2. Objective
At the end of this chapter, the student is expected to:
• Describe basic protein structure and composition
• Define amino acids
• State and describe the four stages of protein structure
• Show how protein properties can be used for their
identification and assay
• List the proteins synthesized by the liver
• Describe the physiological functions of proteins
• Describe how plasma protein abnormalities reflect
severity of hepatic dysfunction
• Describe the methods used for determination of protein
concentration in blood
3. Outline of protein lecture
• Introduction
• Classification of proteins
• Functions of proteins
• The plasma proteins
• Plasma proteins with clinical significance
• Properties of proteins
• Specific methods for the determination of proteins
• Serum protein electrophoresis
• Samples
• Interpretation
• Quality control
• summary
4. Introduction
Proteins are polymers of amino acids that are
linked covalently through peptide bonds.
Aminoacid: an organic cmpound containing both
amino and carboxyl functional groups; simplest
units of proteins
There are 20 different kinds of amino acids,
combined in different proportion and
arrangements to build all protein molecules
When only two amino acids combine by peptide
bond ,it is called dipeptide, when amino acids
involved in the bond formation become 3, 4, 5
they are named as tri-, tetra-, and penta-
peptides respectively.
5. Proteins, continued..
All proteins contain carbon, hydrogen, oxygen,
and nitrogen; some proteins may also contain
sulfur phosphorous, copper, iron, zinc, iodine,
and other elements.
The presence of nitrogen in all proteins sets
them apart from carbohydrates and lipids.
The average nitrogen content of proteins is
approximately 16%.
6. Proteins, continued..
Comprises 50-70% of cell’s dry weight
Found in cells, as well as in all fluids,
secretions, and excretions
more than 300 different types of plasma
proteins are discovered
8. Protein classification, continued….
• Based on their shape proteins can be classified
as fibrous proteins and globular proteins
• Proteins have four levels of structure
-Primary structure
-Secondary structure
-Tertiary structure
-Quaternary structures.
9. Function of Proteins
• used to construct or build our body
• catalyze biochemical reactions as an enzyme
• regulate body metabolism as hormones
• protect our body from foreign body attack as an
antibody and components of complement
• maintain osmotic pressure in plasma
• Transport different lipids, minerals, hormones, vitamins
etc as hemoglobin, apolipoprotein, albumin etc
• assist to arrest bleeding and maintain homeostasis as
coagulation factor
10. Plasma proteins
• Many different proteins are present in the blood,
and collectively known as plasma proteins.
• They include Albumin, Alpha1Acid glycoproteins,
ceruloplasmin, C-reactive protein, complements,
fibrinogen and immunoglobulins.
• Most of plasma proteins are synthesized and
catabolized in the liver.
11. Clinical significance of protein
• The two general causes of alteration of serum
total protein are:
change in volume of plasma water
change in concentration of protien
The relative hypoproteinemia --hemodilution.
The relative hyperproteinemia-hemoconcentration.
12. Plasma proteins with Clinical significance
Albumin
• the most abundant plasma protein extra
vascular body fluids, including CSF, Interstitial
fluid, urine, and amniotic fluid.
• accounts approximately one-half of the plasma
protein mass.
• a globular protein, with molecular mass of 66.3
KD.
• Because of its high net negative charge at
physiological pH, highly soluble in water, but
does not have carbohydrate side chain.
13. Functions of albumin
• maintaining the colloidal osmotic pressure in
both the vascular and extra vascular space with
continuous equilibration in between
• Binding and transportation of large number of
compounds, including free fatty acids,
phospholipids, metallic ions, amino acids, drugs,
hormones and bilirubin.
15. Decreased levels of albumin seen in..
• Edema and ascitis
• Analbuminemia
• Urinary loss
• Inflammatory conditions
• Gastrointestinal loss
• Hepatic disease
• Protein energy malnutrition
16. Alpha 1 –fetoprotein [AFP]
• one of the first α –globulin appear in mammalian
sera during development of the embryo
• Dominant serum protein in early embryonic life
• synthesized primarily by the fetal yolk sac and
liver..
