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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

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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. Bile duct obstruction by gallstones or cancer should be suspected. this reflects liver cell damage or bile duct damage within the liver itself. If the direct bilirubin is low. while the total bilirubin is high. but is not able to excrete it. then the liver is conjugating bilirubin normally.

PRINCIPLE SGOT (AST) catalyses the transfer of amino groups between L-Aspartate and α- ketoglutarte to form oxaloacetate and glutamate. brain. INCREASES Elevated levels are found in myocardial infarction. hepatitis. skeletal muscle. Injury to these tissues results in the release of the enzyme in blood. DECREASES Decreased levels may be found in pregnancy. liver cells. and primary muscle diseases. acute renal diseases. L-Aspartate + α ketoglutarate Oxaloacetate + L-Glutamate Oxaloacetate + NADH + H Malate + NAD . The oxaloacetate formed reacts with NADH in the presence of Malate Dehydrogenase to form NAD. acute pancreatitis. CLINICAL SIGNIFICANCE Serum Glutamic Oxaloacetic Transaminase (SGOT) is an enzyme found mainly in heart muscle. SGOT is also called Aspartate Aminotransferase (AST). Beri Beri and Diabetic ketoacidosis. cirrhosis. kidneys. spleen and lungs. cardiac operations. 4 SGOT The test is for the determination of SGOT (AST) activity in serum. pancreas. 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.

Pipette into a clean dry test tube labeled as test (T): Working reagent: 1ml Sample: 0.200 mmol/L NADH.25 mmol/L L-Alanine. ASSAY PARAMETERS Reaction: UV Kinetic Wavelength: 340nm Zero Settings: Distilled Water Incubation temperature: 37 ْC Factor: 1746 Light path: 1cm PROCEDURE 1.5mmol/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.2000U/L EDTA.1ml . SGOT is stable in serum for 3 days at 2-8 ْC. 5 REAGENT COMPOSTION Tris buffer. Mix gently to allow dissolution. SAMPLE Serum free from hemolysis. This working reagent is stable for at least 4 weeks when stored at 2-8 ْC.15mmol/L MDH.0.

6 2. Samples having very high activity show a very low initial absorbance as most of the NADH is consumed prior to the start of measurement. The procedure is linear upto 450 IU/L at 37 ْC. NORMAL VALUES Serum (males): upto 37 U/L at 37 ْC (females): upto 31 U/L at 37 ْC . Mix well and take the readings from the spectrophotometer. If this is suspected then dilute the sample and repeat the assay.

heparin plasma or EDTA plasma. from a heart attack). It is an enzyme that is normally present in liver and heart cells. liver disease and intensive care patients. 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. from patients with myocardial infarction.18mmol/l SAMPLE Serum. REAGENTS R1: TRIS pH – 7. from viral hepatitis) or with an insult to the heart (for example. The blood SGPT levels are thus elevated with liver damage (for example. .5 100mmol/l L-Alanine 500mmol/l R2: 2-Oxoglutarate 15mmol/l NADH 0. 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. SGPT is released into blood when the liver or heart is damaged. As a liver specific enzyme ALAT is only significantly elevated in hepetobilliary diseases.

Add 250µl of reagent 2. 2. NORMAL VALUES MALE: <41U/L FEMALE: <31U/L . 3. Mix and immediately take the readings. Mix and incubate for approximately 1min. 4. Add 1000µl of reagent 1 to 100µl of sample. Avoid contact with skin and mucous membranes. 8 ASSAY PROCEDURE Wavelength – 340nm Optical path – 1cm Temperature – 37 ْC PROCEDURE 1. PRECAUTIONS The reagent contains sodium azide as preservative.

Caffeine solution (0.Sodium chloride solution (0.9%) SAMPLE PREPARATION Hemolysis interferes with the test.Tartrate solution (0. heparinised plasma or EDTA plasma REAGENTS Reagent 1. SAMPLE MATERIAL Serum. Do not expose samples to excessive light. No caffeine is added when direct bilirubin is determined. Bilirubin reduction in the gut leads to a product called urobilinogen which is then oxidized to urobilin which is excreted in the urine.93mol/l) Reagent 5. METHOD Jendrassik Method TEST PRINCIPLE Bilirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an azo dye.Nitrite solution (25mmol/l) Reagent 3.Sulfanilic acid solution (29mmol/l) Reagent 2. 9 BILIRUBIN Bilirubin (also referred to as hematoidin) is the yellow breakdown product of normal heme catabolism. .26mol/l) Reagent 4. Bilirubin is excreted in bile. biliary obstruction or hemolysis. It is responsible for the yellow color of bruises and the yellow discoloration in jaundice. and its levels are elevated in liver disease (like hepatitis).

