POLYMERASE CHAIN REACTION

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Experiment 1: Agglutination Technique: ABO typing

Aim: Introduction: In 1900 “Karl Landsteiner” first reported the presence of two antigens namely “A” and “B” on the surface of human red blood cells . Based on this discovery he divided human blood cells in to three groups like A,B,O. In 1902, Decastell and Sturil recognize the existence of a fourth group “AB”. The “Rh” factor antibody was identified by Leving and Stetson. About 95% of individuals among Indian population and 85% individual of Caucasian origin possess D(Rh) antigen on their erythrocytes. Human red blood cells are classified as Rh +ve or Rh -ve depending upon the presence or absence of this antigen on their surface. Principle: Human red blood cells possessing A and B antigen will agglutinate in the presence of antibody directed towards the respective antigen. Agglutination of RBC with anti-A monoclonal or anti-B monoclonal is a positive test result and indicates the presence of corresponding antigen A and B respectively. Absence of agglutination of RBC with anti-A monoclonal or anti-B monoclonal is a negative test result and indicates the absence of the corresponding antigen and is termed as blood group “O”. Human RBC possessing “D (Rh)” antigen is agglutinated by the antibody directed against “D (Rh)” antigen. Materials Required:

 Monoclonal Antibodies ( Anti-A, B and D)  Blood Lancet  Alcohol swabs  Tooth picks  Sterile cotton balls  Clean glass slide  Ice tray  Biohazard disposal container

Procedure: 1. Set the table with all the materials required. Remember to place the Monoclonal

Antibody (Mab) kit in an Ice tray.

2. Open an Alcohol swab, and rub it at the area from where the blood will be sampled

(finger tip). (Discard the swab)

3. Open the Lancet cover, put pressure at the tip of the finger from where blood will be

sampled (maintain it). Prick the finger tip with the opened Lancet.(Discard the Lancet)

4. As blood starts oozing out, make 1 drop fall on the three depressions of the glass slide.

(in clinical setup, there will be a fourth well used as a control).

5. Place a cotton ball at the site where it was pricked. Using the thumb, put pressure on the

area to stop blood flow.

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6. Take the Anti-A (blue) bottle, resuspend the content and use the dropper to place a drop

of the Mab in the 1st spot. Place the bottle back in ice.

7. Take the Anti-B (yellow) bottle, resuspend the content and use the dropper to place a

drop of the Mab in the 2nd spot. Place the bottle back in ice.

8. Take the Anti-D (colorless) bottle, resuspend the content and use the dropper to place a

drop of the Mab in the 3rd spot. Place the bottle back in ice.

9. Take a tooth pick and mix the content in each well. Discard the tooth pick after using in

one well (take a new one for the next well).

10. After mixing, wait for a while to observe the result. Result: The given blood group was identified as…………………………. Inference: Clinical Applications: Further Reading:

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ABO blood grouping system: According to the AB0 blood group system there are four different kinds of blood groups: A, B, AB and O (null).

Red blood cell t).'pe Antibodies in plasma Anti cell ens in lood Group A Anti-B A antigen Group B B antigen Group AB None A and B antigens Group O Anti-A and Anti-B None

Rh factor

Rh (Rhesus) factor is found on the RBC's surface in most people. Like A and B, this is also an antigen and those who have it are called Rh+. Those who lack the antigen on the surface of RBCs are called Rh-. A person with Rh- blood does not have Rh antibodies naturally in the blood plasma. But a

person with Rh- blood can develop Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies (as the immune system is triggered by the presence of an unknown antigen in the system). A person with Rh+ blood can receive blood from a person with Rh- blood without any problems. Inheritance of Blood Groups:

