1

LIVER FUNCTION TESTS

Liver function tests (LFTs or LFs), which include liver enzymes, are groups of clinical
biochemistry laboratory blood assays designed to give information about the state of a
patient's liver. Most liver diseases cause only mild symptoms initially, but it is vital that
these diseases be detected early. Hepatic (liver) involvement in some diseases can be of
crucial importance. This testing is performed on a patient's serum or plasma sample
obtained by phlebotomy. Some tests are associated with functionality (eg. albumin);
some with cellular integrity (eg. transaminase) and some with conditions linked to the
biliary tract (gamma-glutamyl transferase and alkaline phosphatase).

LIVER ENZYMES

Albumin (Alb): Albumin is a protein made specifically by the liver, and can be
measured cheaply and easily. It is the main constituent of total protein; the remaining
fraction is called globulin (including the immunoglobulin). Albumin levels are decreased
in chronic liver disease, such as cirrhosis. It is also decreased in nephritic syndrome,
where it is lost through the urine. Poor nutrition or states of protein catabolism may also
lead to hypoalbuminaemia. Albumin is not considered to be an especially useful marker
of liver synthetic function. 3.9 to 5.0 g/dL

Alanine transaminase (ALT): Alanine transaminase (ALT), also called Serum Glutamic
Pyruvate Transaminase (SGPT) or Alanine aminotransferase (ALAT) is an enzyme
present in hepatocytes (liver cells). When a cell is damaged, it leaks this enzyme into the
blood, where it is measured. ALT rises dramatically in acute liver damage, such as viral
hepatitis or paracetamol (acetaminophen) overdose. Elevations are often measured in
multiples of the upper limit of normal (ULN).

Aspartate transaminase (AST): Aspartate transaminase (AST) also called Serum

Glutamic Oxaloacetic Transaminase (SGOT) or aspartate aminotransferase (ASAT) is
similar to ALT in that it is another enzyme associated with liver parenchymal cells. It is
raised in acute liver damage, but is also present in red cells and cardiac and skeletal
 2

muscle and is therefore not specific to the liver. The ratio of AST to ALT is sometimes
useful in differentiating between causes of liver damage. 10-40 IU/L

Alkaline phosphatase (ALP): Alkaline phosphatase (ALP) is an enzyme in the cells

lining the biliary ducts of the liver. ALP levels in plasma will rise with large bile duct
obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. ALP is also
present in bone and placental tissue, so it is higher in growing children (as their bones are
being remodeled) and elderly patients with Paget's disease. || 30 to 120 IU/L

Total bilirubin (TBIL): Bilirubin is a breakdown product of heme (a part of hemoglobin

in red blood cells). The liver is responsible for clearing the blood of bilirubin. It does this
by the following mechanism: bilirubin is taken up into hepatocytes, conjugated (modified
to make it water-soluble), and secreted into the bile, which is excreted into the intestine.

Increased total bilirubin causes jaundice, and can signal a number of problems:

• 1. Prehepatic: Increased bilirubin production. This can be due to a number of

causes, including hemolytic anemias and internal hemorrhage.
• 2. Hepatic: Problems with the liver, which are reflected as deficiencies in
bilirubin metabolism (e.g. reduced hepatocyte uptake, impaired conjugation of
bilirubin, and reduced hepatocyte secretion of bilirubin). Some examples would
be cirrhosis and viral hepatitis.
• 3. Posthepatic: Obstruction of the bile ducts, reflected as deficiencies in bilirubin
excretion. (Obstruction can be located either within the liver or outside the liver.)

Direct bilirubin: The diagnosis is narrowed down further by looking at the levels of
direct bilirubin.

• If direct (i.e. conjugated) bilirubin is normal, then the problem is an excess of

unconjugated bilirubin, and the location of the problem is upstream of bilirubin
excretion. Hemolysis, viral hepatitis, or cirrhosis can be suspected.
 3

• If direct bilirubin is elevated, then the liver is conjugating bilirubin normally, but
is not able to excrete it. Bile duct obstruction by gallstones or cancer should be
suspected.