0.10ml 0. Reagent 4 0.50ml Sample 0.30 ْC Measure against sample blank. 0.10ml Reagent 2 ----. Read absorbance of sample (ATB) against sample blank.00ml Sample 0.50ml Mix and let stand at 20.10ml 0. 10 PROCEDURE Wavelength for total bilirubin: Hg 578nm (560. DIRECT BILIRUBIN Pipette Sample blank Sample Reagent 1 0.50ml 0.10ml 0.02ml Reagent 3 0.550nm) Temperature: 20.600nm) Wavelength for direct bilirubin: Hg 546nm (530.10ml 0.30 ْC for 5 min.10ml Mix and let stand at 20-30 ْC for 5 min.02ml Reagent 3 1.10ml Reagent 2 -----. Read absorbance of sample (ADB) against sample blank.00ml 1. TOTAL BILIRUBIN Pipette Sample blank Sample Reagent 1 0.50ml 0. CALCULATION .10ml Mix and let stand at 20-30 ْC exactly for 5 min.

25mg/100ml = 4. 11 Concentration of bilirubin in the sample Total bilirubin Direct bilirubin Wavelength Hg 578nm Hg 546nm C (mg/100ml) 10.3 µmol/l ALKALINE PHOSPHATASE CLINICAL SIGNIFICANCE .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 0.8 x ATB 14.

biliary tract epithelium and in the bones. A physiological increase is observed in pregnancy. Citrate. oxalate. kwashiorkor and cretinism. Alkaline phosphatase level decreases in severe anemia. androgens. heparinised plasma can also be used. 12 Alkaline Phosphatase (ALP) is an enzyme of the hydrolase class of enzymes and acts in an alkaline medium. PRINCIPLE Alkaline phosphatase hydrolyses p-Nitrophenyl Phosphate (PNPP) into p-Nitrophenol and phosphate. Moderate increases are seen in Hodgkin disease and congestive heart failure. Many drugs cause increase in serum or plasma alkaline phosphatase activity eg. steatorrhea and bone diseases. penothiazines. antibiotics. MAO inhibitors. estrogens. D-Nitrophenol is yellow. scurvy. Elevation of alkaline phosphatase in serum or plasma is found in hepatitis. The color developed by hydrolysis is measured at 405nm and is proportional to the alkaline phosphatase activity. sulphonamides. At the alkaline pH of the buffered medium. hyperparathyroidism. EDTA etc should not be used as they inhibit the activity of alkaline phosphatase. anabolic steroids. Serum is stored at 2-8 ْC. It is found in high concentrations in the liver. tricyclic antidepressants. Normal levels are age dependent and increase during bone development. p-Nitrophenyl Phosphate + H2O p-Nitrophenol + Phosphate SAMPLE COLLECTION Serum is preferred. REAGENTS Reagent1 (substrate) p-Nitrophenyl phosphate – 10mmol/l . anticoagulants. biliary obstructions. diuretics. some immunosuppressant such as azathioprine and anticonvulsants.

13 Reagent 1A (buffer) Diethanolamine – 1mol/l Magnesium chloride – 0. borates and glycine which inhibit the reaction should not be used. Wait for 5mins before using. TOTAL PROTEIN CLINICAL SIGNIFICANCE . 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.5mmol/l REAGENT RECONSTITUION Add 3mL of reagent 1A into one bottle of reagent 1. Mix by gently swirling till completely dissolved. mix and read immediately. The solution will be light yellow in color. As traces of detergents interfere clean glassware must be used. NORMAL VALUES Serum/ Plasma Children – 151-471 U/L (25 ْC) Adults – 60-170 U/L (25 ْC) PRECAUTIONS Buffers using ammonium salts.

DECREASES In renal diseases and terminal liver failure. 14 Total protein is useful for monitoring gross changes in protein levels caused by various disease states. each copper ion complexing with 5 or 6 peptide bonds. 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. Haemolysed specimens are unsuitable because hemoglobin reacts in the assay. The color formed is proportional to the protein concentration and is measured at 546nm.0g/dl higher due to fibrinogen . multiple myeloma and chronic liver diseases. METHODOLOGY The peptide bonds of proteins react with copper II ions in alkaline solution to form blue- violet complex (biuret reaction). INCREASES In dehydration. liver function tests or protein electrophoresis. It is usually performed in conjugation with other tests such as serum albumin. Result obtained with plasma may be upto 4.0g/dl SAMPLE Serum or plasma. . Tartarate is added as a stabilizer while iodide is used to prevent auto reduction of the alkaline copper complex.

