank”. 12. Add 100 µl of Acid Reagent to each of the wells. 13. Let stand for 5 minutes. 14. Measure absorbance as described above.
?. [Urine Collection Protocols]
Data Analysis:1. Calculate the data absorbance: Adelta = Aalkaline picrate — Aalkaline picrate+acid 2. Determine the least squares regression line using delta absorbance versus creatinine concentration for each standard. Do not include blank. 3. Determine the concentration of diluted samples by substituting the respective delta absorbance appropriately. 4. Multiply these values by the reciprocal of the dilution factor to obtain concentration (mg/dL) in undiluted samples.