Rh antigen Rh antibody

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Blood groups for each individual are determined by genes or alleles (small packets of information in cells contained in the DNA) which are inherited from both parents. Genes for the Rh-ve and O groups from one parent are masked (i.e., they are recessive) by the presence of Rh+ve and A or B genes from the other parent. That is, O and Rh negative genes only produce an effect when there is a "double dose" of such genes, i.e., one from each parent (homozygous condition). Thus, people who are apparently A or B Rh+ve may also carry genes for the O and Rh-ve blood groups which can be inherited by their children. Principle behind blood tests: Blood clumping or Agglutination observation. Compatibility between the blood groups of donor and recipient determines the success of a blood transfusion. The AB0 and Rh blood groups are looked at while conducting the test. In a diagnostic lab, Monoclonal antibodies are available for A, B and Rh antigen. Monoclonal antibody against Antigen A (also called Anti-A), comes in a small bottles with droppers; the monoclonal suspension being BLUE in colour. Anti-B comes in YELLOW colour. Anti-D (monoclonal antibody against Rh) is colourless. All the colour codes are universal standards. When the monoclonal antibodies are added one by one to wells that contain the test sample (blood from patient), if the RBCs in that particular sample carry the corresponding Antigen, clumps can be observed in the corresponding wells. A drop of blood is left without adding any of the antibodies; it is used as a control in the experiment. The monoclonal antibody bottles should be stored in a refrigerator. It is recommended to tilt the bottle a couple of times before use in order to resuspend the antibodies that have settled at the bottom of the bottle.

Rh+ Rh-

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Experiment 2: Differential counting of WBC

Aim:

Principle: White blood cells (WBC) are a heterogeneous group of nucleated cells that can be found in circulation for at least a period of their life. Their normal concentration in blood varies between 4000 and 10,000 per microliter. They play a most important role in phagocytosis and immunity and therefore in defense against infection. A white blood cell differential is a medical laboratory test that provides information about the types and amounts of white blood cells in a person's blood. The test, which is usually ordered as part of a complete blood count (CBC), measures the amounts of the five normal white blood cell types – neutrophils, lymphocytes, monocytes, eosinophils and basophils – as well as abnormal cell types if they are present. These results are reported as percentages and absolute values, and then they are compared against reference ranges to determine whether the values are normal, low, or high. Changes in the amounts of white blood cells can aid in the diagnosis of many health conditions, including viral, bacterial, and parasitic infections and blood disorders such as leukemia. To determine the differential, a drop of blood is thinly spread over a glass slide, air dried, and stained with a Romanofsky stain, most commonly the Wright or May-Grunewald-Giemsa technique. Materials Required:

 Blood Lancet  Alcohol swabs  Tooth picks  Sterile cotton balls  Clean glass slide  Wright stain or Leishman stain or Giemsa stain.  Microscopes  Hemocytometer

Procedure:

1. Place a drop of blood from the finger about 2mm in diameter in the central line of a

slide about 1-2 cm from one end.

2. The spreader is placed at an angle of 40 degrees to the slide and then moved back to

make contact with the drop.

3. The drop should spread out quickly along the line of contact of the spreader with the

slide. The moment this occurred the film should be spread by a rapid, smooth, forward, movement of spreader.

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4. The drop should be of such size that the film is 3-4 cm in length. 5. The film should be dried rapidly. 6. A good blood film preparation will be thick at the drop end and thin film at the

opposite end. The thickness of the spread when pulling the smear is determined by the:

 Angle of spreader slide (The greater the angle, The thicker and Shorter the

smear).

 Size of the blood drop.  Speed of spreading (Slowly the thicker).

7. The thin smears are used for describing blood cell, the thick smear are used for

detecting malarial parasites.

8. Pour the stain on the smear and leave for 1-2 minutes. 9. Wash the stain, air dry the slide and observe the cells under the microscope. 10. For differential leukocyte counts choose an area where the morphology of the cells is

clearly visible.

11. Do differential count by moving the slide in area including the central and peripheral

and the smear.

12. A total of 200 cells should be counted in which every white cell seen must be

recorded in a table under the following heading: Neutrophile, Eosinophile, Basophile, Lymphocyte, and Monocyte then find the percentage of each type.

Results: Type of WBCs Number of cells found in the Smear

Percentage of Cells

Neutrophile

Eosinophile

Basophile

Lymphocyte

Monocyte

Inference:

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Clinical Applications:

White Blood Cells (9) Eosinophil Neutrophil Lymphocyte Basophil Monocyte #231140234

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Further Reading: Neutrophile

 Neutrophile: is a cell with acidophilic cytoplasm and fine

pinkish red granules ,the nucleus is usually lobulated (3-5) lobes, connected by thin chromatin filament .

 Percentage 65-70% from blood.  Number 3000-6000 \ mm3.  Disease case: Neutrophilia: increase Neu. In blood

ex: Inflammation, intoxication.

