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Estimation of Serum Total Proteins
By
Biuret Method
By Santhosh Kumar N/ Dept.of Biochem
Principle:
Cu2+ ions of biuret reagent reacts with peptide bond nitrogen of protein in alkaline
medium to form a violet colored complex.
Intensity of the colour is directly proportional to the conc. of protein
present in the serum. Color intensity is measured at 540nm(green filter) by
colorimeter.
By Dr. Santhosh Kumar N/ Dept.of Biochem
Dilution of serum: Dilution factor – 1:10
in 10ml containing 9 ml of normal saline and 1ml of serum
[ volume of sample (1ml) = 0.1ml serum]
Standard protein solution: 6gm /dl
By Santhosh Kumar N/Dept.of
Biochem/SIMSRH
PROCEDURE:
• Label three dry test tubes as Blank (B), Standard (S) & Test (T).
• Pipette 1ml distilled water into ‘B’, 1ml of working standard protein solution into ‘S’ and 1ml of
diluted serum into T’.
• To each test tube add 3ml of biuret reagent
• Mix well & allow to stand for 10min & read optical density by using colorimeter at 540nm
Reagent Blank Standard Test
Distilled water 1.0ml --- ---
Standard protein solution -- 1.0ml ---
Diluted serum --- --- 1.0ml
Biuret reagent 3ml 3ml 3ml
Mix well & allow to stand for 10min & read at 540nm
OD Values
By Santhosh Kumar N/ Dept.of Biochem
Calculation
• Conc. of serum total protein (gm/dl) =
O.D of T – O.D of B Conc. Of standard 1
X X 100 X
O.D of S – O.D of B Volume of Test 1000
T – B 6 100
= X X
S – B 0.1 1000
T – B
= X 6 = ------------gm/dl
S - B
By Santhosh Kumar N/Dept.of Biochem/SIMSRH
Other methods for total proteins:
• Lowry’s method,
• UV–absorption method,
• Alkaline copper sulphate method and
• BCA (Bicinchoninic acid) method
By Dr. Santhosh Kumar N/Dept.of Biochem/
Clinical significance
Total proteins conc. in serum is 6 to 8 gm%
Hypo proteinemia:
Due to Decrease
anabolism
Due to Increase
catabolism
Due to increased
loss
Haemodilution
Malnutrition
(kwashiorkor)
Liver diseases (cirrhosis)
Malabsorption from GIT
Thyrotoxicosis
Uncontrolled DM
Prolonged Febrile
diseases
Trauma
Severe burns,
Hemorrhage
Nephrotic syndrome
Pregnancy
Prolonged steroid
therapy
By Santhosh Kumar N/ Dept.of Biochem
Hyperproteinemia: Multiple myeloma, Rheumatoid arthritis,& Tuberculosis
Estimation of serum Albumin
by
Biuret Method
By Dr.Santhosh Kumar N/Dept.of Biochem
Principle:
The serum is treated with 28% of sodium sulphate it causes precipitation of
globulin and the filtrate contains only albumin.
Cu2+ ions of biuret reagent reacts with peptide nitrogen of albumin in
alkaline medium to form a violet color complex. Color compound which is
measured at 540nm by colorimeter.
By Santhosh Kumar N/Dept.of Biochem
Precipitation of Globulin :
• Take a clean & dry test tube, pipette 3.6ml of 28% Na2SO4 to add 0.4ml of
blood.
• Mix it & let stand for 10mins, Filter into a dry test tube. The filtrate should be
clear & colorless.
• 4ml containing 0.4ml of serum, so [1ml contain 0.1ml of serum]
Albumin Standard: 4gm/dl
PROCEDURE:
• Pipette 1ml distilled water in to ‘Blank’, 1ml of Standard albumin solution into ‘Standard’
and 1ml of Globulin precipitated solution into ‘Test’.
• To each test tube add 3ml of biuret reagent
• Mix well & allow to stand for 10min & read optical density by using colorimeter at 540 nm
.
