Rbc,wbc count
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  • 1. IMMUNOLOGY Experiment Page Date Sign. no.Total count of RBC & WBC differential count ofLeukocyte & blood groupinga) Total RBC countb) Total WBC countc) Differential count of Leukocyted) ABO blood groupingELISASingle Redial Immunodiffusion AssayOuchterlony Double diffusion AssayWestern Blotting1Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 2. Experiment: 1 Date: TOTAL COUNT OF RBC & WBC, DIFFERENTIAL COUNT OF LEUKOCYTES & BLOOD GROUPINGINTRODUCTION:Hematology is the branch of science concerned with the study of blood, blood forming tissues &the disorders associated with them. It includes,1) Analysis of the concentration, structure and functions of the cells and their precursors in thebone marrow.2) Analysis of chemical constituents of plasma or serum intimately, linked with blood cellstructure and functions.3) Study of functions of the platelets and proteins involved in blood coagulation.The hematology laboratory deals with routine determination of total number of cells incirculation, hemoglobin concentration, and differential count of leukocytes based on the study ofthe stained blood smear.Blood:Blood may be described as the specialized liquid connective tissue, which circulates in a closedsystem of blood vessels. The circulating blood consists of suspension of formed elements such aserythrocytes, leukocytes and platelets in a pale yellow colored fluid called “plasma”. In adults,the total volume of blood comprises about 8% of the body weight. The formed elements accountsfor about 46% of total blood volume. Under normal conditions, the production of blood cells iscarried out by the bone marrow.Functions of blood:1. Respiration: Transport of oxygen from the lungs to the tissues and of carbon dioxide from tissues to the lungs.2. Excretion: Transport of metabolic wastes to the lungs, kidneys, skin and intestine for removal.3. Maintenance of normal acid-base balance i.e. pH regulation.4. Nutrition: Transport of absorbed fatty acids, monosaccharides and amino acids.5. Regulation of water balance.6. Regulation of body temperature.7. Transport of hormones, vitamins, salts and metabolites.8. Defense against infection by the white blood cells & the antibodies.9. Coagulation of blood: To stop bleeding by clotting.2Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 3. Physical features of blood: 1. Blood is more viscous & denser than water and feels slightly sticky. 2. Temperature of blood is 38oC, about 10oC higher than oral or rectal body temperature. 3. It has slightly alkaline pH ranging from 7.35 to 7.45Experiment: 1(a) Date: DETERMINATION OF TOTAL COUNT OF (RBC) RED BLOOD CELLSAIM: To determine total count of RBC in the given blood sample.INTRODUCTION:Erythrocytes are elastic, non-nucleated biconcave discs. The cell possesses on its surface anumber of antigens responsible for the blood groups. The function of the red cell is to transportoxygen through hemoglobin in a functional state. Life span of red blood cell is about 120days.The erythrocyte count is performed to determine whether a person has anemia or polycythaemiaand to judge their degree.Clinical Significance:As birth the total erythrocyte count varies from 6.5millions -7.25millions per cubic mm (mm3).There is steady decline after a few hours and at the end of 15 days to one month there is a slowrise to normal adult levels. An increase in total erythrocyte count is observed in conditions suchas haemoconcentration due to burns, cholera etc. In central cyanotic states as seen in chronicheart disease, condition of decreased lung functions such as emphysema and polycythemia.Decrease in erythrocyte count is observed in(a) Old age(b) In pregnancy(c) In group of diseases classified under anemiaNormal values:Male: 4.5 to 6.0 x 106cells /mm3 Female: 4.0 to 4.5 x 106cells/mm3Specimens: EDTA blood or Capillary blood3Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 4. PRINCIPLE:The blood specimen is diluted 1:200 with the RBC diluting fluid and cells are counted underhigh (40x objective) by using a counting chamber. The number of cells in undiluted blood arecalculated and reported as the total number of cells per cubic mm of whole blood.REQUIREMENTS: 1. Neubauer‟s Haemocytometer Slide 3. RBC diluting fluid 2. RBC pipette 4. MicroscopeDescription of the Apparatus: It is a special glass slide. It has two central platforms separated by a horizontal groovewhich joins a vertical groove at each of its ends, thus an „H‟ shaped grooved area is seen on theslide. Outer to vertical groove i.e. on each side of ‟H‟, there is a slide pillar; both the slide pillarsare 1/10mm (0.1mm) lighter than the two central platforms. On each platform lies a ruled area of counting chamber so that two preparations may beset simultaneously. Each chamber is a square of the counting chamber i.e. 1sq mm area ismarked „R‟. This 1 sq mm area is divided into 25 small squares by triple lines i.e. each of thesesmall squares has an area of 1/25 sq mm which is bounded by triple lines. Each of these smallsquares i.e. as in area 1/25 sq mm is further divided into 16 smaller squares each of which has anarea = 1/25 X 1/16 = 1/400 = 0.0025 sq mm For WBC each of the 4 corner square at the counting chamber i.e. 1 sq mm area marked„W‟ and is divided into small squares, which are WBC counting chambers. Each small squarehas an area of 1/16 sq mm.4Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 5. Figure 1.1: Hemocytometer grid red square = 1 mm2, green square = 0.0625 mm2, yellow square = 0.04 mm2, blue square = 0.0025 mm2, at a depth of 0.1 mmRBC PIPETTE (Thoma glass pipette): It consists of capillary tube, central tube and three graduation marks. Capillary tube isopened at both ends. One end of the tube is narrow while the other end is broad. For sucking arubber tube fixed to the broad end. The bubble of the pipette has a red glass bead inside. This is abulb pipette having a long stem with a capillary bore and a pointed tip. The bulb contains a redbead inside. A small rubber tube provided with a mouth piece is connected to the small narrowportion above the bulb for sucking blood and fluid into the pipette. The pipette has threemarkings on in it, „0.5‟ mark in the middle of the stem, „1‟ mark at the junction between stemand bulb, and „101‟ mark above the bulb. The total volume of the pipette is 101 parts, of whichone part is in the stem and 100 parts in the bulb. Blood is drawn to „0.5‟ mark and then dilutingfluid is drawn up