If the direct bilirubin is low, while the total bilirubin is high, this reflects liver cell
damage or bile duct damage within the liver itself.
 4

SGOT

The test is for the determination of SGOT (AST) activity in serum.

CLINICAL SIGNIFICANCE
Serum Glutamic Oxaloacetic Transaminase (SGOT) is an enzyme found mainly in heart
muscle, liver cells, skeletal muscle, kidneys, brain, pancreas, spleen and lungs. Injury to
these tissues results in the release of the enzyme in blood. SGOT is also called Aspartate
Aminotransferase (AST).

INCREASES
Elevated levels are found in myocardial infarction, cardiac operations, hepatitis, cirrhosis,
acute pancreatitis, acute renal diseases, and primary muscle diseases.

DECREASES
Decreased levels may be found in pregnancy, Beri Beri and Diabetic ketoacidosis.

PRINCIPLE
SGOT (AST) catalyses the transfer of amino groups between L-Aspartate and α-
ketoglutarte to form oxaloacetate and glutamate. The oxaloacetate formed reacts with
NADH in the presence of Malate Dehydrogenase to form NAD. The rate of oxidation of
NADH to NAD is measured as a decrease in absorbance which is proportional to the
SGOT (AST) activity in the sample.

L-Aspartate + α ketoglutarate Oxaloacetate + L-Glutamate

Oxaloacetate + NADH + H Malate + NAD

 5

REAGENT COMPOSTION
Tris buffer- 25 mmol/L
L-Alanine- 200 mmol/L
NADH- 0.15mmol/L
MDH- 2000U/L
EDTA- 5mmol/L
α ketoglutarate- 12mmol/L

STORAGE AND STABILITY

Contents are stable at 2-8 ْC till the expiry.

WORKING REAGENT
Reconstitute one vial of enzyme reagent with 10ml of SGOT Diluent. Mix gently to
allow dissolution.
This working reagent is stable for at least 4 weeks when stored at 2-8 ْC.

SAMPLE
Serum free from hemolysis. SGOT is stable in serum for 3 days at 2-8 ْC.

ASSAY PARAMETERS
Reaction: UV Kinetic
Wavelength: 340nm
Zero Settings: Distilled Water
Incubation temperature: 37 ْC
Factor: 1746
Light path: 1cm

PROCEDURE
1. Pipette into a clean dry test tube labeled as test (T):
Working reagent: 1ml
Sample: 0.1ml
 6

2. Mix well and take the readings from the spectrophotometer.

The procedure is linear upto 450 IU/L at 37 ْC.
Samples having very high activity show a very low initial absorbance as most of the
NADH is consumed prior to the start of measurement. If this is suspected then dilute the
sample and repeat the assay.

NORMAL VALUES
Serum (males): upto 37 U/L at 37 ْC
(females): upto 31 U/L at 37 ْC
 7

SGPT

CLINICAL SIGNIFICANCE
Alanine Aminotransferase (ALAT/ALT) also called as Serum Glutamic Pyruvic
Transaminase (SGPT) catalyses the conversion of α ketoacids into amino acids by
transfer of amino groups. As a liver specific enzyme ALAT is only significantly elevated
in hepetobilliary diseases. It is an enzyme that is normally present in liver and heart cells.
SGPT is released into blood when the liver or heart is damaged. The blood SGPT levels
are thus elevated with liver damage (for example, from viral hepatitis) or with an insult to
the heart (for example, from a heart attack). Some medications can also raise SGPT
levels.

PRINCIPLE
L-Alanine + 2 Oxoglutarate L-Glutamate + Pyruvate

Pyruvate + NADH + H D-Lactate + NAD

Addition of pyridoxal-5-phosphate (P5P) stabilizes the transaminates and avoids falsely

low values in samples containing insufficient endogenous P5P eg. from patients with
myocardial infarction, liver disease and intensive care patients.

