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OUR SUPPLIERS
KOMABIOTECH
301,
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Gayang 3 dong,
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Spherotech, Inc.
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Lake Forest, IL 60045
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Exalpha
Biologicals,
Inc.
2 Shaker Road,
Unit B101
Shirley, MA
01464
SCETI K.K
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Laboratories, Inc. Headquarters
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EXBIO Praha, a.s.
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QuantiChromTM
Bilirubin Assay Kit (DIBR-180)
Quantitative Colorimetric Bilirubin Determination at
530nm
DESCRIPTION
BILIRUBIN is one of the degradation products of
hemoglobin formed when red blood cells die. Bilirubin exists in the
insoluble unconjugated form (also indirect bilirubin), or soluble
glucuronide conjugated form bilirubin (also direct bilirubin).
Conjugated bilirubin moves into the bile canaliculi of the liver and
then to the gall bladder. When stimulated by eating, bile (including the
conjugated bilirubin) is excreted into the small intestine, where
bilirubin is converted into urobilinogen. Bilirubin is a key diagnostic
indicator. High levels of bilirubin result when too much hemoglobin is
broken down or the removal of bilirubin does not function properly. The
accumulation of bilirubin in the body causes jaundice. Simple and
automation-ready procedures for quantitative determination of bilirubin
find wide applications in research and drug discovery. BioAssay Systems'
bilirubin assay kit is designed to measure bilirubin in blood specimen
in 96-well or cuvette formats. The improved Jendrassik- Grof method
utilizes the reaction of bilirubin with diazotized sulfanilic acid, in
which a red colored product is formed. The intensity of the color,
measured at 510-550nm, is an accurate measure of the bilirubin level in
the sample. Total bilirubin is assessed using caffeine benzoate to split
bilirubin from the unconjugated bilirubin protein complex.
KEY FEATURES
Sensitive and accurate .
Detection limit 0.16 mg/dL bilirubin in 96-well plate assay.
Simple and high-throughput. The procedure
involves addition of a single working reagent and incubation for 10 min.
Can be readily automated as a high-throughput assay in 96-well plates
for thousands of samples per day.
APPLICATIONS:
Direct Assays:
total and direct bilirubin
in serum or plasma.
Pharmacology: effects of drugs on bilirubin
metabolism.
KIT CONTENTS (180 tests in 96-well plates)
Reagent A: 30 mL Reagent B: 10 mL
Reagent C: 30 mL Saline: 50 mL
Calibrator: 2 mL (equivalent to 5 mg/dL Bilirubin) .
Storage conditions. The kit is shipped at room
temperature. Store all reagents at 4 °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
Hemolysis interferes with the assay. Avoid exposure
of sample to any light. Samples can be store at –20°C for up to 3
months, 2–8°C for 4 days. If turbidity is observed, centrifuge sample
and use clear supernatant for assay.
Procedure using 96-well plate:
1. Reagent Preparation: prepare at least 200
μL/well fresh Working
Reagent as follows,
“Total Bilirubin” is determined with Working Reagent
that contains Reagent C, and “Direct Bilirubin” with Working Reagent
that does not contain Reagent C but saline instead.
2.
Calibrator:
transfer 50 μL
H2O and 50 μL
Calibrator into two wells of clear-bottom 96-well plate, add 200
μL
H2O. The volume in each well 250
μL.
Samples :
transfer 50 μL sample into separate wells, add 200 μL respective Working
Reagent (i.e. for total bilirubin and/or direct bilirubin) and 200
μL
“Blank” Reagent to the sample wells.
3. Incubate 10 min and read OD530nm (510 to 550nm).
Procedure using Cuvet:
1. Prepare at least 800
μL/well
fresh Working Reagent as follows,
2. Transfer 200
μL
H2O and 200 μL
Calibrator into two we lls of clear-bottom 96-well plate, add 800
μL
H2O. Transfer 200 μL
sample, add 800 μL
Working Reagent.
3. Incubate 10 min and read OD530nm (510 to 550nm).
CALCULATION
where ODSAMPLE, ODBLANK, ODCALIBRATOR and ODH2O are
the OD530nm values of the sample, the sample blank, the calibrator and
water. 5 (mg/dL) is the equivalent bilirubin concentration of the
calibrator.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipeting devices and accessories, 96-well plates and
plate reader.
PUBLICATIONS
[1]. Vinchi F et al. 2008. Hemopexin prevents
endothelial damage and liver congestion in a mouse model of heme
overload Am J Pathol. 173(1): 289–299
[2]. Nedredal GI et al. 2009. Optimization of mass
transfer for toxin removal and immunoprotection of hepatocytes in a
bioartificial liver. Biotechnol Bioeng. 104(5):995-1003.
[3]. Beppu F. et al. 2009. Single and repeated oral
dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice.
J. Toxicol. Sci. 34(5): 501-510. |
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