to the mark „101‟. The dilution of blood is 1:200.RBC diluting fluid:Hayem‟s fluid is isotonic with blood. Its composition is 1. Sodium chloride (NaCl) - 0.5 2. Sodium Sulphate (Na2SO4) - 2.5 g 3. Mercuric Chloride (HgCl2) - 0.25 g 4. Distilled water - 100 ml5Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 6. Figure 1.2: Neubauer‟s Haemocytometer Slide, RBC pipette, WBC pipette. Sodium chloride and sodium sulphate together keeps the isotonicity of fluid. Sodiumsulphate also prevents clumping of red cells. Mercuric per chloride fixes the cells and acts as apreservative, so diluting fluid helps in preventing hemolysis, coagulation of blood and fungal andbacterial growth.Cover slip of Haemocytometer slide: It is thicker than the ordinary cover slip used in laboratory. When it is placed on the twocounting chambers the space enclosed between the lower surfaces of the two platforms is 1/10mm deep. This means that the volume of the fluid enclosed in each of the smaller squares havingan area of 1/400 sq mm will be =1/400 X 1/10= 1/4000 mm3.6Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 7. Precautions:1. Counting chamber and pipette should be clean and dry.2. Fingertip and pricking lancet should be sterile.3. Blood should freely come out without squeezing.4. Be careful to prevent clotting of blood inside the pipette.5. While filling the pipette and charging (loading) the counting chamber, no air bubble shouldenter.6. Blood should be taken only up to the „0.5‟ mark and diluting fluid sucked only up to „101‟mark.7. Blood should be properly mixed with the diluting fluid.8. While charging the counting chamber, over filling and spilling should be avoided.9. Cells should be settled down and more or less evenly distributed before counting.10. Count from Left to Right (fig 1) and avoid counting of the same cell.Figure 1.3: Directions to count cells Figure 1.4: Empty hemocytometer grid at 100xpower7Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 8. PROCEDURE: 1. Mix the anticoagulated blood carefully by swirling the bulb. 2. In the case of capillary blood the lancet stab should be sufficiently deep to allow free flow of blood. It is drawn quickly in the RBC pipette. 3. Draw blood up to „0.5‟ mark. 4. Carefully wipe the excess blood outside the pipette by using cotton or a gauze. 5. Draw diluting fluid up to „101‟ mark. 6. The pipette in rotated rapidly by keeping it horizontal during mixing. 7. After two minute, by discarding few drops from the pipette and holding it slightly inclined small volume of the fluid is introduced under the cover slip which is placed on the counting chamber. 8. Allow the cells to settle for 2 to 3 minutes. 9. Place the counting chamber on the stage of the microscope. 10. Switch to low power (10 X) objective. Adjust light and locate the large square in the center with 25 small squares. 11. Now switch to high power (40 X) objective. 12. The red blood cells in the four corners and central Square of „R‟ are counted.Calculation of red blood cells: Total RBC/mm3 = Number of RBC counted × Dilution factor × Depth factor No. of chambers countedRESULT: Total RBC count of the given blood sample is ______________ million/mm38Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 9. Experiment: 1(b) Date: DETERMINATION OF TOTAL COUNT OF WHITE BLOOD CELLS (WBC)AIM: To determine total count of WBC in the given blood sample.INTRODUCTION:The leucocytes or white blood cells are nucleated corpuscles. The various types of white bloodcells are neutrophils, eosinophils, basophils, lymphocyte & monocytes. Leucocytes are neededby the body for its defense against invading organisms like bacteria, viruses and cancer cell.Clinical significance:Increase in total leucocytes count of more than 10,000/mm3 is known as leucocytosis anddecrease less than 3,000/mm3 as leukopeniaCauses of leucocytosis:1. Pathological: It is common for a transient period in infection. The degree of rise in leucocytes dependson the type and severity of the infection and response of the body. The infection may beBacterial, Viral, Protozoan (Malaria), and Parasitic (filarial) hookworm infection. Leucocytosisis also observed in severe hemorrhage and in leukemia2. Physiological: a) Age: At birth total leucocytes count is about 18,000/mm3, later it drops to adult level. b) Pregnancy: At “full term” the total count tends to be about 12,000-15000/mm3. It rises soon after delivery and then gradually returns to normal. c) High temperature d) Severe pain e) Muscular ExerciseCauses of leucopenia:Certain viral and bacterial infection (typhoid) leads to leukopenia rather than leucocytosis.Infection a) Bacterial (Typhoid, Paratyphoid, Tuberculosis etc.) b) Viral (Hepatitis, Influenza, Measles etc) c) Protozoan (Malaria)9Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 10. Some cases of leukemia: 1. Primary bone marrow depressions (Aplastic anemia) 2. Secondary bone marrow depression (due to drugs, radiation etc) 3. Anemia (iron deficiency, Megaloblastic etc)Normal Values: Adult : 4000-10,000/mm3 4 to 7 years : 6,000-15,000/mm3 At birth : 10,000-25,000/mm3 8-12 years : 4,500-13,500/mm3 1 to 3 years : 6000-18,000/mm3Specimens: a) Double oxalated or EDTA blood or b) Capillary bloodREQUIREMENTS: 1. Neubauer Haemocytometer slide with cover slip 2. WBC pipette 3. WBC diluting fluid 4. MicroscopeWBC Pipette: This is a bulb pipette having a long stem with a capillary bore and a pointed tip. The bulbcontains a red bead inside. A small rubber tube provided with a mouth piece is connected to thesmall narrow portion above the bulb for sucking blood and fluid into the pipette.WBC pipette islike RBC pipette but here the bulb has a white glass bead in place of red and the third graduationmark which lies just above the bulb is „11‟ instead of „101‟, this means the WBC pipette has 11volumes from the tip up to „11‟ mark.WBC diluting fluid (Truck’s fluid): 1. 1ml of 3% acetic acid, 1% gelatin violet- 1 or 2 drops or methylene blue. 2. The function of diluting fluid is to destroy RBC and stain the nucleus of WBC to increase the visibility.PROCEDURE: 1. The blood sample is taken up to the „0.5‟ mark, then the diluting fluid is taken up to the „11‟ mark, this gives 1:20 dilution.10Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 11. 2. The mixing was done well for one minute. 3. The counting chamber and cover glass placed on central platform were cleaned thoroughly before use. 4. A drop of diluted blood was discharged into the chamber. 