Eosinophile

 Eosinophile: acidophilic cell ,usually larger than the Neu.

,it is cytoplasm contain bright red granules or orange the nucleus consist usually 2 lobes or (bilobes).

 Per. 2-4 % from blood.  Num. 150-300 \ mm3.  Eosinophilia :ex: Allergy, Asthma

Basophile

 Basophile: it is a small cell granules are black or blue in

color and over cover most of the cell even the nucleus or (S-shaped).

 Percentage 0-1 % from blood  Num. 0-100 \mm3  Bosophilia : ex: Wounds

Lymphocyte

 Lymphocyte: it is also small cell ,the nucleus cover most

of the cells &it is round dark violet in color ,the cytoplasm is usually blue in color with no granules .

 Percentage. 32-40 % from blood.  Num. 1500-4000 \ mm3.  Disease Case: Lymphocytosis: increase lymphocyte in

blood ex: Brucellosis and Tuberculosis.

Monocyte

 Monocyte is the largest mature leukocyte the nucleus is

usually kidney shape or horse shape.

 Percentage. 5-8 % from blood.  Numbers. 300-600 \ mm3.  Monocytosis: ex: Malaria, Typhus.  Function of WBC: it is the first defense line of the body

against bacterial infection.

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Experiment 3: Isolation of lymphocytes from peripheral blood

Aim: Principle: Lymphocytes are present in blood, peritoneal exudates or lymphoid organs mixed with other cells. Human lymphocytes can be isolated most readily from peripheral blood. The Lymphocyte Separation Medium (LSM) is an iso-osmotic, low viscosity medium containing polysucrose and sodium diatrizoate adjusted to a density of 1.0770 +/- 0.0010 g/ml. This medium offers a quick and reliable method for the simple isolation of human mononuclear cells and lymphocytes from defibrinated EDTA human blood. Differential migration following centrifugation results in the formation of several cell layers. Mononuclear cells (lymphocytes and monocytes) and platelets are contained in the banded plasma-LSM interphase due to their density, and the pellet that is formed contains mostly erythrocytes and granulocytes, which have migrated through the gradient to the bottom of the tube. Most extraneous platelets are removed by low speed centrifugation during the washing steps. Lymphocytes or other mononuclear cells (monocytes or mesenchymal stromal cells) or granulocytes are recovered by aspirating the plasma layer and then removing the cells. Excess platelets, LSM and plasma can then be removed by cell washing with isotonic diluent buffer. Materials: Phosphate buffer saline (PBS), Blood sample, Ficoll-Hypaque (LSM: Lymphocyte Separation Medium), Heparin/EDTA, centrifuge, Micropipettes, Sterile tips,

Haemocytometer, Microscopes. Procedure:

1. Collect 5 ml of blood in a centrifuge tube containing anticoagulant, using sterile

syringe and needle.

2. Mix immediately by inverting or vigorously shaking the tube for EDTA to be

uniformly distributed.

3. Dilute the blood by adding 5 ml of diluent buffer. 4. Take 2.5 ml of HiSep LSM 1077 in a new 15 ml centrifuge tube. Overlay the LSM

with 7.5 ml of diluted blood.

5. Centrifuge at 2300 rpm for 30 minutes in a fixed angle rotor. 6. Using a clean glass pasteur pipette carefully remove the lymphocyte layer in a new

collection tube.

7. Add 5 ml of diluent buffer to the lymphocyte layer. Mix by gentle pipetting and

centrifuge at 1900 rpm for 10 minutes.

8. Discard the supernatant, prepare the smear of isolated lymphocytes, stain and observe

under the microscope.

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Result: Inference: Clinical Applications:

Centrifugation Blood Ficoli-paque PLUS plasma Lymphocytes, monocytes, platelets Ficoll-paque PLUS granulocytes, erythrocytes