Reagent Blank Standard Test
Distilled water 1.0ml --- ---
Standard albumin (4gm%) -- 1.0ml ---
Globulin precipitated solution --- --- 1.0ml
Biuret reagent 3ml 3ml 3ml
Mix well & allow to stand for 10min & read at 540nm
OD Values
Calculation
• Conc. of serum total protein (gm/dl) =
O.D of T – O.D of B Conc. Of standard (4gm) 1
X X 100 X
O.D of S – O.D of B Volume of Test (0.1) 1000
O.D of T – O.D of B
= X 4
O.D of S – O.D of B
= ------------gm/dl
Other methods for Albumin: Bromocresol green (BCG) method &
Bromocresol purple (BCP) method and
Electrophoresis and densitometry method
Albumin
• Albumin is a simple, globular & heat coagulable protein with mol wt is 69 kDa and
isoelectric pH is 4.7
• Synthesized in liver (10-15g/day) & has half life is 20 -25 days.
• Consists of a single polypeptide chain having 585 a.a’s with 17 intrachain disulfide
linkages.
• Precipitated by full saturation with ammonium sulphate.
By Santhosh Kumar N/Dept.of Biochem
Functions
1. Maintain colloidal osmotic pressure in vascular & extra vascular spaces
2. Max buffering capacity (histidine): maintain acid-base balance
3. Transport of nutrients(Fatty acids), metabolites(Bilirubin), Ca++, Steroid hormones &
Drugs (Sulfonamides, Penicillin, Aspirin)
4. It exerts low viscosity.
5. After degradation of albumin, all the amino acids are involves in the formation of tissue
proteins, enzymes & hormones etc.
Clinical significance of Albumin
(Normal range-3.5 - 5 gm%)
Hypoalbuminemia
Decreased conc. of alb
in serum.
- Cirrhosis of liver,
- Nephrotic syndrome,
-Malnutrition,
-Burns (-ve nitrogen balance) and
-Protein losing enteropathy
Analbuminemia
(less than 1gm%
Congenital rare autosomal recessive disorder
Defective mutations in the gene is responsible for
absence of albumin synthesis.
A/G ratio is reversed
CALCULATION OF GLOBULINS
Serum Globulins = [Serum Total Proteins – Serum Albumin]
 Normal serum Globulins range is 2.5 – 3.5 gm%
 Increased levels are seen in multiple myeloma and chronic infections
 In the above conditions, the A/G ratio is either lowered or completely reversed
By Dr. Santhosh Kumar N/ Dept.of Biochem
CALCULATION OF ALBUMIN TO GLOBULIN RATIO (A/G ratio)
Conc. of Serum Albumin
A: G ratio =
Conc. of Serum Globulin
Normal A:G ratio is 1.2:1 to 1.5: 1
Reversal of A:G ratio observed in liver cirrhosis, multiple myeloma & nephrotic
syndrome, chronic infections
By Santhosh Kumar N/ Dept.of Biochem
REPORT:
• Concentration of Total Proteins is ----------gm/dl, Albumin is …... gm/dl,
globulin is ------- gm/dl and A:G ratio is ------------- in the given serum
sample
Estimation of serum Albumin
by
Bromocresol green (BCG) Method
By Santhosh Kumar N/Dept.of Biochem/
Principle:
Albumin + BCG Albumin-BCG complex
At pH below 5, albumin is positively charged and it binds with
anionic bromocresol green to produce blue green complex. The color
produced is stable for 10mins. The color is measured at 630nm (600-
650nm)
pH 4.3
Sample:
• Preferable sample: Serum & Plasma
• Anticoagulant –Heparin, EDTA or Potassium oxalate can be used
• Do not use hemolysed sample
Standard conc.
• Standard albumin conc. is 4gm/dL
PROCEDURE:
• Label three dry test tubes as Blank (B), Standard (S) & Test (T).