• contains approximately 4% carbohydrate with a
molecular mass approximately 70KD.
17. Clinical significance
High AFP levels seen in:
• open neural tube or abdominal wall defect in
fetus.
• Multiple fetuses,
• fetal demise,
• fetomaternal bleeding, and
• incorrect estimation of gestational age
Hepatocellular and germ cell carcinomas in
childhood and adults
18. C-reactive protein
• The first APPs to become elevated in inflammatory
diseases
• consists of five identical subunits and is synthesized
primarily by liver.
• C-reactive protein (CRP) found in sera of acutely ill
individuals from S. pneumonia
• CRP activates the classic complement path way
starting at C1q and initiates opsonization,
phagocytosis, and lysis of invading organisms. such
as bacteria and viruses.
19. CRP continues…
• CRP can recognize potentially toxic autogenous
substances released from damaged tissue, to
bind them, and then detoxify or clear them from
the blood.
20. Clinical significance
CRP levels usually rise after
• myocardial infraction, stress, trauma, infection,
inflammation, surgery, or neoplastic proliferation.
CRP is clinically useful for
• Screening for organic disease
• Assessment of the activity of inflammatory
diseases
21. • Detection of inter-current infection in systemic lupus
erythematosus (ALE), in leukemia, or after surgery
• management of neonatal septicemia and meningitis
• Cord blood normally has low CRP concentration,
but in intrauterian infection, the concentration will be
high.
22. properties protein
• Molecular size
• Differential solubility
• Electrical charge
• Adsorption on finely divided inert materials
• Specific binding to antibodies, coenzymes, or
hormone receptors
23. Specific methods for total protein determination.
• Biuret method
• Direct photometric methods.
• Dye-binding methods.
• Turbidimetric and nephelometric methods
24. Biuret method
Principle of the test
• peptide bonds react with Cu2+ ions in alkaline
solutions to form a colored product
• absorbance is measured spectrophotometrically
at 540nm.
25. Biuret, continued….
• The biuret reaction occurs with other
compounds with structural similarity.
• One copper ion probably is linked to 6 nearby
peptide linkage by coordinate bonds.
• Amino acids and di peptides do not react, but tri
peptides, oligo peptides, and polypeptides react
to yield pink to reddish- violet products.
• The intensity of the color produced is
proportional to the amount of protein present in
the reaction system.
• Detect between 1 and 15 mg of protein in the
aliquot measured, an amount present in 15 to
200 μl of a serum containing protein at 7gm/dl.
26. Biuret, continued..
Specimen type, source of errors, and preservation
Either serum or plasma, but serum is preferred.
A fasting specimen may be required to decrease the
risk of lipemia.
Ammonium ions interfere
Hemolysis should be avoided.
Serum samples are stable for atleast 1week at room
temperature and for 1 month at 2 to 4o C.
Specimens that have been frozen and thawed should
be mixed thoroughly before assay.
27. Direct photometric methods.
Principle of the test.
Absorption of UV light at 200-225 nm and 272 –
290 nm is used
Absorption of UV light at 280 nm depend on the
aromatic rings of tyrosine and tryptophan
Peptide bonds are responsible for UV absorption
(70% at A205) ;
Specific absorption by proteins at 200 to 225 nm
10 to 30 times greater than at 280 nm.
28. Limitations of the direct photometric methods
Accuracy & specificity suffer from
uneven distribution of tyrosine and tryptophan
among individual proteins
the presence of free tyrosine and tryptophan, uric
acid, and bilirubin, which also absorb light near
280nm.
interferences from free tyrosine and tryptophan is
significant at 200 to 225nm.
A 1:1000 or 1:2000 dilution of serum with sodium
chloride,0.15 mol/l ,circumvents this interferences.
29. Dye-binding methods
Principle of the test
Based on the ability of proteins to bind dyes
such as amido black 10B and Coomassie
Brilliant Blue.