5. The counting to be done using low power objective lens of the microscope.The four „W‟ corner squares of the both the chambers were counted. The number of WBCpresent give the value of, n-number of WBC counted. Then the total number of WBC per mm3 isobtained by the formulae; Total WBC/mm3 = Number of WBC counted × dilution factor × depth factor No. of chambers countedRESULT:Total WBC count of the given blood sample is ___________ /mm3.11Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 12. Experiment: 1(c) Date:STUDY OF BLOOD SMEAR FOR DIFFERENTIAL LEUKOCYTE COUNTAIM: To determine the differential count of Leukocytes by studying the blood smear.INTRODUCTION: Differential count is the percent distribution of various white cells in the peripheral blood.It is determined from a blood smear stained with a polychromatic stain and after examination ofthe stained smear by using oil immersion objective (total magnification 1000x). The number ofeach type of white cell is then expressed as a percentage of the total number of cells. The stainedblood smear also helps to study abnormal morphology of leucocytes and red cells. Study ofblood smear helps in the diagnosis of various anaemias, leukaemias and detection of bloodparasites. Three major steps involved in differential count are (a) preparation of smear (b)staining of smear and (c) microscopic examination of the stained smear. The leucocytes are themost active and motile constituents of blood. The different types of leucocytes seen in a normalperipheral blood film may be divided in to two broad groups.1. Granulocytes: Granulocytes are the cells containing granules, and a polymorphic nucleus in thecytoplasm. There are three types of granulocytes which derive their names from the stainingreaction of the granules present in the cytoplasm. These cells are(a) Neutrophils or polymorphonuclears(b) Eosinophils and(c) BasophilsNeutrophil (polymorphonuclears): The average diameter is 10 to 12 µm. The nucleus is usually divided into 2-7 lobes. Thecell derives its name from the color of the granules. Neutrophil stains when exposed to acidic, aswell as basic dyes. The number of lobes gives an indication of the age of the cell. Neutrophil aremainly responsible for the protection against infection.Eosinophil: The diameter is about the same as the neutrophil. The cytoplasm contains large, oval orround red-orange (eosinophilic) granules that are stained bright red with eosin, an acid dye. Thenucleus shows fewer lobes, on an average only two. The eosinophils are not markedly motile andonly slightly phagocytic. They play important role in detoxification.12Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 13. Basophil: The diameter is 8 to 10 µm. The nucleus has two to three lobes. It contains fewer coarsegranules, which can be stained with basic dyes, e.g. methylene blue. Basophils are significant inallergic reactions.2. Agranulocytes:Agranulocytes are leucocytes that lack granules and consist of two types of cells: a) Lymphocytes and b) MonocytesMonocyte: The diameter is about 16 to 22 μm. The nucleus is kidney shaped or horse shoe shaped.Sometimes it may be round or oval. It stains pale violet and has fine chromatic arrangement. Thecytoplasm is plentiful and stains pale grayish blue.Lymphocyte: Two forms observed are a) Large lymphocyte b) Small lymphocyte.(a) Large lymphocytes: it is about 12 to 15 µm in diameter. This has abundant clear pale bluecytoplasm and a large round or slightly indented nucleus with dense chromatin.(b) Small lymphocyte: These are small round cells about 10 to12 µm in diameter. It has verylittle blue cytoplasm and often more than just a rim around the nucleus. The nucleus is dark,round and sometimes indented.Clinical significance: Differential count is useful to identify changes in the distribution of white cells whichmay be related to specific type of disorders. It also gives idea regarding the severity of thedisease and the degree of response of the body.Neutrophilia: Increase in the percentage of neutropils is called neutrophilia. All the physiologicalcauses that produce leukocytosis give rise to neutrophilia. The commonest pathological cause isphylogenic bacterial infection. Decrease in neutrophil (Neutropaenia) is observed in infectionssuch as bacterial (typhoid), viral (Measles, influenza etc.) and in other conditions such asanaemias (aplastic, megaloblastic, iron deficiency) and in suppression of bone marrow byvarious drugs and radiation.13Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 14. Lymphocytosis: It may be relative or absoluteRelative lymphocytosis: In this condition the actual number of lymphocytes is unchanged but due to decrease inneutrophils mainly the differential count shows an increase in lymphocytes.Absolute lymphocytosis:It is observed in 1. Children 2. Certain infections such as a. Mumps, b. Cough, c. Measles, d. Influenza, e. Syphilis, f. Tuberculosis, g. Typhoid and other chronic infections 3. Infectious mononucleosis 4. Chronic lymphatic leukemia.Lymphopaenia: In this condition the actual number of lymphocytes is decreased. It is observedin acute stage of infections and in excess irradiation.Eosinophilia: It is defined as an increase in peripheral blood eosinophilic leukocytes to morethan 0.45×109/L (450/μl). It is observed in asthma, hypersensitivity reactions, and parasiticinfections and in chronic inflammatory disease.Monocytosis: It is an increase in the number of monocytes circulating in the blood. It isobserved in tuberculosis, malaria, subacute bacterial endocarditis, typhoid and in kala Azar.Basophilia: It is a condition where the basophil quantity is abnormally elevated (more than 1010basophils per liter of blood). It is usually observed in chronic myeloid leukaemia.Specimen:The blood smears should be preferably prepared immediately after skin puncture or venipuncturebefore mixing with anticoagulant. If EDTA blood is used the smears should be prepared within1to 2 hours after blood drawing. Other anticoagulants do not give satisfactory results. The bloodsmears should be immediately fixed in methanol.14Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 15. PRINCIPLE:The polychromic staining solutions (Wright, Leishman‟s, and Giemsa) contain methylene blueand eosin. These basic and acidic dyes induce multiple colors when applied to cells, methanolacts as fixative and also as solvent. The fixative does not allow any further change in the cellsand makes them adhere to the glass slide. The basic component of white blood cells (i.e.cytoplasm) is stained by acidic dye and they are described as eosinophilic. The acidiccomponents (e.g. nucleus with nucleic acid) take blue to purple shades by the basic dye and theyare called basophilic. The neutral components of the cell are stained by both the dyes.REQUIREMENTS: 1. Blood sample 2. Microscope slides 3. Cedar wood oil 4. Glass Spreader 5. Staining reagent 6. Methanol 7. Distilled water and Miscellaneous. 