15 PROCEDURE ELISA .

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. as well as a quality control check in various industries. . forming a complex with the antigen. or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. In ELISA an unknown amount of antigen is affixed to a surface. The detection antibody can be covalently linked to an enzyme. 16 Enzyme-Linked ImmunoSorbent Assay. After the antigen is immobilized the detection antibody is added. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Enzyme Immunoassay or EIA. in a "sandwich" ELISA). After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal. and then a specific antibody is washed over the surface so that it can bind to the antigen. which indicates the quantity of antigen in the sample. and in the final step a substance is added that the enzyme can convert to some detectable signal. 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. This antibody is linked to an enzyme. 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. Performing an ELISA involves at least one antibody with specificity for a particular antigen. also called ELISA.

17 Triiodothyronine .

In order to accurately measure the total T3 concentration in serum. Measurement of serum levels of T3 is an important adjunct for the assessment of thyroid function. TMB is hydrolyzed to a colored end product. After equilibrium is attained. Purified T3 conjugated to the enzyme Horseradish Peroxidase (HRP) is used to detect T3. Enzyme substrate 3. The concentration of T3 is interpolated from a standard curve. the endogenous binding proteins (i. 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. 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). an elevated serum T3 level. albumin and prealbumin) are blocked by the use of 8-anilino-1-naphthalene sulfonic acid (ANS). . An elevated serum T3 level accompanied by an elevated serum thyroxine (T4) level is a strong indication of hyperthyroidism. 5. The intensity of color is measured spectrophotometrically at 450 nm. 18 Triiodothyronine (T3) Enzyme Immunoassay (EIA) system is for the quantitative determination of T3 in human serum. Microwell plates are coated with rabbit T3 antibody (produced in white rabbits by administration of T3 that was coupled to a protein carrier). In the assay procedure. In the presence of conjugate – antigen – antibody complex. PRINCIPLE Antibody to T3 is the specific antibody immobilized on microwell plates. It is especially useful in the conformation of hyperthyroidism and in the detection of T3 thyrotoxicosis. 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. the antibody – bound fraction is separated from unbound antigen in a washing step by decantation or aspiration.e. combined with a normal serum T4 concentration is evidence for T3 thyrotoxicosis. In patients exhibiting clinical hyperthyroidism. TBG. 3’. 5’ – tetramethybenzidine (TMB) is then added to the microwell and incubated.

6. Complete additions within 5mins. Reconstitute with 1mL of distilled water. Add 50µl each of standards. 5. Drain thoroughly by inverting the plate and tapping vigorously on an adsorbent paper towel to remove excess fluid. After each wash completely empty the wells. 5’– tetra methyl benzidine substrate reagent in a stabilized buffer system and urea hydrogen peroxide. Cover the plate. . containing bovine serum albumin. Wash solution concentrate – buffer solution concentrate with thimerosol added as a preservative. 500 and 1000 ng/dL in T3 free human serum with thimerosol added as a preservative. Standards – T3 standards 0. control serum and test samples in the appropriate wells. Add 2 drops or 100µl of T3 conjugate to each well. 5. 3’. Prepare working wash solution by adding the 15 mL wash solution concentrate to 500 mL of distilled water. 2. inhibitors and preservatives. 100. PROCEDURE 1. Decant or aspirate the supernatant from all the wells. Stop solution – Hydrochloric acid 1N. 50. 19 REAGENTS T3 conjugate – horseradish peroxidase conjugated to triiodothyronine in a buffered solution. 250. Control serum (lyophilized) – contains human serum and preservative. Mix wells by swirling the plate gently for 30sec and incubate at room temp for 1hour. OneBlue TMB Single Substrate – contains 3. Wash the wells five times with diluted wash solution.

8. Cover the plate and incubate at room temperature for 30mins. PRECAUTIONS 1. Samples containing biliubin should not be used. If skin comes in contact with skin. 20 7. Read the absorbance at 450nm. 2. . Avoid splashing. wash the area with copious amount of water. 9. All reagents should be stoppered when not in use. Complete all additions within 5mins. The stop solution contains acid. Avoid contact with skin. Mix wells by swirling the plate gently for 30sec. Tap gently to mix the stop solution. Add 4 drops or 200µl of TMB substrate solution to each well. 3. Add 2 drops or 100µl of stop solution to each well.