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Experiment 4 : Bacterial Agglutination reaction-Widal test (Tube / slide agglutination) Aim: Principle: Widal test is a serological method to diagnose enteric fever or typhoid which is caused by the infection with pathogenic microorganisms like Salmonella typhi, Salmonella paratyphi A, B and C. This method of diagnostic test is based upon a visible agglutination reaction either in a test tube or on a slide between antibodies of patient serum and antigens specifically prepared from Salmonella sp. The organisms causing enteric fever possesses two major antigens namely somatic antigen, O and a flagellar antigen, H along with another surface antigen, Vi. During infection antibodies are produced in patient’s sera against Salmonella typhi O and H and Salmonella paratyphi AH and BH antigens. Antigens specifically prepared from this organism are used in the agglutination test to detect the presence of antibodies in patients’ sera which are elucidated in response to infection by these bacteria. There are some agglutinins that are produced in the patient’s serum during the fever period, which react with somatic antigen O of Salmonella typhi, A or B of Salmonella paratyphi and then with flagellar antigen H which is common in most of the Salmonella species. In this test four specific antigen suspensions are used e.g. Salmonella typhi (H antigen), Salmonella typhi (O antigen), Salmonella paratyphi - A and Salmonella paratyphi - B. If agglutination occurs with O antigen then it is considered positive for Salmonella typhi. If agglutination occurs in A or B antigen then it is confirmed as positive for Salmonella paratyphi. Agglutination will occur in H antigen circle for all the cases of antigens like O, A, and B. Salmonella species are characterized by three antigens present on the cell.

Antigenic Structures of Salmonellae Used in Serologic Typing ViAg O Ag

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Materials Required:

 Salmonella typhi ‘O’ Antigen  Salmonella typhi ‘H’ Antigen  Salmonella paratyphi ‘AH’ Antigen  Salmonella paratyphi ‘BH’ Antigen  Positive control  Negative control  Test Serum Sample  Glass Slide  Disposable Mixing Sticks  Micropipettes, Tips,  Gloves and Masks.

Procedure:

1. Mark the circles of slides as PC (Positive control), NC (Negative control), O, H, AH,

BH as per antigen solutions used for testing

2. Add a drop (25 µl) of positive control into the circle marked as PC of given glass

slide.

3. Then add 25 µl of negative control into the reaction circle marked as NC. 4. Add 25 µl of test sample into each reaction circle labeled as O, H, AH, BH according

to given antigen solution.

5. Add 25 µl of Antigen solution of Salmonella typhi 'H' into PC and NC circle each.

Mix well using new mixing stick for each circle.

6. To circles labeled as O, H, AH, BH in which test samples have been added, add

antigen solutions of Salmonella typhi 'O', Salmonella typhi 'H', Salmonella paratyphi 'AH' and Salmonella paratyphi 'BH', respectively.

7. Mix the content of each reaction circle uniformly with separate mixing stick. 8. Rock the glass slide gently (approximately for one minute) and observe for

agglutination.

Test Sample + 'O' Antigen o Test Sample + Test sample + 'H' Antigen NC Test sample + Negative control Test sample + 'AH' Antigen AH PC Test sample + Positive control

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Result: Inference: Clinical Applications: Further reading: O Antigen: This is a somatic antigen and is present on the outer membrane of the cell. Its specificity is determined by the nature of the repeating units in the outer O-polysaccharide chain. Somatic antigens are heat stable, alcohol resistant and forms compact and granular clumps when mixed with O antisera. Vi antigens: This is a virulence antigen which is a capsular polysaccharide that overlays the O antigen. This capsule is not necessary for infection but it increases the infectivity by making it less detectable by the body’s immune system. It is heat labile and can be detected using Vi antisera. Vi antigen can interfere with O antigen testing. H Antigens: This is a heat labile flagellar antigen which is inactivated both by boiling and alcohol. H antigens rapidly form fluffy clumps when treated with the corresponding antisera. H antigen induces rapid formation of corresponding antibodies as it is strongly immunogenic.

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Experiment 5 : Radial Immunodiffusion (RID) Aim: Principle: Single Radial Immunodiffusion, also known as Mancini technique, is a quantitative immunodiffusion technique used to detect the concentration of antigen by measuring the diameter of the precipitin ring formed by the interaction of the antigen and the antibody at optimal concentration. In this method the antibody is incorporated into the agarose gel whereas the antigen diffuses into it in a radial pattern. Thus the antibody is uniformly distributed throughout the gel. Single Radial Immunodiffusion is used extensively for the quantitative estimation of antigen. Here the antigen antibody reaction is made more sensitive by the addition of antiserum into the agarose gel and loading the antigen sample in the well. As the antigen diffuses into the agarose radially in all directions, it’s concentration continuously falls until the equivalence point is reached at which the antigen concentration is in equal proportion to that of the antibody present in the agarose gel. At this point ring of precipitation (‘precipitin ring’) is formed around the well. The diameter of the precipitin ring is proportional to the concentration of antigen. With increasing concentration of antigen, precipitin rings with larger diameter are formed. The size of the precipitin rings depend on