• Pipette 10 μl of standard albumin solution in to ‘S’, 10 μl of sample into ‘T’ test tube
• To each test tube add 1ml of BCG reagent
• Mix well & incubate for 5min at 37oC & read the absorbance by using colorimeter at 630
nm
Reagent Blank Standard Test
Standard albumin (4gm%) -- 10 μl ---
Sample --- --- 10 μl
Reagent (BCG solution) 1.0ml 1.0ml 1.0ml
Mix well & incubate for 5min at 37oC & read the absorbance
Absorbance at630nm
Calculation
• Conc. of serum albumin (gm/dl) =
Abs of sample
= X conc. of standard (4g/dL)
Abs of standard
= ------------gm/dl
REPORT:
• Concentration of Albumin in the given serum sample is ………….g/dl,
By Dr. Santhosh Kumar N/ Dept.of Biochem

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Estimation of serum total Proteins & Albumin .pptx

  • 1. Estimation of Serum Total Proteins By Biuret Method By Santhosh Kumar N/ Dept.of Biochem
  • 2. Principle: Cu2+ ions of biuret reagent reacts with peptide bond nitrogen of protein in alkaline medium to form a violet colored complex. Intensity of the colour is directly proportional to the conc. of protein present in the serum. Color intensity is measured at 540nm(green filter) by colorimeter. By Dr. Santhosh Kumar N/ Dept.of Biochem
  • 3. Dilution of serum: Dilution factor – 1:10 in 10ml containing 9 ml of normal saline and 1ml of serum [ volume of sample (1ml) = 0.1ml serum] Standard protein solution: 6gm /dl By Santhosh Kumar N/Dept.of Biochem/SIMSRH
  • 4. PROCEDURE: • Label three dry test tubes as Blank (B), Standard (S) & Test (T). • Pipette 1ml distilled water into ‘B’, 1ml of working standard protein solution into ‘S’ and 1ml of diluted serum into T’. • To each test tube add 3ml of biuret reagent • Mix well & allow to stand for 10min & read optical density by using colorimeter at 540nm Reagent Blank Standard Test Distilled water 1.0ml --- --- Standard protein solution -- 1.0ml --- Diluted serum --- --- 1.0ml Biuret reagent 3ml 3ml 3ml Mix well & allow to stand for 10min & read at 540nm OD Values By Santhosh Kumar N/ Dept.of Biochem
  • 5. Calculation • Conc. of serum total protein (gm/dl) = O.D of T – O.D of B Conc. Of standard 1 X X 100 X O.D of S – O.D of B Volume of Test 1000 T – B 6 100 = X X S – B 0.1 1000 T – B = X 6 = ------------gm/dl S - B By Santhosh Kumar N/Dept.of Biochem/SIMSRH
  • 6. Other methods for total proteins: • Lowry’s method, • UV–absorption method, • Alkaline copper sulphate method and • BCA (Bicinchoninic acid) method By Dr. Santhosh Kumar N/Dept.of Biochem/
  • 7. Clinical significance Total proteins conc. in serum is 6 to 8 gm% Hypo proteinemia: Due to Decrease anabolism Due to Increase catabolism Due to increased loss Haemodilution Malnutrition (kwashiorkor) Liver diseases (cirrhosis) Malabsorption from GIT Thyrotoxicosis Uncontrolled DM Prolonged Febrile diseases Trauma Severe burns, Hemorrhage Nephrotic syndrome Pregnancy Prolonged steroid therapy By Santhosh Kumar N/ Dept.of Biochem Hyperproteinemia: Multiple myeloma, Rheumatoid arthritis,& Tuberculosis
  • 8. Estimation of serum Albumin by Biuret Method By Dr.Santhosh Kumar N/Dept.of Biochem
  • 9. Principle: The serum is treated with 28% of sodium sulphate it causes precipitation of globulin and the filtrate contains only albumin. Cu2+ ions of biuret reagent reacts with peptide nitrogen of albumin in alkaline medium to form a violet color complex. Color compound which is measured at 540nm by colorimeter. By Santhosh Kumar N/Dept.of Biochem
  • 10. Precipitation of Globulin : • Take a clean & dry test tube, pipette 3.6ml of 28% Na2SO4 to add 0.4ml of blood. • Mix it & let stand for 10mins, Filter into a dry test tube. The filtrate should be clear & colorless. • 4ml containing 0.4ml of serum, so [1ml contain 0.1ml of serum] Albumin Standard: 4gm/dl
