BioChain BILIRUBIN Bilirubin Assay Kit Quantitative Colorimetric Bilirubin Determination at 530nm DESCRIPTION Procedure using Cuvet: BILIRUBIN is one of the degradation products of hemoglobin formed
1. Prepare at least 800 mL/w el fresh Working Reagent as fol ow s,
w hen red blood cel s 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 smal
intestine, w here bilirubin is converted into urobilinogen. Bilirubin is a key
2. Transfer 200 mL H2O and 200 mL Calibrator into tw o w el s of
diagnostic indicator. High levels of bilirubin result w hen too much
clear-bot om 96-w el plate, add 800 mL H2O. Transfer 200 mL
hemoglobin is broken dow n or the removal of bilirubin does not function
sample, add 800 mL Working Reagent. Incubate 10 min.
properly. The accumulation of bilirubin in the body causes jaundice.
3. Incubate 10 min and read OD530 nm (510 to 550 nm).
Simple and automation-ready procedures for quantitative determination of bilirubin find w ide applications in research and drug discovery.
Biochain’s bilirubin assay kit is designed to measure bilirubin in blood
CALCULATION
specimen in 96-w el or cuvette formats. The improved Jendrassik-Grof method utilizes the reaction of bilirubin w ith diazotized sulfanilic acid, in w hich 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
w here ODSAMPLE, ODBLANK, ODCALIBRATOR and ODH2O are
bilirubin from the unconjugated bilirubin protein complex.
the OD530 nm values of the sample, the sample blank, the
calibrator and w ater. 5 (mg/dL) is the equivalent bilirubin
KEY FEATURES Sensitive and accurate. Detection limit 0.16 mg/dL bilirubin in 96-w el MATERI ALS REQUIRED, BUT NOT PROVIDED
Pipeting devices and accessories, 96-w el plates and plate reader.
Simple and high-throughput. The procedure involves addition of a
single w orking reagent and incubation for 10 min. Can be readily automated as a high-throughput assay in 96-w el plates for thousands of
Bilirubin APPLICATIONS : Direct Assays: total and direct bilirubin in serum or plasma. Pharmacology: effects of drugs on bilirubin metabolism. KIT CONTENTS (180 te sts in 96-well plates) Reagent A: 30 mL Reagent B: 10 m L Reagent C: 30 mL Saline: 50 mL Calibrator: 2 m L (equivalent to 5 mg/dL Bilirubin). Storage conditions. The kit is shipped at room temperature. Store al reagents at 4 °C. Shelf life: 12 months after receipt. Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised w hile using the reagents.
[Bilirubin], mg/dL
Please refer to Material Safety Data Sheet for detailed information.
PROCEDURES Standard Curve w ith Freshly Prepared Bilirubin
Hemolysis interferes w ith 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
Procedure using 96-well plate: LITERATURE
1. Reagent Preparation: prepare at least 200 mL/w el fresh Working
[1]. Mori L. 1978. Modified Jendrassik--Grof method for bilirubins
adapted to the Abbott Bichromatic Analyzer. Clin Chem. 24: 1841-5.
[2]. Garber CC. 1981. Jendrassik--Grof analysis for total and direct
bilirubin in serum w ith a centrifugal analyzer. Clin Chem. 27:1410-6.
[3]. Poon PK. 1981. A Jendrassik-Grof method modified to eliminate
hemoglobin interference w ith assay of total serum bilirubin. Clin
2O and 50 mL Ca librator into tw o w el s of clear-
200 mL Working Reagent. Incubate 10 min.
3. Incubate 10 min and read OD530 nm (510 to 550 nm).
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6. See the discussion in S. Brownlee, “Doctors without Borders,”13. D. Healy, letter to Peter J. Pitts, Associate Commissioner for Ex- Washington Monthly, April 2004. ternal Relations at the Food and Drug Administration, February 19,7. See J.M. Drazen and G.D. Curfman, “Financial Associations ofAuthors,” NEJM 346 (2002): 1901-1902. 14. “Depressing Research,” Lancet 363 (2004
BioChain BILIRUBIN