8. MicroscopeFigure 1c.1: Differential leukocyte staining15Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 16. Normal Values: Types of cell Female/Male Neutrophil 40-75% (mean: 57%) a) Segmented 2 - 6% (mean: 3%) b) Band forms 50-70% (mean: 54%) Eosinophils 1 - 4% (mean: 2%) Basophils 0 - 1% Lymphocytes 20 –45% (mean37%) 2-8% (mean: 6%) MonocytesPROCEDURE: 1. A thin smear is prepared by spreading a small drop of blood evenly on a slideMaking the film: a. Take clean, dry (grease free) slide. b. Transfer a small drop of blood near the edge of the slide. c. Place the spreader slide at an angle of 300 to 350. Pull back the spreader until touches the drop of blood. d. Let the blood run along the edge of the spreader. e. Push the spreader forward to the end of the slide with a smooth movement. f. Dry the blood smear at room temperature. g. Adequate drying is essential to preserve quality of the film. 2. Fixing the Smear: a. The slide should be stained after making the smear. b. Methanol present in the stain fixed the smear. c. If the staining is to be done later, the blood smear must be fixed with methanol for 2 to 3 minutes to prevent distortion of cells.Field Stain: For the rapid screening of blood smears and also for the screening of thick films formalarial parasites, field stain is used. The blood smears are fixed with methanol for 2 to 3minutes before the staining. Following staining solutions are used.16Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 17. Potassium dihydrogen phosphateSOLUTION –A (anhydrous) - 6.25g Azrure-I- 0.5g Methylene blue - 0.8g Disodiumhydrogen phosphate Distilled water -500 (anhydrous) - 5.0gSOLUTION – B Eosin (Yellow, Water soluble) -1.0g Potassium dihydrogen phosphate Disodium hydrogen phosphate (anhydrous)-6.25g (anhydrous) -5.0g Distilled water-500mlPreparation of the solution: 1. The phosphate salts are dissolved first in distilled water and afterwards the stains are added. 2. Azrure I ground in a mortar with phosphate solution. 3. Keep the solutions for at least 24 hours and filter before use. 4. Store it in coping jars.Staining Procedure: 1. Dip the fixed smear in Field Stain „B‟ for 5 seconds. 2. Wash in the tap water. 3. Dip now in Field Stain „A‟ for 5 seconds. 4. Wash in the tap water. 5. Drain the water. 6. Place it vertically against a rack. 7. Examine the dry smear under oil immersion objective exactly in the same way as described earlier (Figure 1c.1).RESULT:CONCLUSION:17Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 18. Experiment: 1(d) Date: A B O - BLOOD GROUPING AND Rh TYPINGAIM: To determine the ABO blood group and Rh typing of the given blood sample.INTRODUCTION: Immunohaematology is an application of the principles of immunology to the study ofred cell antigens and their corresponding antibodies in blood. Karl Landsteiner in 1900discovered the fundamental principles of blood grouping. He discovered ABO-blood groupsystem in human beings. All people of ABO system can be divided into 4 major groups. Theyare „A‟ group, „AB‟ group, „B‟ group and „O‟ group. This depends on the presence of antigen onthe RBCs. A person with blood group "A" will have antigen "A" and person with blood group"B" will have antigen "B" and with “O” group neither “A” or “B” antigen. There are nearly 300blood group systems so far discovered, such as Rh, Diego, Lewis, MNs, Luthera " Kell, Duffyetc.,.A B O Blood group system: The A B O antigens are detectable early in fetal life but are not well developed untilabout 3 years of age. A unique feature of this blood group system is the fact that antibodies tothese A &/or B antigens arise naturally in virtually all individuals. People with only „A‟ antigenson their cells naturally have anti-B in their serum. People with „B‟ Ag on their cells have anti-Ain their serum, those of group O have both anti-A and anti-B and those of group AB have neitherantibody. These facts are known as „Landsteiner‟s rule‟. It is found that the production of free antibodies is an acquired characteristic i.e. the bodymust be antigenically stimulated before it will release the corresponding antibody from the Blymphocytes. It so happens that the glycoprotein specificities A & B found on red cellmembranes are also widely distributed in nature from bacteria to pollens and animal denders.Rh Typing: Rhesus (Rh) blood group system is clinically the second most important blood groupsystem in humans after ABO blood group system. It was discovered in 1940 by Landsteiner &Wiener. Rh antibodies are not naturally occurring but develop only after exposure to Rh antigen.These are of IgG type.PRINCIPLE:18Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 19. To detect the group an antiserum having a high concentration of antibodies against thespecific antigen is employed. This antiserum when mixed with whole blood shows agglutinationif the RBCs possess the specific antigen in its surface e.g. antiserum A will show agglutinationwith RBCs having A antigen and hence persons blood group will be indicated as "A". There aretwo methods of blood grouping.TILE TEST (SLIDE TEST):REQUIREMENTS: 1. Glass slides 2. Match sticks 3. Lancet 4. Anti-A, Anti-B and Anti-D Monoclonal agglutinating antisera.PROCEDURE: 1. Surfaces sterilize the finger for pricking. 2. Prick the finger and put 2 drops of blood on 1 slide & 1 drop on the other slide. 3. Add 1 drop of antisera „A‟, antisera „B‟ & antisera „D‟ separately on drop of blood. 4. Mix well with pricking stick individually and observe for agglutination after few seconds/ minute. 5. Blood group is indicated as agglutination of RBCs with specific antisera. 