 Antigen concentration in the sample well  Antibody concentration in the agarose gel  Size of the sample well  Volume of the sample

Thus, by having various concentrations of a standard antigen, standard curve can be obtained from which one can determine the amount of an antigen in an unknown sample. Thus, this is a quantitative test. If more than one ring appears in the test, more than one antigen/antibody reaction may have occurred. This could be due to a mixture of antigens or antibodies. Materials Required: Standard Antigens, Antiserum, Agarose, 1X TBE, Conical flask, Measuring cylinder, Beaker, Distilled water, 70% alcohol, Microwave. Procedure: 1. Prepare 10 ml of 1% agarose gel 2. Add 80 l of antiserum Mix well for uniform distribution of the antibody. 3. Pour agarose solution containing the antiserum on to a grease free glass plate placed on a horizontal surface. Allow the gel to set for 30 minutes. 4. Place the glass plate on the template provided. 5. Punch wells with the help of gel puncher corresponding to the markings on the template. Use gentle suction to avoid forming rugged wells

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6. Add 10 l of the given standard antigen and test antigen samples to the wells. A. Standard Antigen A (3.75 mg/ml) B. Standard Antigen B (7.5 mg/ml) C. Standard Antigen C (15 mg/ml) D. Standard Antigen D (30 mg/ml) E. Test Antigen 1 F. Test Antigen 2

Ab in get Ag Concentration

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Result: Inference: Clinical Applications:

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Experiment 6: Ouchterlony Double Diffusion (ODD) Aim: Principle: Immunodiffusion in gels encompasses a variety of techniques, which are useful for the analysis of antigens and antibodies. Gel immunodiffusion can be classified into two groups: 1. Single Immunodiffusion 2. Double Immunodiffusion In the Ouchterlony double diffusion, both the antigen and the antibody diffuse toward each other in a semisolid medium to a point till their optimum concentration is reached. A band of precipitation occurs at this point. The qualitative Ouchterlony Test can simultaneously monitor multiple Antibody-Antigen system and can be used to identify particular antigens in a preparation. This procedure was developed by Örjan Ouchterlony in 1948. Principle: When soluble antigen and antibody samples are placed in adjacent wells in agarose gel, they diffuse radially into the agarose gel and set up two opposing concentration gradients between the wells. Once the gradients reach to an optimal proportion, interactions of the corresponding molecules occur and a line of precipitation will form. Using such a technique, the antigenic relationship between two antigens can be analyzed. Distinct precipitation line patterns are formed against the same anti-sera depending on whether two antigens share all antigenic epitopes or partially share their antigenic epitopes or do not share their antigenic epitopes at all. The Ouchterlony test also can be used to estimate the relative concentration of antigens. When an antigen has a relatively higher concentration, the equivalent zone will be formed a little bit away from the antigen well. When an antigen has a relatively lower concentration, the equivalent zone will be formed a little bit closer the antigen well. The pattern of lines that form can be interpreted to determine the relationship between the antigens and antibodies.

Pattern of Identity Pattern of Non Identity Pattern of Partial Identity

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Materials Required: Standard Antigens, Antiserum, Agarose, 1X TBE, Conical flask, Measuring cylinder, Beaker, Distilled water, 70% alcohol, Microwave. Procedure: 1. Prepare 10 ml of 1% agarose 2. Cool the solution to 55-60o C and pour 5 ml/plate on to grease free glass plates placed on a horizontal surface. Allow the gel to set for 30 minutes. 3. Place the glass plate on the template provided. 4. Punch wells with the help of the gel puncher corresponding to the markings on the template. Use gentle suction to avoid forming of rugged wells. 5. Add 10 l each of the antiserum and the corresponding antigens to the wells 6. Keep the glass plate in a moist chamber overnight at 37o C. 7. After incubation, observe for opaque precipitin lines between the antigen and antiserum wells.

