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. 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 0.16mg/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.
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 200ul/well fresh Working Reagent as follows,A B C Saline H2OTotal 50 uL 20 uL 130 uLDirect 50 uL 20 uL-130 uLBlank 50 uL-- 130 uL 20 uL“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 50uL H2O and 50uL Calibrator into two wells of clear-bottom 96-well plate, add 200uL H2O. The volume in each well 250uL
Samples: transfer 50uL sample into separate wells, add 200uL respective Working Reagent (i.e. for total bilirubin and/or direct bilirubin) and 200uL “Blank” Reagent to the sample wells
3. Incubate 10 min and read OD530nm (510 to 550nm).
Procedure using Cuvet:1. Prepare at least 800uL/well fresh Working Reagent as follows,A B C Saline H2OTotal 200 uL 80uL 520uL--Direct 200uL 80uL-520uL -Blank 200uL-- 520uL 80uL2. Transfer 200uL H2O and 200uL Calibrator into two cuvets, add 800uL H2O. Transfer 200uL sample into cuvet, add 800uL Working Reagent
3. Incubate 10 min and read OD530nm (510 to 550nm).
Calculation:Bilirubin =ODSAMPLE-ODBLANKODCALIBRATOR–ODH2OX 5 (mg/dL)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.