REAGENTS
R1: TRIS pH – 7.5 100mmol/l
L-Alanine 500mmol/l
R2: 2-Oxoglutarate 15mmol/l
NADH 0.18mmol/l

SAMPLE
Serum, heparin plasma or EDTA plasma.
 8

ASSAY PROCEDURE
Wavelength – 340nm
Optical path – 1cm
Temperature – 37 ْC

PROCEDURE
1. Add 1000µl of reagent 1 to 100µl of sample.
2. Mix and incubate for approximately 1min.
3. Add 250µl of reagent 2.
4. Mix and immediately take the readings.

PRECAUTIONS
The reagent contains sodium azide as preservative. Avoid contact with skin and mucous
membranes.

NORMAL VALUES

MALE: <41U/L
FEMALE: <31U/L
 9

BILIRUBIN

Bilirubin (also referred to as hematoidin) is the yellow breakdown product of normal

heme catabolism. Bilirubin is excreted in bile, and its levels are elevated in liver disease
(like hepatitis), biliary obstruction or hemolysis. It is responsible for the yellow color of
bruises and the yellow discoloration in jaundice. Bilirubin reduction in the gut leads to a
product called urobilinogen which is then oxidized to urobilin which is excreted in the
urine.

METHOD
Jendrassik Method

TEST PRINCIPLE
Bilirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an
azo dye. No caffeine is added when direct bilirubin is determined.

SAMPLE MATERIAL
Serum, heparinised plasma or EDTA plasma

REAGENTS
Reagent 1- Sulfanilic acid solution (29mmol/l)
Reagent 2- Nitrite solution (25mmol/l)
Reagent 3- Caffeine solution (0.26mol/l)
Reagent 4- Tartrate solution (0.93mol/l)
Reagent 5- Sodium chloride solution (0.9%)

SAMPLE PREPARATION
Hemolysis interferes with the test.
Do not expose samples to excessive light.
 10

PROCEDURE
Wavelength for total bilirubin: Hg 578nm (560- 600nm)
Wavelength for direct bilirubin: Hg 546nm (530- 550nm)
Temperature: 20- 30 ْC
Measure against sample blank.

TOTAL BILIRUBIN

Pipette Sample blank Sample

Reagent 1 0.10ml 0.10ml
Reagent 2 ----- 0.02ml
Reagent 3 0.50ml 0.50ml
Sample 0.10ml 0.10ml

Mix and let stand at 20-30 ْC for 5 min.

Reagent 4 0.50ml 0.50ml

Mix and let stand at 20- 30 ْC for 5 min.

Read absorbance of sample (ATB) against sample blank.

DIRECT BILIRUBIN

Pipette Sample blank Sample

Reagent 1 0.10ml 0.10ml
Reagent 2 ------ 0.02ml
Reagent 3 1.00ml 1.00ml
Sample 0.10ml 0.10ml

Mix and let stand at 20-30 ْC exactly for 5 min.

Read absorbance of sample (ADB) against sample blank.

CALCULATION
 11

Concentration of bilirubin in the sample

Total bilirubin Direct bilirubin
Wavelength Hg 578nm Hg 546nm
C (mg/100ml) 10.8 x ATB 14.4 x ADB
C (µmol/l) 185 x ATB 246 x ADB

NORMAL VALUES IN SERUM

Total bilirubin: upto 1mg/100ml = 17 µmol/l
Direct bilirubin: upto 0.25mg/100ml = 4.3 µmol/l