Antiserum X Antiserum Y Antiserum Z o o o o o o o o AgZ1 o AgZ2

Interpretation:

When antigen and antibody meet in optimal proportions a precipitation line is formed. In Ouchterlony Double Diffusion (Antigen Antibody Pattern), three patterns of precipitin lines can be observed. 1. If pattern X or pattern of identity is observed between the antigens and the antiserum, it indicates that the antigens are immunologically identical. 2. If pattern Y or pattern of partial identity is observed, it indicates that the antigens are partially similar or cross-reactive. 3. If pattern Z or pattern of non-identity is observed, it indicates that there is no cross- reaction between the antigens. i.e. the two antigens are immunologically unrelated. Result:

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Inference: Clinical Applications:

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Experiment 7: Rocket Immunoelectrophoresis (RIEP) Aim: Principle: Rocket Immunoelectrophoresis, also known as electro-immunodiffusion, is a simple, quick and reproducible method for determining the concentration of antigen in an unknown sample. In Rocket Immunoelectrophoresis, negatively charged antigen samples are electrophoresed in an agarose gel containing antibody which is specific to that antigen. As the antigen moves out of the well and enters the agarose gel, it combines with the antibody to form immune complex which is visible as white precipitin arcs. Because the antigen is migrated through the gel under the influence of an applied electric current, it moves in one direction. During the initial phase there is considerable antigen excess over antibody and no visible precipitation occurs. However, as the antigen sample migrates further through the agarose gel, more antibody molecules are encountered that interact with the antigen to form immune complex. When this immune complexes become large enough to be retained within the gel, movement of the antigen stops. The area of precipitin has the shape of a rocket and its height is proportional to the concentration of antigen in the corresponding well.

Materials Required: Standard Antigens, Antiserum, Agarose, 1X TBE, Conical flask, Measuring cylinder, Beaker, Distilled water, 70% alcohol, Microwave. Procedure: 1. Prepare 15 ml of 1 % agarose (as given in important instructions). 2. Cool the solution to 55-60o C and add 250 l of antiserum to 13 ml of agarose solution. Mix well for uniform distribution of antibody. 3. Pour agarose solution containing the antiserum on to a grease free glass plate placed on a horizontal surface. Allow the gel to set for 30 minutes 4. Place the glass plate on the template provided.

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5. Punch wells with the help of gel puncher. Use gentle suction to avoid forming rugged wells. 6. Add 10 l of the given standard antigen and test antigen samples to the wells. A. Standard Antigen A (1.87 mg/ml) B. Standard Antigen B (0.94 mg/ml) C. Standard Antigen C (0.47 mg/ml) D. Standard Antigen D (0.23 mg/ml) E. Test Antigen 1 F. Test Antigen 2 7. Pour 1X TBE buffer into the electrophoresis tank such that it just covers the gel. 8. Electrophorese at 80-120 volts and 60-70 mA, until the blue dye travels 3-4 cm from the well. 9. Incubate the glass plate in a moist chamber overnight at 370C.

Sample Standard Antigen Concentration (in mg/ml)

Rocket height (in mm)

Plot a graph of the rocket height (on Y-axis) versus the concentration of antigen (on X-axis) on a semi-log graph sheet. Determine the concentration of the unknown from the graph by finding the concentration against the rocket height.

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Interpretation: The height of the precipitin peak depends on the concentration of antigens loaded in the corresponding wells. By plotting the graph of concentration of antigens versus length of the precipitin peaks one can calculate the concentration of test antigen. Result: Inference: Clinical Applications:

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Experiment 8: Counter Current Immunoelectrophoresis (CCIEP)

Aim:

Principle: Counter current immunoelectrophoresis is a modification of immunoelectrophoresis in which antigen and antibody migrate towards opposite directions and form a visible white precipitate in the area between the wells. In this method, immunoprecipitation occurs when antigen at the cathode (negative pole) is caused to migrate in an electric field through a suitable medium of diffusion against a stream of antibody migrating backward from the anode (positive pole) because of endosmotic flow. When an electrical current is applied through the alkaline buffer, the negatively charged antigen molecules migrate toward the positive electrode and thus towards the wells filled with antibody and the positively charged antibodies are migrated toward the negative electrode. At some point between the wells, a zone of equivalence occurs and the antigen-antibody complex precipitates as a visible white line.