6. Agglutination of patient is as shown in figure.RESULT: The ABO blood group of the given sample is _____________. The Rh typing of the given blood sample is ___________.19Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 20. Experiment: 2 Date: WESTERN BLOTTINGAIM: To perform western blotting for the given protein sample.INTRODUCTION: Western blotting method was devised by Tocobin in 1979 to find out the protein encodedby the cloned gene in transformed cells. Western blotting is a method that takes advantage of thespecificity of antibodies to specifically stain one protein in complex biological samples. The blotis a membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel isplaced next to the membrane and application of an electrical current induces the proteins in thegel to move to the membrane where they adhere. The membrane is then a replica of the gel‟sprotein pattern, and is subsequently stained with an antibody. Proteins are first separated byelectrophoresis (SDS-PAGE in the current experiment). After the gel has been run, the separatedproteins are transferred to a "filter". The filter is "stained" for the protein of interest byincubation with a specific antibody that is conjugated to a "tag" followed by localization of thetag on the filter. In this experiment we will run samples of Glutathione-S-Transferase (GST) fusionprotein on our SDS gel along with a standard protein marker on a polyacrylamide gel. Followingelectrophoresis, the lysate and marker will be stained to know the electrophoretic mobility of theGST fusion protein, while the other electrophoresed lysate sample will be transferred byelectroblotting onto nitrocellulose membrane. The electroblotted sample will then be detectedusing anti- GST IgG as primary antibody and secondary antibody labeled with Horse RadishPeroxidase (HRP). HRP is then detected using hydrogen peroxide as a substrate andTetramethylbenzidine (TMB) as a chromogen. HRP acts on hydrogen peroxide to releaseoxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited wherever enzymeis present and appears as a blue band on the NC membrane.OBJECTIVE: To learn the technique of Western Blotting this involves the following experiments Electrophoresis of the protein (SDS-PAGE) Electro transfer of protein onto nitrocellulose membrane (Western Blotting) Immunodetection of the transferred protein (Blot development)20Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 21. PRINCIPLE: Western blotting (also known as protein blotting or immunoblotting) is a rapid &sensitive assay for detection & characterization of proteins. Western Blotting technique exploitsthe inherent specific antigens by polyclonal or monoclonal antibodies.SDS-PAGE: Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) is carriedout in discontinuous buffer system wherein the reservoir buffer is of a different pH & ionicstrength from the buffer used to cast the gel. The SDS-polypeptide complexes in the sampleapplied to the gel are swept along by a moving boundary created when an electric current ispassed between the electrodes. After migrating through the stacking gel of high porosity,complexes get deposited in a very thin zone on the surface of the resolving gel. On furtherelectrophoresis, polypeptides get resolved based on their size in the resolving gel.WESTERN BLOTTING: Blotting is transfer of resolved proteins from the gel onto a surface of a suitablemembrane, done commonly by electrophoresis and referred to as electro blotting. The gel isplaced in contact with nitrocellulose membrane which is then sandwiched between filter paper,two porous pads and two plastic supports. The entire set up is then placed in an electrophoretictank containing blotting buffer. The proteins get transferred to the corresponding position on themembrane as resolved on the polyacrylamide gel, forming a mirror image of the gel. Protein ofinterest on the membrane is further located by immunodetection. The band on the nitrocellulose membrane indicates the GST protein detected by the antibody (anti-GST) The position of the band on the membrane indicates its electrophoretic mobility during electrophoresis. The transferred proteins bound to the surface of nitrocellulose membrane are detectedusing immunological reagents. This process is known as immunodetection. All the unoccupiedsites on the membrane are first blocked with an inert protein, a detergent or any other suitableblocking agent. The membrane is then identified using an enzyme-labeled secondary antibodyand a suitable substrate to the enzyme, which results in a colored band on the nitrocellulosemembrane.21Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 22. Figure 2.1: Diagrammatic representation of PAGE (a) (b)Figure 2.2:(a) Membrane is in close contact with polyacrylamide gel containing proteins for electro blotting.(b) At the end of electro transfer, all proteins migrate to nitrocellulose (NC) membrane.22Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 23. Figure 2.3: Immunodetection on blotted NC membrane.Figure 2.4: Notch Base plate & Spacers Assembled for casting gelIMMUNODETECTION: The transferred proteins bound to the surface of nitrocellulose membrane are detectedusing immunological reagents. This process is known as immunodetection. All the unoccupiedsites on the membrane are first blocked with an inert protein, a detergent or any other suitableblocking agent. The membrane is then probed with a primary antibody specific to the protein ofinterest. The Ag-Ab complex formed on the membrane is then identified using an enzyme-labeled secondary antibody and a suitable substrate to the enzyme, which results in a coloredband on the nitrocellulose membrane, referred to as blot development.23Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 24. MATERIALS: The list below provides information about the materials supplied in the kit. The productsshould be stored as suggested. Use the kit within 6 months of arrival.Materials Provdide Store @ Blocking Agent 4 ˚CSDS Separating Gel Mix 4 ˚C 10X Diluent Buffer 4 ˚CSDS Stacking Gel Mix 4 ˚C 10X Assay Buffer 4 ˚CAmmonium persulphate (APS) RT 25X Wash Buffer 4 ˚C10X Reservoir Buffer RT Primary Antibody 4 ˚CSample Loading Buffer 4 ˚C 1000X HRP Conjugate 4 ˚CProtein Marker 4 ˚C 10X TMB/H2O2 4 ˚CProtein Sample 4 ˚C Nitrocellulose(NC) membrane with RT Filter Paper20X Blotting Buffer Component A RT Ezee Blue RT20X Blotting Buffer Component B RTMaterials required:1. Equipment: Gel rocker (optional)2. Glassware: Conical flasks, Measuring cylinder, Petri dishes, staining tray.3. Distilled water4. Other equipment: Micropipettes, Tips, Water bath.Note:• Read the entire procedure before starting the experiment.• Wear gloves while handling the gel and membrane.• Prepare 1X TMB/H2O2, 1X secondary antibody, blocking buffer just before use.• Resuspend an aliquot of APS in 1 ml of distilled water. Store at 4°C. Use within 2 months.