The method is simple, easy, and linear up to 150
mg/dl.
assay of total protein in CSF and urine uses
CBB G-250
30. Limitation of dye binding methods
unequal affinities and binding capacities of
individual proteins for dyes
inability to define a consistent material for use
as a calibrator.
31. Turbidimetric and nephelometric methods
Principle of the test
Protein in the sample is precipitated with
addition of sulfosalicylic acid alone, with
sulfosalicylic acid in combination with sodium
sulfate or trichloroacetic acid (TCA), or with TCA
alone to produce turbidity.
Degree of turbidity measured with
Turbidometeric or nephelometric methods
32. Assay Techniques for serum albumin
Dye-binding methods
Salt fractionation or the 'salting-out'
procedure
By difference
Electrophoresis
Immunochemical techniques.
33. Dye binding method
BCG Method
Test principle: Albumin and BCG are allowed to bind
at pH 4.2, in succinate buffer,
absorption of the BCG-albumin complex is measured
at 628 nm.
At pH 4.2, albumin acts as a cation to bind the
anionic dye.
The reaction is extremely fast and goes to
completion in only a few seconds.
34. Reference Range
Adult serum albumin
Recumbent: 3.5 - 5.0 g/dl
In the upright position levels are about 0.3 g/dl
higher because of hemoconcentration.
35. Source of error and remedy
hyperlipemia
hyperbilirubinemia
hemolysis
can generally be eliminated (minimized) by
dilution of serum 1:250
36. BCP Method
Test principle:
Yellow BCP dye, buffered at pH 5.2 with
acetate
turns green when complexed with albumin.
Absorbance of the green complex is
measured at 603 nm.
37. Specimen
serum is recommended
Results tend to be erroneous if the overall
serum protein pattern is abnormal
38. Methods for the determination of total globulins
Methods for the quantitative determination of
total globulins
Colorimetric method
Globulin by difference
Electrophoresis
Immunochemical technique
39. Colorimetric method
Test principle:
glyoxylic acid reacts with tryptophan residues
of proteins to form a purple color.
Copper sulphate is added to enhance color
formation.
human globulins are known to contain 2 - 3%
tryptophan
40. Serum Protein Electrophoresis
• Electrophoresis is widely used in clinical
laboratories to study and measure the protein
content of biological fluids- serum, urine or csf.
• Screening tool for prtein abnormalities
• Electrophoresis techniques include:
– Cellulose acetate electrophoresis
– Gel and capillary electrophoresis
– Specialized techniques termed western
blotting, immunofixation, and two-dimensional
electrophoresis
41. Methodology for Protein Electrophoresis
Patient’s specimen is placed into a sample
trough within agarose gel, is placed in an
alkaline buffer solution
a standardized voltage is applied and
allowed to run for 1hr
the agarose gel is processed in acetic
acid and alcohol washes to fix the
proteins in the agarose.
the protein fractions are stained with
Coomassie Brilliant Blue protein stain.
42. After a second wash, fixed protein bands
can be visualized and quantified with
densitometry.
In normal serum electrophoresis 5-6
bands are visible:
Albumin
Globulins: α1-, α2-, β-, and γ-
43. Materials and procedures of protein
electrophoresis
Buffer: barbital with an ionic strength of 0.05 and
pH 8.6
Sample volume: 3 to 5 µl
Power supply: 1.5 mA per 2-cm width of cellulose
acetate medium; 10mA per 1-cm width of agarose
medium
Run time:40 to 60 min producing a 5- to 6-cm
migration distance for aalbumin
46. Interpretation of Results
Reference Range of total protein
– Serum---------------------------6-8 g/dl
– CSF----------------------------- 8-32 mg/dl
For electrophoresis
-serum: albumin-----------------3.9-5.1 g/dl
α1-globulin------------0.2-0.4 g/dl
α2-globulin------------0.4-0.8 g/dl
β-globulin--------------0.5-1.0 g/dl
γ-globulin---------------0.6-1.3 g/d
Compare the patient results with the reference range to
assess for hyper- or hypoglycemia
47. Quality Control
A normal & abnormal quality control sample should be
analyzed along with patient samples, using Westgard or
other quality control rules for acceptance or rejection of
the analytical run.