Ag

Materials Required: Standard Antigens, Antiserum, Agarose, 1X TBE/TAE, Conical flask, Measuring cylinder, Beaker, Distilled water, 70% alcohol, Microwave.

Procedure:

1. Prepare 10 ml of 1.5% agarose. 2. Mark the end of the slide that will be towards negative electrode during the

electrophoresis.

3. Cool the solution to 55-60o C and pour on to grease free glass plate placed on a

horizontal surface. Allow the gel to set for 30 minutes.

4. Place the glass plate on the template provided. 5. Punch wells with the help of gel puncher corresponding to the markings on the

template. Use gentle suction to avoid forming rugged wells.

6. Add 10 μl of antigen sample to the wells that will be placed towards the negative

electrode and 10 μl of antiserum samples to the wells towards the positive electrode.

7. Pour 1X TAE into the electrophoresis tank such that it just covers the gel. 8. Electrophorese at 80-120 volts and 55-60 mA, until precipitin lines are observed. 9. Place the glass plate in a moist chamber and incubate overnight at 37oC.

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Interpretation:

Negative Electrode Antigen Positive Electrode Positive Control (Antiserum) Test Sample I Test Sample 2 Test Sample 3

The precipitin line indicates the presence of antibody specific to the antigen while the absence of precipitin line indicates absence of corresponding antibody in the test antiserum to the given antigen. The presence of more than one precipitin line indicates the heterogeneity of the antibody for the antigen in the test sera.

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Result: Inference:

Clinical Applications:

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Experiment 9 : Dot ELISA

Aim:

Principle:

Enzyme linked immunosorbent assay or ELISA is a sensitive immunological technique to detect the presence of a specific antigen (Ag) or antibody (Ab) in a biological sample. There are various forms of ELISA for the detection of antigen or antibody based on antibody- antigen interactions. Dot ELISA, a qualitative ELISA test, can be performed very quickly with the end detection done visually. Because of its relative speed and simplicity, the dot ELISA is an attractive alternative to standard ELISA. In Dot-ELISA, small volumes of antibodies are immobilized on a protein binding membrane (Nitrocellulose) and the other antibody is linked to an enzyme Horse radish perxoidase (HRP). The test antigen at first reacts with the immobilized antibody and later with the enzyme-linked antibody. The amount of enzyme linked antibody bound is determined by incubating the strip with an appropriate substrate (Hydrogen peroxide, H2O2) and a chromogen [Tetramethylbenzidine (TMB)]. HRP acts on H2O2 to release nascent oxygen, which oxidizes TMB to TMB oxide, which gives, a blue coloured product. The latter precipitates onto the strip in the area of enzyme activity and appears as a coloured dot, hence the name Dot-ELISA. The results can be visualized in naked eye. The enzyme activity is indicated by intensity of the dot, which is directly proportional to the antigen concentration.

•J Substrate Coloured product Labeled antibody Immobilized antibody 1 Antigen Menrbrane

In Dot ELISA, an antibody is immobilized on a membrane and the test antigen is first allowed to react with immobilized antibody and then to the HRP-labeled antibody. The amount of HRP-labeled antibody bound is measured by treating the membrane strip with an

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appropriate chromogenic substrate which is converted to a coloured precipitate and appears as a dot on the membrane

Materials required:

1X Assay Buffer, HRP conjugated antibody, TMB/H2O2, Dot-ELISA strip, Test tubes, Distilled water, Micropipette, Tips.

Procedure: 1. Take 2 ml of 1X Assay Buffer in a test tube and add 2 μl of the test serum sample. Mix thoroughly by pipetting. Insert a Dot-ELISA strip into the tube. 2. Incubate the tube at room temperature for 20 minutes. Discard the solution. 3. Wash the strip two times by dipping it in 2 ml of 1X Assay Buffer for about 5 minutes each. Replace the buffer each time. 4. Take 2 ml of 1X Assay Buffer in a fresh test tube, add 2 μl of HRP conjugated antibody to it. Mix thoroughly by pipetting. Dip the ELISA strip into it and allow the reaction to take place for 20 minutes. 5. Wash the strip as in step # 3 for two times. 6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H2O2 and dip the ELISA strip into this substrate solution. 7. Observe the strip after 5 - 10 minutes for the appearance of a blue spot. 8. Rinse the strip with distilled water. Interpretation: Spot in the positive control zone and no spot in the negative control zone indicates proper performance of test. In the negative control zone the immobilized antibody is not present and the region is blocked with an inert protein. Therefore, there is no reaction when the reagents are added and no spot can be seen. In the test zone an antibody (specific to the test antigen, serum) is immobilized on it and then blocked with an inert protein. The test serum binds to this region and the HRP-labeled antibody binds to serum which when reacts with substrate develops blue dot. In the positive control zone, the test serum binds to the immobilized antibody and the HRP-labeled antibody binds to serum which when reacts with substrate develops blue dot.