• Destaining step is not required on staining the gel with Ezee blue stainer (Coommassie brilliantblue).Prepare the reagents as indicated below before starting each experiment:Preparation of 1X Assay Buffer: To 2 ml of 10X assay buffer add 18 ml of distilled water toget 20 ml of 1X assay buffer.24Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 25. Preparation of Protein Sample: Resuspend protein sample in 25 μl of distilled water.Preparation of Primary Antibody: Resuspend an aliquot of primary antibody in 1 ml of 1Xassay buffer. Transfer to a test tube and make up the volume to 10 ml with 1X assay buffer.Preparation of Blotting Buffer: Mix 25 ml each of blotting buffer components A and B with450 ml of distilled water.Preparation of 1X Reservoir Buffer: To 25 ml of 10X Reservoir Buffer add 225 ml of distilledwater to get 250 ml of 1X reservoir buffer.Preparation of 1X Secondary Antibody: Pipette 10 μl of 1000X HRP conjugate and add 9.90ml of 1X assay buffer. Mix thoroughly.Preparation of Diluent Buffer: Dilute 1 ml of 10X diluents buffer to 10 ml with distilled waterjust before use.Preparation of Blocking Buffer: Weigh 300 mg of blocking agent, suspend in 10 ml of 1Xdiluent buffer.Kit description: In this kit, bacterial lysate having Glutathione-S-Transferase (GST) fusion protein isprovided, which will be electrophoresed in duplicates along with a standard protein marker on apolyacrylamide gel. Following electrophoresis, the lysate and marker will be stained to know theelectrophoretic mobility of the GST fusion protein, while the other electrophoresed lysate samplewill be transferred by electro blotting onto nitrocellulose membrane. The electro blotted samplewill then be detected using anti-GST lgG as primary antibody and secondary antibody labeledwith Horse Radish Peroxidase (HRP). HRP is then detected using hydrogen peroxide as asubstrate & Tetramethylbenzidine (TMB) as chromogen. HRP acts on hydrogen peroxide torelease oxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited whereverenzyme is present & appears as a blue band on the NC membrane.25Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 26. PROCEDURE:DAY-1SDS – PAGE 1. Assemble the plates for casting gel: 2. Clamp the assembly of plates to fix it in a gel casting apparatus. Ensure the assembly is leak proof by filling water between the plates. Silicon grease can be applied to spacer to make a water- tight seal. 3. Add 50µl of APS solution to 5ml of SDS separating gel mix & pour the gel solution between the plates till the level is 2cm below the top edge of notched plate. 4. Add 200 to 250ml of water to make the surface even. 5. After the gel is set (approximately 20-30 min.), was the top of the separating gel with distilled water & drain off the water completely. 6. Add 20 µl of APS solution to 2ml of stacking gel mix and pour directly onto the polymerized separating gel. 7. Insert the comb into the gel solution carefully without trapping any bubbles, about 1cm above the separating gel. The stacking gel will set in approximately 10min. 8. Add 25 µl of Sample Loading Buffer to Protein sample. 9. Add 25 µl of Sample Loading Buffer to 25 µl of protein marker. 10. Place it in a boiling water bath for 5 minutes. 11. After the stacking Gel has set, carefully remove the comb and the bottom spacer. Wash the wells immediately with distilled water to remove non-polymerized acryl amide. Fill the bottom reservoir with 1X Reservoir Buffer and carefully fix the plate to the apparatus without trapping any air bubbles between the buffer and the bottom of the gel. Fix the plates to PAGE apparatus. Fill the top reservoir with 1X Reservoir Buffer. 12. Load 30 µl protein marker in well 1, 40 µl of protein sample in well 2 & 10 µl of protein sample in well 4. Note down the order of loading. Connect the cords to the power supply according to the convention red: anode, black: cathode. 13. Set voltage at 100 V & switch on the power supply. 14. When the dye front comes to 0.5 cm above the bottom of the gel, turn off the power. This will take approximately 1 to 1.5 hours. 15. Remove the gel plates and gently pry the plates apart using a spatula or similar tool, not at the notch. 16. Transfer the gel to a tray containing water; wash the gel for 1-2 minutes at room temperature. 17. Decant water, cut the gel along lane 3. 18. Transfer lane 4 i.e., Protein Sample in 10ml of blotting buffer taken in a Petri dish. Keep at room temperature for 10 minutes. Following incubation, proceed for electro blotting as described in step 22. 19. To the gel piece (lanes 1&2) add minimum of 20ml water. 20. Decant the water; add minimum 20ml of Ezee Blue Stain. Stain at room temperature for 1-2hours.Note: For uniform staining & washing, place the tray on a rocker or shake intermittently every10 to 15 minutes. 21. Decant the staining solution add minimum quantity of water cover the gel.26Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 27. Note: Cover the tray & leave it overnight at room temperature.ELECTRO BLOTTING 22. Assemble the blotting sandwich within the blotting cassette as shown in the figure. Take care to avoid air bubbles between the gel and NC membrane. 23. Insert the cassette into the apparatus filled with blotting buffer and connect blotting unit to power supply as per the convention. 24. Red: anode, black: cathode 25. Electrophoreses the sample at 50 V for 2 hours for blotting to occur. 26. Remove the NC membrane gently from the cassette and place the membrane in 10ml of freshly prepared blocking buffer taken in a Petri dish. Leave it overnight at 4oC.DAY: 2IMMUNODETECTION: 27. Discard Blocking Buffer. 28. Immerse blot in 10ml of Primary Antibody Solution & mix gently for 30 minutes. Discard the Primary Antibody Solution 29. Wash the blot by immersing in 10ml Wash Buffer for 3-5 minutes. Repeat the wash two times. Discard the buffer each time. 30. Immerse the blot in 10ml of 1X HRP labeled Antibody. Mix gently for 30 minutes. Discard the HRP labeled Antibody. 31. Wash the blot by immersing in 10ml Wash Buffer for 3-5 minutes. Repeat the wash process four to five times. Discarding the buffer each time. 32. Immerse the washed blot in 10ml of Substrate Solution, mix gently for 5-10 minutes, within this time colored band will appear. 33. Remove the blot; wash with distilled water, discard & dry. Note: Although the colored band fades with time, the rate of color loss can be retarded if the blots are kept in dark. 34. Compare the SDS-Polyacrylamide gel with the developed NC membrane.OBSERVATION: 35. On staining SDS-Polyacrylamide gel, different proteins will appear as dark blue bands against a light blue background. 