– Assayed known samples
– Commercially manufactured
Validate patient results
Detects analytical errors.
48. Documentation of protein
Results
• Record patient results in result logbook
• Record QC results in QC logbook
• Retain records for recommended time
49. summary
Proteins are polymers of amino acids that are linked covalently
through peptide bonds.
The presence of nitrogen in all proteins sets them apart from
carbohydrates and lipids.
Proteins are classified based on the number of amino acid
molecules ,composition of amino acids.
Protein have four structural levels;10,20,30,and 40.
Properties of proteins include molecular size, differential
solubility, electrical charge, adsorption on finely divided inert
materials, and specific binding to antibodies, coenzymes, or
hormone receptors
50. Summary, continued…
Proteins function includes building our body , serving as
enzymes, as antibody. etc..’
Major plasma proteins include Albumin, Alpha1Acid
glycoproteins, ceruloplasmin, C-reactive protein, complements,
fibrinogen and immunoglobulins
Increase level of protein caused by acute dehydration and has
no clinical significance; decreased levels of proteins seen in
edema and ascitis, analbuminemia, urinary loss, inflammatory
conditions, gastrointestinal loss, hepatic disease, protein
energy malnutrition.
Specific methods for total protein determination include Biuret
method, direct photometric methods, dye-binding methods,
turbidimetric and nephelometric methods
Serum protein electrophoresis used to fractionate proteins
51. Reference
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. Philadelphia,
2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis
3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
textbook of Clinical Chemistry. Philadelphia, 1999.
simple proteins (made from amino acids only)
complex proteins (contain protein and non-protein parts; protein part called apoproteins, and non-protein part called conjugated or prostatic proteins).
eg. -Metallo-proteins eg, ferritin (with iron),
-complex structure (hemoglobin)
-glycoproteins (having 10-16% carbohydrates),
- mucoproteins (high carbohydrate concentration in proteins)
Although fibrous proteins, such as fibrinogen, troponine, collagen, keratin, and myosin, are of clinical interest, most proteins of clinical interest are soluble globular proteins, such as hemoglobin, enzymes, peptide hormones, and plasma proteins.
Most globular proteins retain their biological activates within narrow ranges of temperature or pH. When exposed to high temperatures or extremes of pH causes the molecules of protein to “denature” and loss their solubility and biological activities.
The modular nature of protein synthesis and folding are embedded in the concept of orders of protein structure; primary structure, the sequence of amino acids in a polypeptide chain; secondary structure, the folding of short(3-30 residues) contiguous segment of polypeptide into geometrically order units; tertiary structure, the three dimensional assembly of secondary structural units to form larger functional units such as the mature polypeptide and its components domains; and quaternary structure, the number and types of polypeptide units of oligomeric proteins and their spatial arrangement.
Analbuminemia:- a genetic defect manifested by abnormal lipid transport
Inflammation:- acute and chronic inflammations, result from hemodilution, loss into extravascular spaces, decrease synthesis are most Common causes of hypoalbunimeia
Urinary loss :-Normal urine may contain up to 20mg albumin per gram of creatinine. Excretion above this level suggests either increase molecular filtration, tubular damage, hematuria, or combination of these. Mildly increased excretion (20 to 300mg/l) or microalbuminemia, appears to predict future development of clinical renal diseasein individuals with hypertension or diabetic mellitus.
Edema and ascities :- Plasma levels of albumin are decreased in the presence of edema and ascites. However, edema and ascities are usually secondary to increased vascular permeability, rather than to hypoalbuminemia per se. The albumin levels in these fluids vary from very low to higher than those in plasma.
