Certificate of Analysis
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1. Bilirubin 0 2 4 6 8 10 Bilirubin mg dL Standard Curve with Freshly Prepared Bilirubin in 5g dL Bovine Serum Albumin in 96 well plate assay 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 Active Date 091 720122. 3 gt J BioChain www biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com User s Manual and Instructions Bilirubin Assay Kit Z5030053 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 Biochain s 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 w ith 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
3. 130 pL Total Bilirubin is determined with Working Reagent that contains Reagent C and Direct Bilirubin w ith Working Reagent that does not contain Reagent C but saline instead 2 Calibrator transfer 50 uL H2O and 50 uL Calibrator into two wells of clear bottom 96 w ell plate add 200 uL H2O The volume in each well 250 uL F 753 3UM RevA 25030053UA Samples transfer 50 uL sample into separate wells add 200 uL respective Working Reagent i e for total bilirubin and or direct bilirubin and 200 uL Blank Reagent to the sample w ells 3 Incubate 10 min and read OD530nm 510 to 550nm Procedure using Cuvet 1 Tepa al e at least 800 TB on Reagent as follows CC Saline HO Total 200p 80pl 520 ee Direct 200uL 80uL 520 Blank 200uL 520uL 2 Transfer 200 uL H O and 200 uL Calibrator into two wells of clear bottom 96 w ell plate add 800 uL H O Transfer 200 uL sample add 800 uL Working Reagent 3 Incubate 10 min and read OD530nm 510 to 550nm CALCULATION ODsamete ODe tank Bilirubin _ _ _ __ ODcatiprator ODu20 X 5 mg dL where ODsanete ODsLank ODcatisrator and ODhzo are the ODs3onm 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
4. split bilirubin from the unconjugated bilirubin protein complex KEY FEATURES Sensitive and accurate Detection limit 0 16 mg dL bilirubin in 96 w ell plate assay Simple and high throughput The procedure invoves 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 12 months after receipt 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 uLWwell fresh Working Reagent as follows ee ae 130pL Direct 50uL 20L 130pL Blank 50L
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