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Principle:
• Measures total protein based on the refractive index of serum/plasma
REFRACTOMETRY
What’s the concept of Refractometry?
The higher the protein concentration, the greater the ________________?
light refraction
Disadvantages of Refractometry:
• Affected by other molecules.
• Less accurate than spectrophotometric
Albumin remains soluble, while globulins precipitate in the presence of salts (sodium sulfate, ammonium sulfate)
Most widely used methods for determining Albumin.
DYE-BINDING
Principle:
• Albumin binds to dyes, causing color change proportional to concentration
DYE-BINDING
What are the Dyes under DYE-BINDING for Albumin Testing?
• Methyl orange
• 2,4’-hydroxyazobenzene-benzoic acid (HABA)
• Bromocresol Green (BCG)
• Bromocresol Purple (BCP)
• Nonspecific for albumin
• Beta-lipoproteins, alpha-1 and alpha-2 globulins also bind to dye
Methyl Orange
• Has low sensitivity but more specific for albumin
• Salicylates, penicillin, conjugated bilirubin, and sulfonamides -interfere with the binding of albumin to this dye
2,4’-hydroxyazobenzene-benzoic acid (HABA)
Formula for Bromocresol Green (BCG):
Albumin + BCG → Green-blue complex (628 nm)
is an anionic dye that binds to albumin in an acidic environment, forming a green-blue complex (628 nm)
Bromocresol Green (BCG)
Limitations of Bromocresol Green (BCG):
• Hemoglobin binds to BCG (for every 100 mg/dL of Hgb, albumin increased by 0.1 g/dL)
• Has been reported to overestimate low albumin values (nephrotic syndrome or end-stage renal disease)
Formula for Bromocresol Purple (BCP):
Albumin + BCP → Purple complex (603 nm)
is a more specific anionic dye that binds only to albumin, forming a purple complex.
Bromocresol Purple (BCP)
Limitations of Bromocresol Purple (BCP):
• Underestimates albumin in renal insufficiency.
• Bilirubin interferes with BCP binding, leading to falsely low albumin results in jaundiced patients.
Principle:
• Glyoxylic acid, in the presence of copper ions (Cu²⁺) and an acidic medium (acetic acid & sulfuric acid, H₂SO₄), reacts with tryptophan residues in globulins
TOTAL GLOBULIN: DIRECT COLORIMETRIC
What is the formula of TOTAL GLOBULIN: DIRECT COLORIMETRIC?
Globulin (Tryptophan) + Glyoxylic Acid + Cu²⁺ (Acid Medium) → Purple Complex (Measured at 540 nm)
What is the Indirect calculation of Albumin using Total Globulin?
Albumin (g/dL)=Total Protein (g/dL)−Total Globulin (g/dL
What are the 5 bands in ELECTROPHORESIS?
5 bands:
Albumin
Alpha-1 globulins
Alpha-2 globulins
Beta-globulins
Gamma-globulins
in ELECTROPHORESIS the Width of the band = ?
number of proteins present in that fraction.
Computation for protein fractions:
• Specialized scanner: Densitometer
• Uses dye to measure a specific wavelength (Coomassie Blue at 595 nm)
Absorbance Measurement and Strip-Chart Recorder