36. On immunodetection, a single blue band will be observed on NC membrane.INTERPRETATION: On electrophoresis of bacterial lysate on SDS-PAGE, many bands indicating the differentproteins present in the crude sample are seen. A predominant band among these is that of GSTfusion protein corresponding to 26 kD protein of the marker. Following transfer &immunodetection, one observes a predominat band corresponding to GST protein bound to anti-27Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 28. GST antibody. However, few light bands may be seen indicating the proteins with which anti-GST antibody cross reacts.RESULT:CONCLUSION:28Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 29. Experiment: 3 Date: SINGLE RADIAL IMMUNODIFFUSION ASSAYAIM: To perform the single radial immunodiffusion assay.INTRODUCTION: Single Radial Immunodiffusion (RID) is used extensively for the quantitative estimationof antigens. RID technique used for estimating the concentration of plasma protein likeimmunoglobulin and also for the estimation of antibody concentration in sera samples.PRINCIPLE: When antigen diffuses radially forming a concentration gradient disk in gel containinguniformly distributed antibody, the Ag-Ab complex precipitates in the gel where the diffusingantigen reaches equivalence with the antibody concentration presenting the gel. The precipitateformed in the gel appears as an opaque ring. The ring diameter is directly proportional to theantigen concentration.The Technique: In this method antibody is incorporated before the gel is made. Thus, the antiserum isuniformly distributed throughout the agarose gel. Antigen is then allowed to diffuse from wellscut into the gel. Initially, as the antigen diffuses out of the well, its concentration is relativelyhigh and it forms soluble antigen-antibody adducts. However, as it diffuses farther and fartherfrom the well, its concentration decreases. When its concentration becomes equivalent to that ofthe antibody in the gel; antigen-antibody precipitates and a precipitin ring is formed. Greater theconcentration of antigen greater is the diameter of the precipitin ring. Thus, by running a range ofknown antigen concentration on the gel and by measuring the diameter of their precipitin rings, acalibration graph can be constructed. The antigen concentration of unknown samples run on thesame gel can then be determined by simple interpolation, having measured the diameters of therespective precipitin rings.REQUIREMENTS: 1. Agarose 7. Glass Plates 2. 10X assay Buffer (To be diluted 10 8. Template times before use.) 9. Semi log Graph Sheet 3. Standard Antigens. 10. Distilled Water 4. Test Antigen 11. Micropipette. 5. Antiserum 12. A box to keep the gel plate in moist 6. Gel Punch with syringe chamber29Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 30. Precautions: The glass plate should be wiped grease free with cotton for the even spreading of agarose. The wells should be cut neatly without rugged margins to get a perfect ring of precipitation. The antiserum should be added to the agar only after it cools to 550C. Higher temperature will inactivate the antibody. After the addition of antiserum to the agar proper mixing is essential for uniform distribution of antibodyReconstitutions: Reconstitute each vial containing lyophilized antigen and antisera with 1ml of 1X assaybuffer. Mix it well and allow it to stand for 30 minutes.PROCEDURE: 1. 1gm Agarose is dissolved in 100 ml of 1X assay buffer by heating. 2. The solution is then allowed to cool to 550C and 120 µl of antiserum mixed with 6ml of the solution. 3. The agarose solution containing the antiserum is poured onto a grease free glass plate that had previously been set on a horizontal level surface; Cool to form a gel. 4. Using a gel punch, wells of 4mm diameter are cut into the gel as show figure1. 5. Add 20µl of the given standard antigens and samples to the wells as shown in figure (two test samples provided with the kit); Avoid overflow. 6. The gel plate is left in a box containing wet cotton and incubated overnight at room temperature. 7. The diameter of the disk can be measured by marking the edges of the circle. 8. A standard graph is constructed by plotting the diameters of the disk against the concentration of antigen in a semi log graph sheet as shown in figure 2. The concentration of unknown is then determined by reading the concentration against the ring diameter.30Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 31. Figure 3.1: Pattern of wells for Antigen and tests. 1 2 Standard Antigen A-0.25mg/ml Standard Antigen B-0.5mg/ml Standard Antigen C-1.0mg/ml 3 4 Standard Antigen D-2.0mg/ml Test Antigen –1 5 6 Test Antigen –2Observation table: Sr. No. Std Conc. mg/ml Ring Diameter mm Test Conc. mg/ml. 1 0.25 6 2 0.5 8 3 1.0 10 4 2.0 12 5 Test-1 ? 6 Test-2 ?RESULT:CONCLUSION:31Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 32. Experiment: 4 Date: OUCHTERLONY DOUBLE DIFFUSION ASSAYAIM: To determine the titter value of the antiserum using ouchterlony double diffusion assay.INTRODUCTION: Interaction between antigen (Ag) and antibody (Ab) in the molecular level forms thebasis for several techniques that are useful in modern day scientific studies and in routine clinicaldiagnosis. These techniques are either based on use of labeled reagents, a tracer or byimmunoprecipitation. Ouchterlony double diffusion (ODD) is one of the simplest techniqueextensively used to check antisera for the presence and specificity of antibodies for a particularAg. This technique is also known as double immunodiffusionPRINCIPLE: The technique is based on the phenomenon that when Ag and Ab mix in optimalproportion, the Ag-Ab complex precipitates. The precipitate formed in agarose gel appears as anopaque line.The Technique: In this technique, solutions of Ag and Ab placed in adjacent wells cut in agarose geldiffuse radially. Initially as the Ag and Ab diffuse out of the respective wells, the concentrationsare relatively high nearer to the well. However as they diffuse further from the wells, theconcentration decreases. At one point their concentrations become equivalent and Ag-Abcomplex precipitates to form a precipitin line at this point. The relative position of the precipitinline yields an estimate of Ag (or Ab) concentration, i.e. more concentrated the immunoreactionssolution, the precipitin line formed is farther from the well.REQUIREMENTS: 1. Agarose 9. Tubes 2. 10X Assay buffer 10. Distilled water 3. Antigen 11. A box to keep the gel plate in moist 4. Test antisera chamber. 5. Glass plates 6. Gel punch with syringe 7. Template 8. Micropipettes32Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contribution tostudents community.
