24.4 Reagents........................................
1.Diazo reagent
Sol. A–Dissolve 100 mg sulfanilic acid in 100 ml of 1.5% HCl.
Sol. B–0.5% aqueous solution of sodium nitrite. Diazo reagent is prepared by
mixing 10 ml of solution A with 0.3 ml of solution B. Prepare just before use.
2.Diazo Blank: 1.5% v/v aqueous solution of HCl.
3.Bilirubin standard (10 mg %): Dissolve 10 mg of bilirubin powder in 5 ml of
0.1 N NaOH, and add 80 ml of 2% BSA solution in saline. Then add 5 ml of 0.1 N
HCl, and mix and make the volume 100 ml with 2% BSA solution. Store in
brown bottle at 4C.
26.5 Procedure
- Take a set of 12 test tubes in duplicate and label as blank, standard (S1-S7), total
(T) total control (Tc), direct (D), and direct control (Dc). Also take another six
tubes for standard controls. - Add 20, 40, 60, 80, 100, 150, and 200μl of 10 mg % bilirubin standard into the
tubes labelled as S1-S7, respectively, and standard controls. - Pipette out 0.1 ml of serum to all the tubes labelled as T, Tc, D, and Dc. Make
volume to 2 ml with distilled water in each tube except D and Dc. Add 4.9 ml
distilled water to the tubes labelled as D and Dc. - Add 0.5 ml diazo reagent to all the tubes except blank, Tc and Dc, and standard
controls. 0.5 ml of diazo blank was added to the tubes blank, Tc, Dc, and standard
controls. - Add 3 ml of methanol to all the tubes except D and Dc. Mix and keep in dark for
30 min at room temperature and read absorbance at 540 nm.
24.5 Procedure 99