PRINCIPLE Antibody to T4 is a specific antibody immobilized on polystyrene microwells.03% of T4 is in the free. Diseases affecting thyroid function may present a wide array of confusing symptoms. After 60mins incubation at room temperature the wells are washed 5 times by water to remove unbound T4 conjugate. During incubation T4 and conjugated T4 compete for the limited binding sites on the anti – T4 antibody. Greater than 99% of T4 is reversibly bound to three plasma proteins in blood – thyroxine binding globulin (TBG) binds 70%. 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 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). 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. albumin and prealbumin are blocked by the use of 8 – aniline – 1 – naphthalene sulphonic acid (ANS).e. chronic thyroiditis (Hashimoto’s disease) and with some genetic abnormalities. 21 Thyroxine L – Thyroxine (T4) is a hormone that is synthesized and stored in the thyroid gland. thyroxine binding pre – albumin (TBPA) binds 20% and albumin binds 10%. TBG. Low levels of T4 have been associated with congenital hypothyroidism. Purified T4 conjugated to the enzyme horseradish peroxidase (HRP) is used to detect T4. . Approximately 0. In order to accurately measure the total T4 concentration in serum the endogenous binding protein i. unbound state in blood at any one time. myxedema. Proteolytic cleavage of follicular thyroglobulin releases T4 into the bloodstream. The intensity of the color formed is proportional to the amount of unlabeled T4 in the sample.

T4 conjugate – horseradish peroxidase conjugated to T4 in a buffered solution containing bovine serum albumin. All reagents should be stoppered when not in use. Complete additions within 5mins. 2.Complete all additions within 5mins. . After each wash completely empty the wells. Tap gently to mix the stop solution. Mix wells by swirling the plate gently for 10sec. Add 2 drops or 100µl of stop solution to each well. Add 2 drops or 100µl of T4 conjugate to each well. Do not let the wells dry out. control serum and test samples in the appropriate wells. The stop solution contains acid. 7. Mix wells by swirling the plate gently for 10secs. Cover the plate and incubate at room temperature for 30mins. 2. 22 REAGENTS 1. Standards 5. Drain thoroughly by inverting the plate and tapping vigorously on absorbent paper towel to remove excess fluid. Wash the wells five times with distilled water. OneBlue TMB Single Substrate 3. inhibitors and preservatives. Decant or aspirate the supernatant from all the wells. 3. Add 2 drops or 100µl of TMB substrate solution to each well. Avoid contact with skin. PRECAUTIONS 1. Samples containing bilirubin should not be used. Control Serum PROCEDURE 1. 4. Read the absorbance at 450nm. Avoid bubble formation. 2. Stop Solution 4. Add 10µl each of standards. 6. 3. 5. Cover the plate and incubate at room temperature for 30mins.

Stop Solution 4. The diagnosis of overt hypothyroidism by the finding of a low total or free T4 value is readily confirmed by a raised TSH level. TSH production and release is stimulated by the hypothalamic thyrotropin releasing hormone (TRH). TSH conjugate – horseradish peroxidase conjugated to purified anti – TSH IgG in a buffered red solution containing bovine serum albumin. Standards . OneBlue TMB Single Substrate 3. 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. REAGENTS 1. Upon exposure to the enzyme a color change will take place. In hyperthyroidism levels of T4 and T3 are elevated and TSH secretion is suppressed. After stopping the reaction the resulting color is measured using a spectrophotometer at 450nm. TSH is the primary regulator of the functional state of the thyroid gland. PRINCIPLE The immunoassay is based on the sandwich principle. Serum TSH levels are raised in cases of primary hypothyroidism. Then the unbound conjugate is removed by washing and the color development reagents (substrates) are added. So an Ab – Ag – Ab enzyme complex is formed on the microwell surface. 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. TSH suppression is controlled by levels of the thyroid hormones thyroxine and triiodothyronine via feedback at the pituitary gland and hypothalamus. inhibitors and preservatives. 23 Thyroid Stimulating Hormone Thyroid stimulating hormone (TSH) or thyrotropin is a glycoprotein hormone consisting of two peptide chains designated as alpha and beta. 2.

