10.7 Estimation of Albumin in Serum
Method Bromocresol Green (BCG) Binding
10.8 Principle........................................
Many colored anionic dyes such as methyl orange and BCG bind with albumin. The
use of BCG is free from pigment interference. Albumin at a pH below its pI is
positively charged and has affinity for anionic dye (BCG) which on combining with
it undergoes a change in color from yellow to blue-green which is measured at
620 nm. The color produced is directly proportional to albumin concentration.
10.9 Reagents........................................
1.BCG solution
Sol A:Succinate buffer (0.5 M, pH 4.1): Dissolve 0.56 g succinic acid in
approximately 80 ml distilled water. Adjust pH to 4.1 with 1 N NaOH, and
makefinal volume up to 100 ml with distilled water and store at 4C.
Sol B:BCG dye (10 mM): Dissolve 10 mg BCG dye in 1 ml of 1 N NaOH, and
make thefinal volume 10 ml with distilled water.
Sol C:Sodium azide solution: Dissolve 4 g sodium azide in distilled water, and
makefinal volume up to 100 ml.
Sol D:Brij 35: Prepare 30% in distilled water.
Prepare BCG solution by mixing 50 ml of solution A, 4 ml of solution B, 1.25 ml
of solution C, and 1.25 ml of solution D. Make the volume to 500 ml with
distilled water, and adjust the pH to 4.10.05 by adding 1 N NaOH.
Sodium azide solution is added as a preservative. Brij 35 is added to prevent the
turbidity produced when sample is mixed with reagent. It also causes a slight
change in absorbance maxima of dye-albumin complex.
2.Stock BSA standard: 5 g/dl in normal saline.
3.Working BSA standardof concentration 1, 2, 3, 4, and 5 g/dl by diluting the
stock BSA standard in normal saline.
4.0.9% aqueous solution of normal saline.
10.10 Procedure
- Take a set of seven test tubes in duplicate and label as blank, standard (S 1 – S 5 ),
and test (T). - Add 1 ml of normal saline in all the tubes.
- Add 0.02 ml of working standard of concentration 1, 2, 3, 4, and 5 g/dl in
respective test tubes.
46 10 To Estimate the Amount of Total Protein and Albumin in Serum and to Find...