ALKALINE PHOSPHATASE

CLINICAL SIGNIFICANCE
 12

Alkaline Phosphatase (ALP) is an enzyme of the hydrolase class of enzymes and acts in
an alkaline medium. It is found in high concentrations in the liver, biliary tract epithelium
and in the bones. Normal levels are age dependent and increase during bone
development. Elevation of alkaline phosphatase in serum or plasma is found in hepatitis,
biliary obstructions, hyperparathyroidism, steatorrhea and bone diseases. A physiological
increase is observed in pregnancy. Moderate increases are seen in Hodgkin disease and
congestive heart failure.
Alkaline phosphatase level decreases in severe anemia, scurvy, kwashiorkor and
cretinism.
Many drugs cause increase in serum or plasma alkaline phosphatase activity eg.
androgens, anabolic steroids, estrogens, sulphonamides, penothiazines, tricyclic
antidepressants, MAO inhibitors, antibiotics, diuretics, anticoagulants, some
immunosuppressant such as azathioprine and anticonvulsants.

PRINCIPLE
Alkaline phosphatase hydrolyses p-Nitrophenyl Phosphate (PNPP) into p-Nitrophenol
and phosphate. At the alkaline pH of the buffered medium, D-Nitrophenol is yellow. The
color developed by hydrolysis is measured at 405nm and is proportional to the alkaline
phosphatase activity.

p-Nitrophenyl Phosphate + H2O p-Nitrophenol +

Phosphate

SAMPLE COLLECTION
Serum is preferred, heparinised plasma can also be used. Citrate, oxalate, EDTA etc
should not be used as they inhibit the activity of alkaline phosphatase. Serum is stored at
2-8 ْC.

REAGENTS
Reagent1 (substrate)
p-Nitrophenyl phosphate – 10mmol/l
 13

Reagent 1A (buffer)
Diethanolamine – 1mol/l
Magnesium chloride – 0.5mmol/l

REAGENT RECONSTITUION
Add 3mL of reagent 1A into one bottle of reagent 1. Mix by gently swirling till
completely dissolved. Wait for 5mins before using. The solution will be light yellow in
color.

ASSAY PARAMETERS
Reaction type – Kinetic
Wavelength – 405 nm
Path length – 1cm
Factor – 1826
Zero setting with – distilled water

PROCEDURE
Dispense into test tube reconstituted reagent 1mL and sample 30µL. mix and read
immediately.

NORMAL VALUES
Serum/ Plasma
Children – 151-471 U/L (25 ْC)
Adults – 60-170 U/L (25 ْC)

PRECAUTIONS
Buffers using ammonium salts, borates and glycine which inhibit the reaction should not
be used. As traces of detergents interfere clean glassware must be used.
TOTAL PROTEIN
CLINICAL SIGNIFICANCE
 14

Total protein is useful for monitoring gross changes in protein levels caused by various
disease states. It is usually performed in conjugation with other tests such as serum
albumin, liver function tests or protein electrophoresis.

INCREASES
In dehydration, multiple myeloma and chronic liver diseases.

DECREASES
In renal diseases and terminal liver failure.

METHODOLOGY
The peptide bonds of proteins react with copper II ions in alkaline solution to form blue-
violet complex (biuret reaction), each copper ion complexing with 5 or 6 peptide bonds.
Tartarate is added as a stabilizer while iodide is used to prevent auto reduction of the
alkaline copper complex. The color formed is proportional to the protein concentration
and is measured at 546nm.

REAGENT COMPOSITION
Reagent 1: Total Protein Reagent
Copper II Sulphate 19mmol/L
Potassium Sodium Tartarate 43mmol/L
Potassium Iodide 30mmol/L
Sodium Hydroxide 600mmol/L

Total Protein Standard

Protein Standard 6.0g/dl

SAMPLE
Serum or plasma. Result obtained with plasma may be upto 4.0g/dl higher due to
fibrinogen . Haemolysed specimens are unsuitable because hemoglobin reacts in the
assay.
 15

PROCEDURE

ELISA
 16

Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme Immunoassay or

EIA, is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in
medicine and plant pathology, as well as a quality control check in various industries. In
ELISA an unknown amount of antigen is affixed to a surface, and then a specific
antibody is washed over the surface so that it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step a substance is added that the enzyme can
convert to some detectable signal.

