Estimation of Serum Total proteins and Albumin and A:G ratio.
1. Estimation of Serum Total proteins by biuret method
Principle: proteins present in the serum react with biuret reagent to form a violet coloured
complex. The colour obtained is measured in colorimeter at 540nm along with a blank and a
standard.
Reagents required:
1. Normal Saline
2. Biuret reagent
3. Protein Standard solution – 6 gm/dl
Procedure: take three test tubes and label them as Blank(B), Standard(S) and Test(T). Pipette
the reagents as follows:
Normal Protein Standard Serum Biuret Mix well OD at
Saline solution reagent and allow 540nm
to stand
B 3.0 ml 3.0 ml for 15
mins
S 2.9 ml 0.1 ml 3.0 ml
T 2.9 ml 0.1 ml 3.0 ml
Read the OD of S, T after adjusting the OD to Zero with B at 540nm.
Calculation:
Serum Total protein concentration = OD of Test X concentration of standard x 100 gm/dl
OD of Standard Volume of sample
= OD of Test X 0.006 x 100 gm/dl
OD of Standard 0.1
= OD of Test X 6 gm/dl
OD of Standard
Report:Serum Total protein concentration is ____________________.
Other methods of estimation of serum Total Proteins
Lowry’s method, Kjeldahl’s method,
2. Estimation of Serum Albumin by BCG dye method.
Principle: Albumin binds with Bromocresol green (BCG) at pH 4.2 to form a blue-green
coloured complex, which is proportional to the concentration of albumin present in the serum,
measured in a colorimeter at 630nm.
Reagents required:
1. BCG reagent
2. Albumin Standard solution – 3.6 gm/dl
Procedure: take three test tubes and label them as Blank(B), Standard(S) and Test(T). Pipette
the reagents as follows:
Distilled Albumin Serum BCG Mix well OD at
water Standard reagent and allow 630nm
solution to stand
B 20 µl 2.0 ml for 5 mins
S 20 µl 2.0 ml
T 20 µl 2.0 ml
Read the OD of S, T after adjusting the OD to Zero with B at 630nm.
Calculation:
Serum Total protein concentration = OD of Test X concentration of standard x 100 gm/dl
OD of Standard Volume of sample
= OD of Test x 3.6 gm/dl
OD of Standard
Report: Serum Albumin concentration is __________________g/dl
[Link] of Albumin: Globulin ratio = Serum Albumin
Serum Globulin
Serum Globulin = Serum Total Protein – Serum Albumin
Interpretation:
Normal Serum Total protein – 6 to 8 gm/dl
Normal Albumin – 3.5 to 5gm/dl
Globulin -2.5 to 3.5g/dl
Normal Fibrinogen – 200 to 400 mg/dl
Normal A/G ratio – 1.2:1 to 1.5: 1
Functions of Albumin are maintenance of colloidal osmatic pressure in blood, transport of
various substances in blood, acts important blood buffer and nutritive role.
Clinical significance:
Hyperproteinemia/ increased serum total protein:
Dehydration due to loss of fluids from body
Increase serum globulins
Chronic infections like TB, leprosy, kalaazar,
Multiple myeloma
Leukaemia
Amyloidosis
Hypoproteinemia:
Cirrhosis of liver due to decreased synthesis
Nephrotic syndrome due to loss of proteins/albumin in urine
Malnutrition due to deficiency of proteins
Protein loosing enteropathy due to loss of proteins
Burns due to loss of proteins through exposed skin surface
Uncontrolled DM due to loss of albumin
Important consequence of hypoproteinemia is edema. Edema occurs when serum total protein is
below 5 gm/dl and Serum albumin below 2 gm/dl
