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QuantiChromTM Creatinine Assay Kit
(DICT-500)
Quantitative Colorimetric Creatinine
Determination at 510nm
DESCRIPTION
Creatinine is synthesized in the body at
a fairly constant rate from creatine, which is produced
during muscle contractions from creatine phosphate. In
the blood, creatinine is removed by filtration through
the glomeruli of the kidney and is secreted into urine.
In healthy individuals, creatinine secretion is
independent of diet and is fairly constant. The
creatinine clearance test has become one of the most
sensitive tests for measuring glomerular filtration
rate. In kidney disease, creatinine levels in the blood
are elevated, whereas the creatinine clearance rate and
hence the urine levels are diminished. Creatinine test
is most widely used to assess kidney function. Simple,
direct and automation-ready procedures for measuring
creatinine concentration in biological samples are
becoming popular in Research and Drug Discovery.
BioAssay Systems' creatinine assay kit is designed to
measure creatinine directly in biological samples
without any pretreatment. The improved Jaffe method
utilizes picrate that forms a red colored complex with
creatinine. The intensity of the color, measured at
510nm, is directly proportional to creatinine
concentration in the sample. The optimized formulation
substantially reduces interference by substances in the
raw sample.
KEY FEATURES
Sensitive and accurate. Use 30 μ L
samples. Detection limit 0.10 mg/dL (8μM)
creatinine in 96-well plate assay.
Simple and high-throughput. The
procedure involves addition of a single working reagent
and incubation for 5 min. Can be automated as a
high-throughput assay for thousands of samples per day.
Improved reagent stability and
versatility. The optimized formulation has greatly
enhanced reagent and signal stability. Assays can be
executed in 96-well plate or cuvet.
Low interference in biological samples.
No pretreatments are needed. Assays can be directly
performed on raw biological samples.
APPLICATIONS
Direct Assays: urine, serum, plasma
and biological preparations.
Drug Discovery/Pharmacology: effects
of drugs on creatinine metabolism.
KIT CONTENTS (500 tests in 96-well
plates)
Reagent A: 50 mL Reagent B: 50 mL
Creatinine Standard: 1 mL 50 mg/dL
Storage conditions. The kit is
shipped at room temperature. Store all components at
2-8°C. For long-term storage, keep standard at –20°C.
Shelf life: at least 6 months (see expiry dates on
labels).
Precautions:
reagents are for research use
only. Normal precautions for laboratory reagents should
be exercised while using the reagents. Please refer to
Material Safety Data Sheet for detailed information.
PROCEDURES
Equilibrate reagents to room temperature
prior to use. Please note the difference in
standard/sample volume and Working Reagent strength for
blood and urine assays. This assay is based on a kinetic
Jaffe reaction. To ensure identical incubation time,
addition of Working Reagent to standard and samples
should be quick and mixing should be brief but thorough.
Use of a multi-channel pipettor is recommended.
Procedure using 96-well plate:
BLOOD ASSAY (LOW CREATININE LINEAR UP TO
50 mg/dL) :
1. Dilute standard to 2 mg/dL by mixing
5
μL
50 mg/dL standard stock and 120
μL
distilled water. Transfer 30
μL
diluted standard and serum/plasma in duplicate into
wells of a clear bottom 96-well plate.
2. Prepare enough
Working Reagent by mixing per well reaction at least 100
μL
Reagent A and 100 μL
Reagent B. Add 200 μL
Working Reagent quickly
to
all wells. Tap plate briefly to mix.
3. Read optical density immediately
(OD0) and then at 5 min (OD5) at 490-530nm (peak
absorbance at 510nm).
URINE ASSAY (HIGH CREATININE LINEAR UP
TO 300 mg/dL) :
1. Transfer 5
μL
50 mg/dL standard and urine in duplicate into wells of a
clear bottom 96-well plate.
2. Prepare enough Working Reagent by
mixing per well reaction 50
μL
Reagent A, 50 μL
Reagent B and 100 μL
water. Add 200 μL
Working Reagent quickly
to
all wells. Tap plate briefly to mix.
3. Read optical density immediately
(OD0) and then at 5 min (OD5) at 490-530nm (peak
absorbance at 510nm).
Procedure using cuvette:
1. Transfer 100
μL
2 mg/dL Standard and serum/plasma samples (Urine
Assay: 15 μL
50 mg/dL Standard and 15
μL
urine)
to cuvets.
2. Prepare appropriate Working Reagent
as above for the 96-well plate procedures. Add 1000
μL
Working Reagent to each cuvet and pipet
briefly
to
mix (avoid bubble formation).
3. Read OD immediately (OD0) and at 5
min (OD5) at 490-530nm.
CALCULATION
Creatinine concentration of the sample is calculated
as
OD SAMPLE5,
ODSAMPLE0,
ODSTD5
and ODSTD0
are OD510nm values of sample and standard at 5 and 0
min, respectively. [STD] is 2 mg/dL for blood assay and
50 mg/dL for urine assay.
Conversions: 1 mg/dL creatinine equals
88.4 μ M,
0.001% or 10 ppm.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipeting devices and accessories (e.g.
multi-channel pipettor). Clear bottom 96-well plates
(e.g. Corning Costar) and plate reader for the plate
procedure. Spectrophotometer and cuvets for measuring OD
510nm for the cuvette procedure.
EXAMPLES
Samples were assayed in duplicate (n =
2) using the 96-well plate protocol. The creatinine
concentration (mg/dL) was 0.38
±
0.01 for rat serum, 0.71 ±
0.02 for rat plasma, 0.79 ±
0.00 for human serum, 0.89 ±
0.04 for human plasma, 1.20 ± 0.04 for goat serum and
136.4 ± 2.9 in a fresh human urine sample.
PUBLICATIONS
1. Wang, J.J. et al (2006). Salutary
effect of pigment epithelium– derived factor in diabetic
nephropathy evidence for antifibrogenic activities.
Diabetes 55: 1678-1685.
2. Zhang, S.X. et al (2006). Therapeutic
potential of angiostatin in diabetic nephropathy. J Am
Soc Nephrol 17: 475–486.
3. Davalos-Misslitz, A.C.M. et al
(2007). Generalized multi-organ autoimmunity in
CCR7-deficient mice. Eur. J. Immunol. 37: 613–622. |
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