  • 33. PRECAUTIONS:1. The glass plates should be wiped grease free with cotton for the even spreading of theagarose.2. Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.RECONSTITUTIONS: Reconstitute each vial containing lyophilized antigen and antisera with 1 ml of 1 Xassay buffer. Mix it well and allow it to stand for 30 minutes.PROCEDURE: 1. Prepare 1 % agarose solution in 1 X assay buffer by heating to dissolve the agarose completely. 2. Cool the solution to about 55-60°C and pour 5ml of the solution onto grease free glass plate that had previously been set on horizontal level surface. Allow the gel to set for 20-30 minutes. 3. Serially dilute the test antisera up to 1:32 dilution. Take 20 μl of 1 X assay buffer in five eppendorf tubes. To the first tube add 20 μl of test antiserum mix well. The dilution of antiserum in the first tube is 1:2. From the first tube take out 20 μl of 1:2 diluted antiserum and add to the second tube. The dilution in the second tube is 1:4; repeat the dilution up to the fifth tube as shown in Figure 1. Add 20μl test antiserum in first well. 4. Keep the gel plate on the template provided. Punch wells in the gel with the help of a gel punch corresponding to the marking on the template with gentle suction so that the wells are cleanly formed as the resultant agarose plugs are sucked out. 5. Add 20 μl of the Ag into the centre well and 20 μl of each of neat, test serum, 1 :2, 1:4, 1:8, 1:16, 1:32, dilutions into the surrounding wells as shown in the following figure.33Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contributionto students community.
  • 34. Figure 4.1: The pattern of wells and additions of Ag and test serum dilutions 6. Keep the plate in a moist chamber overnight at room temperature. 7. After incubation observe for opaque precipitation lines between the Ag and antisera wells.INTERPRETATION:1. Precipitin line indicates presence of Ab in the antiserum to the Ag added.2. The titter of the antiserum is the highest dilution showing the precipitin line. For exampleif the line of precipitation is seen up to 1:16 dilution, the titter of the antiserum is consideredas 1:16.RESULT:DISCUSSION:34Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contributionto students community.
  • 35. Experiment: 5 Date: ENZYME LINKED IMMUNOSORBENT ASSAY (DOT ELISA)AIM: To perform sandwich Dot ELISA test for antigen.INTRODUCTION: ELISA is being extensively used as a tool in research as well as analytical/diagnosticlaboratories. The specificity, sensitivity and ease to perform these techniques have madethese methods popular. It is therefore necessary to have hands on experience of this techniquealong with the theoretical knowledge. The kit is designed to understand the principle andworking of a direct sandwich ELISA test using human IgG as a model. Such methods can beused for estimating any type of multivalent antigen using the appropriate antibodies. The kitis safe to handle, it does not involve handling of any type of hazardous materials. It is easy toperform the test and understand the reactions.PRINCIPLE: In direct sandwich Dot-ELISA the antigen is sandwiched directly between twoantibodies. For this to happen, the two antibodies used must be able to react (bind) with twodifferent epitope on the same antigen. One antibody is immobilized on a solid support and theother one is linked to an enzyme. Then the bound antigen is allowed to react with the secondenzyme linked antibody. Then the substrate for the linked enzyme is added. The enzymelinked conjugate oxidizes the substrate. The oxidized substrate gives a blue color spot (hencethe name Dot-ELISA). The enzyme activity (color intensity of the spot) is directlyproportional to the antigen concentration. TMB (Tetramethyl benzidine) / H2O2 (horseradish peroxidase) is used for thedetection of HRP enzyme linked conjugate. Here H2O2 is the substrate and TMB is achromogen. HRP act on H2O2 to release oxygen, which oxidizes the TMB to TMB oxide.This TMB oxide is deposited whenever enzyme is present and appears as a blue spot. That is35Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contributionto students community.
  • 36. If the test sample does not contain the antigen specific to the antibody, there will beno enzyme reaction and no spot develops.REQUIREMENT:1. ELISA strip for human IgG test.2. Assay buffer at 10x concentration.3. Goat anti human IgG peroxidase conjugate (antibody-HRP)4. TMB/H2O2 10 x concentration (substrate)5. Serum samples for human IgG test 3 x 0.5ml.6. Micropipette.7. Distilled water.8. Test tubes of size 10 x 75 mm or 1.5 ml eppendorf tubes.PRECAUTIONS: Reconstitute test samples with 0.5 ml distilled water and store at 4 - 8˚c. Prepare 1x assay buffer by diluting 10x assay buffer 10 times with distilled water. Use the diluted buffer on the same day. Do not contaminate reagents with each other. Dilute only required amount of reagents. Do not leave the reagents at room temperature. Ensure all three zones on strip are immersed in solution.PROCEDURE:1. To one drop (50μl) of the sample add 1ml of 1x assay buffer and insert an ELISA strip. Keep for 20 minutes at room temperature2. Wash the strip by dipping it in 1 ml of 1x assay buffer for about 5 minutes and replace the buffer. Repeat two more times.3. In 1ml 1x assay buffer add 10μl antibody-HRP conjugate and dip the strip. Keep for 20 minutes.4. Wash the strip as in step 2.5. In a fresh tube take 0.1 ml TMB/H2O2, 10X concentrate, 0.9 ml distilled water and dip the strip.6. Observe the strip in 10-20 minutes for the appearance of blue/gray spot on the strip.36Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contributionto students community.
  • 37. 7. Rinse the strip with distilled water.INTERPRETATION:1. A spot on the lower portion and no spot on the upper portion indicate the proper performance of the test.2. A spot in middle portion indicates presence of the antigen in the sample.3. Intensity of the spot is proportional to antigen concentration in the sample, i.e. human IgG.RESULT:CONCLUSION:37Prepared & compiled by,K.P.Senthil Kumar.,MSc.,M.phil.,ADABFor practical use and not for any other commercial purposes; All the scientific community members are acknowledged for their contributionto students community.