Performing an ELISA involves at least one antibody with specificity for a particular
antigen. The sample with an unknown amount of antigen is immobilized on a solid
support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to
the surface) or specifically (via capture by another antibody specific to the same antigen,
in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is
added, forming a complex with the antigen. The detection antibody can be covalently
linked to an enzyme, or can itself be detected by a secondary antibody which is linked to
an enzyme through bioconjugation. Between each step the plate is typically washed with
a mild detergent solution to remove any proteins or antibodies that are not specifically
bound. After the final wash step the plate is developed by adding an enzymatic substrate
to produce a visible signal, which indicates the quantity of antigen in the sample.

ELISA is a useful tool both for determining serum antibody concentrations (such as with
the HIV test) and also for detecting the presence of antigen.
 17

Triiodothyronine
 18

Triiodothyronine (T3) Enzyme Immunoassay (EIA) system is for the quantitative

determination of T3 in human serum.
Measurement of serum levels of T3 is an important adjunct for the assessment of thyroid
function. It is especially useful in the conformation of hyperthyroidism and in the
detection of T3 thyrotoxicosis. An elevated serum T3 level accompanied by an elevated
serum thyroxine (T4) level is a strong indication of hyperthyroidism. In patients
exhibiting clinical hyperthyroidism, an elevated serum T3 level, combined with a normal
serum T4 concentration is evidence for T3 thyrotoxicosis. A reduced serum level of T3 is
not a clear indicator of primary hypothyroidism as a reduced serum level of T4 or an
elevated level of thyroid stimulating hormone (TSH).

PRINCIPLE
Antibody to T3 is the specific antibody immobilized on microwell plates. Purified T3
conjugated to the enzyme Horseradish Peroxidase (HRP) is used to detect T3. In order to
accurately measure the total T3 concentration in serum, the endogenous binding proteins
(i.e. TBG, albumin and prealbumin) are blocked by the use of 8-anilino-1-naphthalene
sulfonic acid (ANS).
In the assay procedure, the T3 standard and/or patient serum is added along with T3-
horseradish peroxidase conjugate to the antibody coated wells. A competition reaction
results between the native antigen in the serum and the enzyme – antigen conjugate for a
limited number of solid phase binding sites. After equilibrium is attained, the antibody –
bound fraction is separated from unbound antigen in a washing step by decantation or
aspiration. Enzyme substrate 3, 3’, 5, 5’ – tetramethybenzidine (TMB) is then added to
the microwell and incubated. In the presence of conjugate – antigen – antibody complex.
TMB is hydrolyzed to a colored end product. The enzyme activity in the antibody –
bound fraction as measured by the intensity of the color development is inversely
proportional to the native antigen concentration. The intensity of color is measured
spectrophotometrically at 450 nm. The concentration of T3 is interpolated from a
standard curve.
Microwell plates are coated with rabbit T3 antibody (produced in white rabbits by
administration of T3 that was coupled to a protein carrier).
 19

REAGENTS
T3 conjugate – horseradish peroxidase conjugated to triiodothyronine in a buffered
solution, containing bovine serum albumin, inhibitors and preservatives.

OneBlue TMB Single Substrate – contains 3, 3’, 5, 5’– tetra methyl benzidine substrate
reagent in a stabilized buffer system and urea hydrogen peroxide.

Stop solution – Hydrochloric acid 1N.

Standards – T3 standards 0, 50, 100, 250, 500 and 1000 ng/dL in T3 free human serum
with thimerosol added as a preservative.

Control serum (lyophilized) – contains human serum and preservative. Reconstitute

with 1mL of distilled water.

Wash solution concentrate – buffer solution concentrate with thimerosol added as a

preservative. Prepare working wash solution by adding the 15 mL wash solution
concentrate to 500 mL of distilled water.

