Determination of Serum Protein by Biuret Method
Clinical Significance
An increase in total proteins may in dehydration. Both albumin and globulin are increased due to hemo concentration. A decrease in total protein is always due to a low albumin level, accompanied either by no increase in globulin or an increase in globulin so that the ratio of A/G is changed. A low serum albumin may due to:
- A heavy loss of albumin in urine (as in nephritis).
- Malabsorption of amino acids from the alimentary tract (as in steatorrhea).
- Decreased formation in the liver (as in cirrhosis of liver).
- Increased catabolism of proteins (as in fever).
- Insufficient intake of proteins in the food (malnutrition).
A reduction in the total proteins is one of the cause of edema. It may take place when total protein fall below about 5.0 g/dl and albumin below about 2.5g/dl. In this condition, water from blood passes into tissue spaces. Plasma protein may also be decrease in acute or chronic hemorrhage.
Increase in Globulin occurs in-
- Advanced liver disease
- Multiple Myeloma
- In number of chronic infections such as tuberculosis, rheumatoid arthritis, sub-acute bacterial endocarditis, lupus erythematosus disseminates etc.
Biuret Test for Protein Principle
Proteins react with cupric ions in alkaline medium to form a violet colored complex. The intensity of the color produced is the directly proportional to proteins present in the specimen and can be measured on a photometer at 530 nm by using green filter.
Protein Normal Range
Sarum Proteins : 6-8 g/dl
Protein Method Name
Biuret Method
Requirements
- Test Tube
- Serological Pipette
- Test Tube stand
- Push Button Pipettes or Serological Pipettes
- Photometer
Specimen
Serum
Protein Reagent
Protein Reagent Preparation
- Stock Biuret Reagent :
- Rochelle salt : 45 g
- 0.2 N Sodium hydroxide : 400 ml
- Copper Sulphate : 15 g
- Potassium Iodide : 5 g
- Add 0.2 N Sodium hydroxide (Make up to a Liter)
- Working Protein Reagent :
- Stock Biuret Reagent : 200 ml
- Dilute with 0.2 N Sodium hydroxide
- Potassium iodide : 5 g/L
- Protein Standard :
- Bovine Albumin : 6 g
- Normal Saline : 100 ml
- Sodium azide : 0.1 g/dl
- Sample Blank Reagent :
- Rochelle salt : 9.0 g
- Potassium iodide : 5 g
- 0.2 N Sodium hydroxide : 1000 ml
Stability of the regents
Reagents 1 and 2 are stable at room temperature for one year. Reagent 3 protein standard is stable at 2-8°C for one year.
Protein Procedure
Mono step method
Pipetting in three tubes labelled as follows –
| Reagent | Test | Standard | Blank |
| Protein Reagent, ml | 5.0 | 5.0 | 5.0 |
| Serum, ml | 0.05 | – | – |
| Protein Standard, ml | – | 0.05 | – |
| Distilled Water, ml | – | – | 0.05 |
Mix thoroughly and keep at room temperature for exactly 10 minutes. Measure the intensities and of the test and standard by setting blank at 100% T, by using 530 nm by using green filter.
Calculations
Serum Proteins, g/dl = O.D. Test / O.D. std × 6
Direct Coombs Test ?
Electrophoresis