α1-fetoprotein (AFP) is one of the first α –globulin to appear in mammalian sera during development of the embryo and is the dominant serum protein in early embryonic life. AFP contains approximately 4% carbohydrate with a molecular mass approximately 70KD. It is a major protein in fetal serum, synthesized primarily by the fetal yolk sac and liver.
Elevated maternal serum or amniotic fluid AFP indicates the possibility of an open neural tube or abdominal wall defect in fetus. Multiple fetuses, fetal demise, fetomaternal bleeds, and incorrect estimation ofgastational age also may elevatematernalserumlevel. Hepatocellular and germ cell carcinomas in childhood and adults are the other causes for the elevated AFP.
It is one of the first APPs to become elevated in inflammatory diseases and also the one exhibiting the most dramatic increases in concentration. It consists of five identical subunits and is synthesized primarily by liver. C-reactive protein (CRP) found in sera of acutly ill individuals from S. pneumniae infection, in which the protein binds with C-polysaccharide found on the bacterial cell wall.
In the presence of Ca2+, CRP binds not only the polysaccharides present in Bacteria, fungi, and protozoal parasites but also phosphorylcholines;such as phosphotidyal cholines,, such as lecithin; and polyanions, such as nucleic acids. In the absence of Ca2+, CRP binds polycathions, such as histones.
Once complexed, CRP activates the classic complement path way starting at C1q. Similar to antibodies, CRPthus initatesopsonization, phagocytosis, and lysis of invading organisms such as bacteria and viruses. CRp also can recognize potentially toxic autogenious substances released from damaged tissue, bind them, and then detoxify them or from the blood. CRP itself is catabolized after opsonization.
CRP levels usually rises after myocardial infraction, stress, trauma, infection, inflammation, surgery, or neoplastic ptoliferation. The incre. The increase begans within 6 to 12 hours after onset of the problems.
Additionally CRP value important for
Screening for organic disease
Assessment of the activity of inflammatory diseases
Detection of intercurrent infection in systemic lupus erythematosus (ALE), in leukemia, or after surgery
management of neonatal septicemia and meningitis ,when specimen collectionfor bacteriological investigations may be difficult
Cord blood normally has low CRP concentration, but in intrauterian infection, the concentration will be high. Levels in infancy normally rise for a few days after vaginal delivery,fall to very low levels, and then gradually rise over several weeks to adult levels
Molecular size:- Most proteins because of their size (most are macromolecules)can be separted from small molecules by dialysis, ultrafiltration, molecular exclusion gelfiltrationchromatography, and density gradient ultracentrifugation.,
Differential solubility Proteins solubility is affected by the pH, ionic strength, and temperature. when these parameters are varied, protein molecules become either more or less soluble. For example, through variations in the ionic strength of solution, proteins become either more soluble (“salting in”) or less soluble (“salting out”).
Electrical charge Proteins based on change of their surface charge, or pH, can be separted by using methods like electrophoresis, isoelectric focusing, and ion-exchange chromatography.
Adsorption on finely divided inert materials these materials offer large surface areas for interaction with proteins. These interactions are either hydrophobic, absorptive, ionic, or molecular (hydrogen bond).
Specific binding to antibodies, coenzymes, or hormone receptors Based on proteins specific binding properties with antibodies, coenzymes, or hormone receptors , different methods, such as immunochemical assay techniques, and affinity chromatography can be used for separation and identification.
This method depends on the presence of peptide bonds in all proteins. These peptide bonds react with Cu2+ ions in alkaline solutions to form a colored product, the absorbance of which is measured spectrophotometrically at 540nm.
The buret reagent contains sodium potassium tartarate to form a complex with cupric acid and maintain their solubility in alkaline solution. Iodine is included as an antioxidant. IN the biuret reaction a colored chelate is formed between the cupper ion and the carbonyl oxygen and amide nitrogen atoms of the peptide bonds. The reaction occurs with any compound containing at least two H2N-C-, H2N-CH2, CH2-, H2N-CS-, or similar groups joined together directly or through a carbon or nitrogen atom. One copper ion probably is linked to 6 nearby peptide linkage by coordinate bonds. Amino acids and di peptides do not react, but tri peptides, oligo peptides, and polypeptides react to yield pink to reddish- violet products.