PROCEDURE
1. Add 50µl each of standards, control serum and test samples in the appropriate
wells.
2. Add 2 drops or 100µl of T3 conjugate to each well. Complete additions within
5mins. Cover the plate. Mix wells by swirling the plate gently for 30sec and incubate
at room temp for 1hour.
5. Decant or aspirate the supernatant from all the wells. Drain thoroughly by
inverting the plate and tapping vigorously on an adsorbent paper towel to remove
excess fluid.
6. Wash the wells five times with diluted wash solution. After each wash completely
empty the wells.
 20

7. Add 4 drops or 200µl of TMB substrate solution to each well. Mix wells by
swirling the plate gently for 30sec. Complete all additions within 5mins. Cover
the plate and incubate at room temperature for 30mins.
8. Add 2 drops or 100µl of stop solution to each well. Tap gently to mix the stop
solution. Avoid splashing.
9. Read the absorbance at 450nm.

PRECAUTIONS
1. All reagents should be stoppered when not in use.
2. The stop solution contains acid. Avoid contact with skin. If skin comes in contact
with skin, wash the area with copious amount of water.
3. Samples containing biliubin should not be used.
 21

Thyroxine

L – Thyroxine (T4) is a hormone that is synthesized and stored in the thyroid gland.
Proteolytic cleavage of follicular thyroglobulin releases T4 into the bloodstream. The
release of T4 and T3 from the thyroid gland is influenced by the pituitary thyroid
stimulating hormone (TSH) that is in turn influenced by the hypothalamic thyrotropin
releasing hormone (TRH). Greater than 99% of T4 is reversibly bound to three plasma
proteins in blood – thyroxine binding globulin (TBG) binds 70%, thyroxine binding pre –
albumin (TBPA) binds 20% and albumin binds 10%. Approximately 0.03% of T4 is in
the free, unbound state in blood at any one time.
Diseases affecting thyroid function may present a wide array of confusing symptoms.
Increased levels of T4 have been found in hyperthyroidism due to Grave’s disease and
Plummer’s disease and in acute and sub – acute thyroiditis. Low levels of T4 have been
associated with congenital hypothyroidism, myxedema, chronic thyroiditis (Hashimoto’s
disease) and with some genetic abnormalities.

PRINCIPLE
Antibody to T4 is a specific antibody immobilized on polystyrene microwells. Purified
T4 conjugated to the enzyme horseradish peroxidase (HRP) is used to detect T4. In order
to accurately measure the total T4 concentration in serum
the endogenous binding protein i.e. TBG, albumin and prealbumin are blocked by the use
of 8 – aniline – 1 – naphthalene sulphonic acid (ANS). During incubation T4 and
conjugated T4 compete for the limited binding sites on the anti – T4 antibody. After
60mins incubation at room temperature the wells are washed 5 times by water to remove
unbound T4 conjugate. A solution of TMB reagent is then added and incubated for
20mins resulting in the development of blue color. The color development is stopped
with the addition of Stop Solution and the absorbance is measured spectrophotometrically
at 450nm. The intensity of the color formed is proportional to the amount of unlabeled T4
in the sample.
 22

REAGENTS
1. T4 conjugate – horseradish peroxidase conjugated to T4 in a buffered solution
containing bovine serum albumin, inhibitors and preservatives.
2. OneBlue TMB Single Substrate
3. Stop Solution
4. Standards
5. Control Serum

PROCEDURE
1. Add 10µl each of standards, control serum and test samples in the appropriate
wells.
2. Add 2 drops or 100µl of T4 conjugate to each well. Mix wells by swirling the
plate gently for 10secs. Avoid bubble formation. Complete additions within
5mins. Cover the plate and incubate at room temperature for 30mins.
3. Decant or aspirate the supernatant from all the wells. Drain thoroughly by
inverting the plate and tapping vigorously on absorbent paper towel to remove
excess fluid.
4. Wash the wells five times with distilled water. After each wash completely empty
the wells. Do not let the wells dry out.
5. Add 2 drops or 100µl of TMB substrate solution to each well. Mix wells by
swirling the plate gently for 10sec.Complete all additions within 5mins. Cover the
plate and incubate at room temperature for 30mins.
6. Add 2 drops or 100µl of stop solution to each well. Tap gently to mix the stop
solution.
7. Read the absorbance at 450nm.

