BILIRUBIN DIRECT BILIRUBIN ASSAY:
(Mod. Jendrassik & Grof's method) Pipette into clean dry test tubes labeled as Blank (B) and Test (T)
For the determination of Direct & Total Bilirubin in serum)
Addition Sequence BD (µl) T (µl)
(For In vitro Diagnostic Use Only)
Direct Reagent (A1) 1000 1000
Direct Nitrite Reagent (A2) - 25
SUMMARY:
Mix well and proceed - -
Bilirubin is mainly formed from the heme portion of aged or damaged RBC's. It then combines
with albumin to form a complex, which is not water soluble. This is referred to as indirect or Sample 50 50
unconjugated Bilirubin. In the liver this Bilirubin complex is combined with glucuronic acid into Mix well and incubate 10 min at R.T or 5 min at 37°C. Measure the absorbance of the Test
a water soluble conjugate. This is referred to as conjugated or direct Bilirubin. samples (Abs.T) immediately against their respective Blanks.
PRINCIPLE: TOTAL BILIRUBIN ASSAY:
Bilirubin + Diazotized Sulphanilic acid Azobilirubin Compound Pipette into clean dry test tubes labeled as Blank (B), and Test (T):
Addition Sequence BT(µl) T (µl)
CONTENTS:
Total Reagent (AI) 1000 1000
DIRECT BILIRUBIN
Total Nitrite Reagent (AII) - 25
PACK SIZE DIRECT REAGENT(A1) DIRECT NITRITE REAGENT (A2)
Mix well and proceed - -
50+50T 100ml 5ml Sample 50 50
TOTAL BILIRUBIN Mix well and incubate 10 min at R.T or 5 min at 37°C. Measure the absorbance of the Test
PACK SIZE TOTAL REAGENT(AI) TOTAL NITRITE REAGENT (AII) samples (Abs.T) immediately against their respective Blanks.
50+50T 100ml 5ml
CALCULATIONS:
STORAGE AND STABILITY: Direct Bilirubin in mg/dl = (Abs.T-Abs.BD)× 26.30(factor)
All reagents are stable at R.T. till the expiry mentioned on the labels. Total Bilirubin in mg/dl = (Abs.T-Abs.BT) × 26.30(factor)
REAGENT PREPARATION: NORMAL VALUE:
Reagents are ready to use. Do not pipette with mouth. Serum (Direct) : upto 0.2 mg/dl
(Total) : upto 1.0 mg/dl
SAMPLE : It is recommended that each laboratory establish its own normal range representing its
Serum Bilirubin is reported to be stable in the sample for 4 days at 2-8°C and protected from patient population.
light as it is photosensitive.
LINEARITY:
GENERAL SYSTEM PARAMETER: The procedure is linear upto 25 mg/dl. If values exceed this limit, dilute the sample with
Reaction End Point Sample Volume 50 µl distilled water and repeat the assay. Calculate the value using the proper dilution factor.
Wavelength 546nm Reagent Volume 1025 µl
NOTE:
Blank Sample Blank Factor 26.30
1. In case of cuvette Volume is more than 1.0 ml requisite volume of reagents and
Incubation 37°C 5 min/R.T 10 min Reaction Slope Increasing sample can be multiplied keeping reagent to sample ratio same.
Delay Time 5 Sec Linearity 25 mg/dl 2. Sequence of reagent addition should be followed strictly as per the procedure.
Read Time 5 Sec Units mg/dl
REFERENCE:
ASSAY PROCEDURE: 1.Jendrassik L and Grf.P. (1938) BIOCHEM 2:297.81.
Wavelength / filter : 546 nm / Yellow Green 1. Doumas, B.T.Watson, W.A.(1971) Clin.Chem.Acta.31:
Temperature : R.T
Light path : 1 cm