The intensity of the color produced is proportional to the number of peptide bonds that are reacting and therefore to the amount of protein present in the reaction system.
Although small peptide present in serum also reacts, their concentration in serum is so low that they contribute little to the biurets color.
Ammonium ions interfere, but not at concentrations that occur in serum. Most biuret methods detect between 1 and 15 mg of protein in the aliquot measured, an amount present in 15 to 200 μl of a serum containing protein at 7gm/dl.
Either serum or plasma may be used for a biuret assay, but serum is preferred. A fasting specimen is not required but may be desirable to decrease the risk of lipemia. Hemolysis should avoided. Tightly stopper samples of serum are stable for one week or more atroom temperature and for 1 month at 2 to 4o C. Specimens that have been frozen and thawed should be mixed thoruoghly before assay.
Absorption of UV light at 200-225 nm and 272 – 290 nm has been used to measure the protein content of biological samples. Absorption of UV light at 280 nm depend chiefly on the aromatic rings of tyrosine and triptophan at pH 8. Accurate and specific suffer from un even distribution of these amino acids among individual proteins in a mixture, as well as from the presence in body fluids of free tyrosine and triptophan, uric acid, and bilirubin, which as\lso absorb light near 280nm. Peptide bonds are responsible chiefly for UV absorption (70% at A205) ; specific absorption by proteins at 200 to 225 nm 10 to 30 times greater than at 280 nm. Interference from free tyrosine and triptophan is significant at these short wavelengths. How ever, a 1:1000 or 1:2000 dilution of serum with sodium chloride,0.15 mol/l ,circumvents this interferences. The method has been used for CSF after removal of small interfering molecules by gel filtration. This approach is sensitive and simple but requires an appropriate spectrophotometer and highly-quality cuvetes with high transmition of light at 220nm.
The method has been used for CSF after removal of small interfering molecules by gel filtration. This approach is sensitive and simple but requires an appropriate spectrophotometer and highly-quality cuvetes with high transmition of light at 220nm.
Dye-binding methods are based on the ability of proteins to bind dyes such as blck 10B and The method is simple,east,and linear up to 150 mg/dl.
CBB.The unequal affinities and binding capacities of individual proteins for dyes are a limitration in all these application, which are complicated further by the inability to defin a consistent material for use as a calibrator. The dye-binding method of greatest contemporary interest, particularly for assay of total proein in CSF and urine uses CBB G-250. CBB binds to protonated amine group of amino acid resiues in the bound species’ of the dye decreases at 465 nm and increase at 595 nm.
Precipitation of protein for turbid metric or nephelometric assays is achieved with sulfosalicylic acid alone, with sulfosalicylic acid in combination with sodium sulfate or trichloroacetric acid (TCA), or with TCA alone. precipitation method for total protein assay depend on formation of a fin precipitate of uniform , and insoluble protein particles which scatter incident light in suspension.
Assay by biuret technique after removal of globulins by precipitation.
Salt fractionation or the 'salting-out' procedure:
Removal of globulins by salt precipitation followed by the quantitation of residual albumin in solution by the biuret or kjeldahl techniques was the standard procedure for many years. Different salts have been used alone or in combination to precipitate out the globulin out of sample solution- the so-called 'salting-out' procedure. These include ammonium sulphate, which was later replaced by other salts because of ammonium ion interference with both the biuret and kjeldahl reactions; sodium sulphate (at least 27 g/dl of the salt is required for complete precipitation of the globulins, an amount that can not be kept in solution); sodium sulfite, 28.3 g/dl, which is used instead of the sulphate because of its high solubility; and sodium sulphate/ sodium sulphite mixture (20.8% Na2SO4 and 7%Na2SO3). The sulphate/sulphite mixture is the recommended procedure because of its solubility as well as efficiency in removing the globulins out of solution.