  • 11. PROCEDURE: • Pipette 1ml distilled water in to ‘Blank’, 1ml of Standard albumin solution into ‘Standard’ and 1ml of Globulin precipitated solution into ‘Test’. • To each test tube add 3ml of biuret reagent • Mix well & allow to stand for 10min & read optical density by using colorimeter at 540 nm . Reagent Blank Standard Test Distilled water 1.0ml --- --- Standard albumin (4gm%) -- 1.0ml --- Globulin precipitated solution --- --- 1.0ml Biuret reagent 3ml 3ml 3ml Mix well & allow to stand for 10min & read at 540nm OD Values
  • 12. Calculation • Conc. of serum total protein (gm/dl) = O.D of T – O.D of B Conc. Of standard (4gm) 1 X X 100 X O.D of S – O.D of B Volume of Test (0.1) 1000 O.D of T – O.D of B = X 4 O.D of S – O.D of B = ------------gm/dl Other methods for Albumin: Bromocresol green (BCG) method & Bromocresol purple (BCP) method and Electrophoresis and densitometry method
  • 13. Albumin • Albumin is a simple, globular & heat coagulable protein with mol wt is 69 kDa and isoelectric pH is 4.7 • Synthesized in liver (10-15g/day) & has half life is 20 -25 days. • Consists of a single polypeptide chain having 585 a.a’s with 17 intrachain disulfide linkages. • Precipitated by full saturation with ammonium sulphate. By Santhosh Kumar N/Dept.of Biochem
  • 14. Functions 1. Maintain colloidal osmotic pressure in vascular & extra vascular spaces 2. Max buffering capacity (histidine): maintain acid-base balance 3. Transport of nutrients(Fatty acids), metabolites(Bilirubin), Ca++, Steroid hormones & Drugs (Sulfonamides, Penicillin, Aspirin) 4. It exerts low viscosity. 5. After degradation of albumin, all the amino acids are involves in the formation of tissue proteins, enzymes & hormones etc.
  • 15. Clinical significance of Albumin (Normal range-3.5 - 5 gm%) Hypoalbuminemia Decreased conc. of alb in serum. - Cirrhosis of liver, - Nephrotic syndrome, -Malnutrition, -Burns (-ve nitrogen balance) and -Protein losing enteropathy Analbuminemia (less than 1gm% Congenital rare autosomal recessive disorder Defective mutations in the gene is responsible for absence of albumin synthesis. A/G ratio is reversed
  • 16. CALCULATION OF GLOBULINS Serum Globulins = [Serum Total Proteins – Serum Albumin]  Normal serum Globulins range is 2.5 – 3.5 gm%  Increased levels are seen in multiple myeloma and chronic infections  In the above conditions, the A/G ratio is either lowered or completely reversed By Dr. Santhosh Kumar N/ Dept.of Biochem
  • 17. CALCULATION OF ALBUMIN TO GLOBULIN RATIO (A/G ratio) Conc. of Serum Albumin A: G ratio = Conc. of Serum Globulin Normal A:G ratio is 1.2:1 to 1.5: 1 Reversal of A:G ratio observed in liver cirrhosis, multiple myeloma & nephrotic syndrome, chronic infections By Santhosh Kumar N/ Dept.of Biochem
  • 18. REPORT: • Concentration of Total Proteins is ----------gm/dl, Albumin is …... gm/dl, globulin is ------- gm/dl and A:G ratio is ------------- in the given serum sample
  • 19. Estimation of serum Albumin by Bromocresol green (BCG) Method By Santhosh Kumar N/Dept.of Biochem/
  • 20. Principle: Albumin + BCG Albumin-BCG complex At pH below 5, albumin is positively charged and it binds with anionic bromocresol green to produce blue green complex. The color produced is stable for 10mins. The color is measured at 630nm (600- 650nm) pH 4.3
  • 21. Sample: • Preferable sample: Serum & Plasma • Anticoagulant –Heparin, EDTA or Potassium oxalate can be used • Do not use hemolysed sample Standard conc. • Standard albumin conc. is 4gm/dL
  • 22. PROCEDURE: • Label three dry test tubes as Blank (B), Standard (S) & Test (T). • Pipette 10 μl of standard albumin solution in to ‘S’, 10 μl of sample into ‘T’ test tube • To each test tube add 1ml of BCG reagent • Mix well & incubate for 5min at 37oC & read the absorbance by using colorimeter at 630 nm Reagent Blank Standard Test Standard albumin (4gm%) -- 10 μl --- Sample --- --- 10 μl Reagent (BCG solution) 1.0ml 1.0ml 1.0ml Mix well & incubate for 5min at 37oC & read the absorbance Absorbance at630nm
  • 23. Calculation • Conc. of serum albumin (gm/dl) = Abs of sample = X conc. of standard (4g/dL) Abs of standard = ------------gm/dl REPORT: • Concentration of Albumin in the given serum sample is ………….g/dl, By Dr. Santhosh Kumar N/ Dept.of Biochem