PRECAUTIONS
1. The stop solution contains acid. Avoid contact with skin.
2. Samples containing bilirubin should not be used.
3. All reagents should be stoppered when not in use.
 23

Thyroid Stimulating Hormone

Thyroid stimulating hormone (TSH) or thyrotropin is a glycoprotein hormone consisting

of two peptide chains designated as alpha and beta. TSH is the primary regulator of the
functional state of the thyroid gland. TSH production and release is stimulated by the
hypothalamic thyrotropin releasing hormone (TRH). TSH suppression is controlled by
levels of the thyroid hormones thyroxine and triiodothyronine via feedback at the
pituitary gland and hypothalamus. Serum TSH levels are raised in cases of primary
hypothyroidism. The diagnosis of overt hypothyroidism by the finding of a low total or
free T4 value is readily confirmed by a raised TSH level. In hyperthyroidism levels of T4
and T3 are elevated and TSH secretion is suppressed.

PRINCIPLE
The immunoassay is based on the sandwich principle. The TSH present in the test sample
reacts simultaneously with one antibody immobilized on the microwell surface and with
another antibody conjugated to horseradish peroxidase enzyme. So an Ab – Ag – Ab
enzyme complex is formed on the microwell surface.
Then the unbound conjugate is removed by washing and the color development reagents
(substrates) are added. Upon exposure to the enzyme a color change will take place. The
intensity of the color reflects the amount of bound anti – TSH enzyme conjugate and is
proportional to the concentration of TSH in the specimen within the dynamic range of the
assay. After stopping the reaction the resulting color is measured using a
spectrophotometer at 450nm.

REAGENTS
1. TSH conjugate – horseradish peroxidase conjugated to purified anti – TSH IgG in
a buffered red solution containing bovine serum albumin, inhibitors and
preservatives.
2. OneBlue TMB Single Substrate
3. Stop Solution
4. Standards
 24

5. Control Serum (lyophilized)

PROCEDURE
1. Add 1 drop or 50µl each of standard, control serum and test samples in the
appropriate wells.
2. Add 2 drops or 100µl of TSH conjugate to each well. Mix well by swirling the
plate gently for 10secs. Avoid bubble formation. Complete additions within
5mins. Gently shake the wells for 20secs and seal with paraffin. Incubate at 37ْC
in a water bath for 1 hour.
3. Remove and discard the cover seal. Decant or aspirate the supernatant from all the
wells. Drain thoroughly by inverting the plate and tapping vigorously on an
absorbent paper towel to remove excess fluid.
4. Wash the wells five times with diluted wash solution. After each wash completely
empty the wells. Do not let the wells dry out.
5. Add 2 drops or 100µl of TMB Substrate solution to each well. Mix
well by swirling the plate gently for 10secs. Complete all additions
within 5mins. Cover the plate and incubate at room temperature for 30mins.
6. Add 2 drops or 100µl of Stop Solution to each well. Tap gently to mix the stop
solution.
7. Read the absorbance at 450nm.