The accepted reference method is the one which uses sulphite/sulphate mixture. In the procedure an aliquot of the sample (0.2 ml) is added to a tube containing the salt solution (4ml) followed by addition of 4ml diethyl ether to the mixture. The tube is stoppered and gently inverted 8 times. It is then centrifuged at 2000 r.p.m. for 5 minutes. After the centrifugation, globulin precipitate forms a pellet between the aqueous and the ether phases; the aqueous phase is carefully removed by inserting a pipette and tested by the biuret method.
By difference, i.e., calculating the albumin value from known values of the total protein and total globulins. (Total protein = albumin + Total globulin)
Assay after electrophoretic fractionation of the serum proteins
Immunochemical techniques. Specific antiserum to the protein to be assayed is added to the sample and the amount of or the rate of formation of antigen/antibody complex is measured using either nephelometry or turbidimetry
Assay Techniques
The following approaches are commonly employed for the quantitative determination of serum albumin:
Assay by biuret technique after removal of globulins by precipitation. Salt fractionation or the 'salting-out' procedure
By difference, i.e., calculating the albumin value from known values of the total protein and total globulins. (Total protein = albumin + Total globulin)
Dye-binding methodsDye-binding methods
Most clinical laboratories assay albumin by dye-binding methods, using bromcresol green(BCG) or purple(BCP) dyes.
BCG Method
Test principle: Albumin and BCG are allowed to bind at pH 4.2, in succinate buffer, and absorption of the BCG-albumin complex is measured at 628 nm. At pH 4.2, albumin acts as a cation to bind the anionic dye. The reaction is extremely fast and goes to completion in only a few seconds.
Procedure: 5 ml of the dye reagent is added into tubes containing 0.02 ml of sample, standard and control; absorbance of the respective tubes is read against the reagent blank 30 seconds later.
Linearity: 1-6 g/dl
Reference Range: Adult serum albumin
Recumbent (lying down): 3.5 - 5.0 g/dl
In the upright position levels are about 0.3 g/dl higher because of hemoconcentration.
Note: Interference from hyperlipemia, hyperbilirubinemia, and hemolysis can generally be eliminated (minimized) by dilution of serum 1:250.
Assay after electrophoretic fractionation of the serum proteins
Immunochemical techniques. Specific antiserum to the protein to be assayed is added to the sample and the amount of or the rate of formation of antigen/antibody complex is measured using either nephelometry or turbidimetry
Interference from hyperlipemia, hyperbilirubinemia, and hemolysis can generally be eliminated (minimized) by dilution of serum 1:250
BCP Method
Test principle: Yellow BCP dye, buffered at pH 5.2 with acetate, turns green when complexed with albumin. Absorbance of the green complex is measured at 603 nm.
the use of serum is recommended because these assays overestimate albumin in the presence of fibrinogen and heparin
The methods tend to be erroneous if the overall serum protein pattern is abnormal
Methods for the quantitative determination of total globulins
The total globulin levels in serum can be determined in any of the following ways:
Globulin by difference: Subtracting the albumin value from total protein value if both are known.
Colorimetric procedure using globulin reagent.
Electrophoresis
Immunochemical technique: use of specific antisera as a reagent.
Colorimetric procedure for total globulins
Test principle: In the presence of strong acids, glyoxylic acid reacts with tryptophan residues of proteins to form a purple color. Copper sulphate is added to enhance color formation.
Since human globulins are known to contain 2 - 3% tryptophan, it is possible to derive an empirical factor relating the concentration of N-acetyl tryptophan to serum globulin levels.
Reagents
Globulin reagent. Glyoxilic acid, 800 mg; copper sulphate, 600 mg; acetic acid, 15 mol; sulfuric acid, 2.2 mol; dissolved and made up to 1 liter.
Standard: 220 mg/dl N-acetyl-DL-tryptophan equivalent to 4 g/dl globulin.
