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Assay Kit
Quantitative Colorimetric Bilirubin Determination at 530nm
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
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
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:
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). 2007 Ó by BioChain · 3517 Breakw ater Ave., Hayw ard, CA 94545, USA Email: [email protected], [email protected] · Website: www .biochain.com

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