PRECAUTIONS
1. The Stop Solution contains acid. Avoid contact with skin.
2. Pipette all reagents into the bottom of the microwell.
3. All reagents should be stoppered when not in use.
 25

Immunoglobin E

Of all the immunoglobins, immunoglobins E (IgE) is present in the serum in the lowest
concentration. It is responsible for the clinical signs and symptoms of an immediate type
allergic reaction and IgE concentration in serum is generally correlated with the intensity
of allergic exposure and the severity of the allergic symptoms. The IgE is produced by
the regional lymph nodes as well as the respiratory and the gastrointestinal mucosa in
response to a specific allergen. IgE then fixes to circulating basophils and tissue mast
cells which subsequent to contact with the allergen, release potent vasoactive substances.
These compounds largely histamine and slow reacting substances of anaphylaxis, cause
capillary vasodilation with leakage of fluid and colloid to the tissues, thereby leading to
the primary symptoms of allergic disease.
Increased IgE production has been demonstrated in a variety of atopic conditions
including allergic rhinitis, eczema, asthma etc. The determination of IgE can aid in the
diagnosis of allergic diseases.

PRINCIPLE
It is a solid phase enzyme immunoassay based on the sandwich principle. Two separate
antibodies directed against distinct antigenic determinants of the IgE molecule are
utilized in the assay. During the first incubation the IgE present in the test sample reacts
and binds with anti – IgE antibody immobilized on the microwell surface. During second
incubation the bound IgE reacts with and to another anti – IgE antibody conjugated to
horseradish peroxidase enzyme. So an Ab – Ag – Ab enzyme complex is formed on the
microwell surface.
Then the unbound conjugate is removed by washing and the color development reagents
(substrates) are added. Upon exposure to the enzyme, a color change will take place. The
intensity of the color reflects the amount of bound anti – IgE enzyme conjugate and is
proportional to the concentration of IgE in the specimen. After stopping the reaction, the
resulting color is measured using a spectrophotometer at 450nm.
 26

REAGENTS
1. Enzyme conjugate – Anti – IgE antibody conjugated with horseradish peroxidase
in a buffer with protein stabilizer and preservative.
2. Standards
3. Test buffer – protein buffer solution with preservative
4. Dilution buffer – buffered protein solution with preservative.
5. Washing buffer – phosphate buffered
6. Substrate reagent A – 0.05M acetate buffer containing 0.02% hydrogen peroxide.
7. Substrate reagent B – 3,3’,5,5’ – tetramethybenzidine in a stabilizing buffer.
8. Stopping Solution – 1N HCl

PROCEDURE
1. Dispense 25µl of each standard, control and test sample into the appropriate well.
Complete pipetting within 5mins.
2. Dispense 2 drops of test buffer into each well.
3. Gently rock the wells for 20secs then seal by covering with parafilm or other film
sealant.
4. Incubate at room temperature for 15mins.
5. Remove and discard cover seal. Decant the incubation mixture thoroughly by
flicking into a sink containing disinfectant.
6. Rinse or wash the microwells five times with diluted washing buffer.
7. Dry the wells by firmly tapping the plate on a clean paper towel to remove excess
washing solution.
8. Dispense two drops of enzyme conjugate into each well. Gently rock the wells for
20secs and then seal by covering with parafilm or other film sealant.
9. Incubate at room temperature for 20mins.
10. Remove and discard cover seal. Decant the incubation mixture thoroughly by
flicking into a sink containing disinfectant.
11. Rinse and wash the microwells five times with diluted washing buffer.
12. Dry the wells by firmly tapping the plate to remove excess washing solution.
 27

13. Dispense one drop of substrate reagent A and one drop of substrate reagent B into
each well. Gently rock the wells for 20secs. Incubate at room temperature for
15mins.
14. Stop the reaction by adding one drop of stop solution to each well and gently rock
the wells.
15. Read the absorbance at 450nm.

PRECAUTIONS
1. Do not ingest the reagents. Avoid contact with eyes, skin, mucosa. Wear
protective clothing and disposable gloves. Do not allow smoking or eating where
antigen containing materials are being handled.
2. Preclude any pipetting by mouth.
3. Handle all patients sample and test components as though capable of transmitting
infection.
4. Avoid splashing or aerosol formation.
5. Avoid contacting stop solution with the skin or other mucous membranes.
28