Procedure
StandardControlTestGlobulin reagent4 ml4 ml4 mlStandard20 l--Control-20 l-Serum--20 l
Mix, cover tubes and heat at 100 0C for 5 minutes. Cool in tap water for 3 minutes, remix. Read absorbance of all tubes against globulin reagent at 550 nm.
Calculate test value using Beer's formula.
Linearity: up to 7.5 g/dl globulin.
Separation of serum proteins on cellulose acetate or agarose gel is almost always performed on serum to avoid the complication of a fibrinogen band in a region to which proteins of greater interest migrate.
The Reaction Principle of Protein Electrophoresis
Patient’s serum or concentrated urine or CSF is placed into a sample trough within agarose gel, placed in an alkaline buffer solution and a standardized voltage is applied to allow separation of the major protein groups by electrophoresis. The result is 6 or more fractions of separated proteins including the main fractions of albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma globulins from anode to cathode. Following electrophoresis the agarose gel is processed in acetic acid and alcohol washes to fix the proteins in the agarose. Following a wash step the protein fractions are stained with Coumassie Brilliant Blue protein stain. After a second wash, fixed protein bands can be visualized and quantified with densitometry.
Normal pattern of serum protein electrophoresis using cellulose acetate strip.
Lower picture: stained electrophoretogram with distinct stained bands
Upper picture: densitometric scanning output of the stained bands
A normal and decreased or increased (depending on the assay) quality control sample should be analyzed along with patient samples, using Westgard or other quality control rules for acceptance or rejection of the analytical run.
If at all possible, use commercially prepared quality control material as it has been preserved to allow for long term storage. If commercially prepared quality control assayed samples are not available, that is, those with established mean, standard deviation and preparation and storage information, laboratory personnel can prepare control materials for their own use.
For the normal control, a large number of samples from similar patients free from most obvious disease, is pooled together in a large container. This is usually salvaged from regular specimens, rejecting those that are haemolysed, lipemic and jaundiced. Specimens are generally collected over a time period and should be frozen at -20 degrees C, tightly covered and held there until ready to determine statistical values. At least 100 mL of serum will be needed to prepare the quality control material.
Upon removing the frozen serum pool from freezer for analysis the container should immediately be placed in a 37 degree C water bath for thawing, if possible. After thawing, the pool should be mixed thoroughly and filtered if needed, prior to analysis. 1.0 mL aliquots of the pool should be placed in test tubes, covered and labelled with a batch number and date. Retain 6-8 of the samples and place the remaining samples back in the freezer.
Mean and standard deviation of the control material is obtained by repeated analysis. A minimum of twenty samples must be analyzed for the particular tests needing quality control information, thawing only what samples will be analyzed immediately. Each sample should be mixed thoroughly prior to analysis. For most accurate representative results, 20 specimens should be run over a three-day period. This will permit more variance to be associated with the results therefore, a more representative of true testing conditions. In order to maximize on specimen availability and eliminate differences the specimen should be run by all methods at the same time, that is automated and manual for which the quality control results will be used.
Once mean and standard deviation is established and recorded, the remaining samples should be labelled with a date of implementation and quality control charts should be prepared to record mean, standard deviation and other identifying information. Aliquots for most analytes will be stable for up to 1 month. So the process is generally repeated monthly in order to have adequate acceptable quality control samples.
For preparation of an abnormal (high or low control), a similar process is used for collection, storage, analysis and aliquotting samples. However, it may be more difficult and take longer to obtain enough abnormal pool. It may be possible to add calibration or standard solution in order to obtain an abnormal high sample or to add appropriate diluent to prepare an abnormally low quality control sample. However, this method of adding to the serum pool or diluting is less desirable.
If unassayed commercially quality control samples are purchased, several vials should be prepared according to the manufacturer’s directions and assayed 20 times over a three day period. Aliquots should be frozen for use during the three day period. Mean, standard deviation can be determined as described above and quality control charts made. This is a desirable alternative, especially for obtaining abnormal quality control samples.