Control Serum (lyophilized) PROCEDURE 1. . Add 2 drops or 100µl of Stop Solution to each well. Decant or aspirate the supernatant from all the wells. The Stop Solution contains acid. Do not let the wells dry out. 2. Cover the plate and incubate at room temperature for 30mins. Gently shake the wells for 20secs and seal with paraffin. After each wash completely empty the wells. 5. Drain thoroughly by inverting the plate and tapping vigorously on an absorbent paper towel to remove excess fluid. Complete all additions within 5mins. Incubate at 37ْC in a water bath for 1 hour. Add 1 drop or 50µl each of standard. Mix well by swirling the plate gently for 10secs. Tap gently to mix the stop solution. Add 2 drops or 100µl of TMB Substrate solution to each well. 4. Avoid bubble formation. Avoid contact with skin. 3. Add 2 drops or 100µl of TSH conjugate to each well. Remove and discard the cover seal. Wash the wells five times with diluted wash solution. Mix well by swirling the plate gently for 10secs. control serum and test samples in the appropriate wells. 24 5. Complete additions within 5mins. Read the absorbance at 450nm. 2. All reagents should be stoppered when not in use. PRECAUTIONS 1. Pipette all reagents into the bottom of the microwell. 7. 6. 3.

immunoglobins E (IgE) is present in the serum in the lowest concentration. eczema. These compounds largely histamine and slow reacting substances of anaphylaxis. Two separate antibodies directed against distinct antigenic determinants of the IgE molecule are utilized in the assay. So an Ab – Ag – Ab enzyme complex is formed on the microwell surface. Increased IgE production has been demonstrated in a variety of atopic conditions including allergic rhinitis. a color change will take place. During second incubation the bound IgE reacts with and to another anti – IgE antibody conjugated to horseradish peroxidase enzyme. release potent vasoactive substances. 25 Immunoglobin E Of all the immunoglobins. the resulting color is measured using a spectrophotometer at 450nm. cause capillary vasodilation with leakage of fluid and colloid to the tissues. Upon exposure to the enzyme. 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. After stopping the reaction. . asthma etc. IgE then fixes to circulating basophils and tissue mast cells which subsequent to contact with the allergen. The IgE is produced by the regional lymph nodes as well as the respiratory and the gastrointestinal mucosa in response to a specific allergen. PRINCIPLE It is a solid phase enzyme immunoassay based on the sandwich principle. During the first incubation the IgE present in the test sample reacts and binds with anti – IgE antibody immobilized on the microwell surface. Then the unbound conjugate is removed by washing and the color development reagents (substrates) are added. 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. thereby leading to the primary symptoms of allergic disease. The determination of IgE can aid in the diagnosis of allergic diseases.

Enzyme conjugate – Anti – IgE antibody conjugated with horseradish peroxidase in a buffer with protein stabilizer and preservative. 5.05M acetate buffer containing 0.3’. Dispense 25µl of each standard. 5. . Stopping Solution – 1N HCl PROCEDURE 1. Substrate reagent A – 0.5’ – tetramethybenzidine in a stabilizing buffer. Dispense two drops of enzyme conjugate into each well. 10. 8. Incubate at room temperature for 15mins. Gently rock the wells for 20secs then seal by covering with parafilm or other film sealant. 8. Substrate reagent B – 3. 9. Complete pipetting within 5mins. 7. 11.02% hydrogen peroxide. 3. control and test sample into the appropriate well. 2. 2. 7. Standards 3. Decant the incubation mixture thoroughly by flicking into a sink containing disinfectant.5. Dry the wells by firmly tapping the plate to remove excess washing solution. Remove and discard cover seal. Dilution buffer – buffered protein solution with preservative. Remove and discard cover seal. Dry the wells by firmly tapping the plate on a clean paper towel to remove excess washing solution. Test buffer – protein buffer solution with preservative 4. Rinse and wash the microwells five times with diluted washing buffer. Incubate at room temperature for 20mins. Dispense 2 drops of test buffer into each well. Gently rock the wells for 20secs and then seal by covering with parafilm or other film sealant. Rinse or wash the microwells five times with diluted washing buffer. Washing buffer – phosphate buffered 6. 26 REAGENTS 1. 4. 12. Decant the incubation mixture thoroughly by flicking into a sink containing disinfectant. 6.

Gently rock the wells for 20secs. skin. PRECAUTIONS 1. Wear protective clothing and disposable gloves. mucosa. Do not ingest the reagents. Do not allow smoking or eating where antigen containing materials are being handled. Avoid contact with eyes. Dispense one drop of substrate reagent A and one drop of substrate reagent B into each well. Preclude any pipetting by mouth. Incubate at room temperature for 15mins. 5. 4. 3. Stop the reaction by adding one drop of stop solution to each well and gently rock the wells. . Handle all patients sample and test components as though capable of transmitting infection. Avoid splashing or aerosol formation. Read the absorbance at 450nm. 2. 14. 15. Avoid contacting stop solution with the skin or other mucous membranes. 27 13.

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