Look for the appearance of the blue dot as shown below:

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Record your observations as follows:

Zone Spot

Positive Zone

Negative Zone

Test Zone

Result: Inference:

Clinical Applications:

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Experiment 10: Coomb’s test

Aim: Principle:

A Coombs test, also known as antiglobulin test (AGT) is either of two blood tests used in immunohematology. They are the direct and indirect Coombs tests. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells.[1] Since these antibodies sometimes destroy red blood cells, a person can be anemic and this test can help clarify the condition. The indirect Coombs detects antibodies that are floating freely in the blood.[1] These antibodies could act against certain red blood cells and the test can be done to diagnose reactions to a blood transfusion.

The direct Coombs test is used to test for autoimmune hemolytic anemia—that is, a condition where the immune system breaks down red blood cells, leading to anemia. The direct Coombs test is used to detect antibodies or complement proteins attached to the surface of red blood cells. To perform the test, a blood sample is taken and the red blood cells are washed (removing the patient's own plasma and unbound antibodies from the red blood cells) and then incubated with anti-human globulin ("Coombs reagent"). If the red cells then agglutinate, the direct Coombs test is positive, a visual indication that antibodies or complement proteins are bound to the surface of red blood cells and may be causing destruction of those cells.

The indirect Coombs test is used in prenatal testing of pregnant women and in testing prior to a blood transfusion. The test detects antibodies against foreign red blood cells. In this case, serum is extracted from a blood sample taken from the patient. The serum is incubated with foreign red blood cells of known antigenicity. Finally, anti-human globulin is added. If agglutination occurs, the indirect Coombs test is positive Materials Required: Test cells, Coombs reagent, Anti human globulin, anti D sera, Test tubes, Pipettes, Centrifuge, Isotonic saline. Procedure: 1. Prepare 5% cell saline suspension of the cells to be tested. 2. Label 3 tubes as T, PC and NC. 3. In the tube labeled as T (Test), take 2 drops of 5% saline cell suspension to be tested. 4. In the test tube labelled as PC (Positive control), take 1 drop of anti D sera and 1 drop of

Rh +ve pooled cells.

5. In the test tube labelled as NC (Negative control), take 1 drop of normal saline and one

drop of Rh +ve pooled cells.

6. Add 2 drops of Anti human globulin to each of the tubes. 7. Mix well and centrifuge for 1 minute at 1500 rpm. 8. Resuspend the cells by gentle agitation and examine macroscopically and

microscopically for agglutination.

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The patient's washed immune mediated haemolytic anaemia: RBCs are incubated with antibodies are shown attached to antigens on the RBC surface. irect Coombs test Direct antiglobulin test Positive test result Antigens on red blood cell's Human anti-RBC Y Antih antibody ( CN.•nbs reagent) Blood sample from a patient with 00 antihuman antibodies (Coombs reagent), RBCs agglutinate: antihuman antibodies form links between RBCs by binding to the human antibodies on the RBCs. test Jn rect anthgjobulin test Positive test result Recipient's serum is obtained, Containing antibodies (Ig•s). Donor's blood sample is added to the tube with Recipient's Ig's that target the donor's red blood cells form antibody-antigen complexes. Anti•human Ig's (Coombs antibodies) are added to the solution. Agglutination of red blood cells occurs, because human Ig's are attached to red blood cells.

Result:

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Inference: Clinical Applications: Further Reading: The Coomb’s test (also known as Antiglobulin Test or AGT) refers to two clinical blood tests used in immunohematology which are done to find certain antibodies that cause autoimmune haemolysis of red blood cells (erythrocytes). In certain diseases or conditions, an individual’s blood may contain IgG antibodies that can specifically bind to antigens on the red blood cell (RBC) surface membrane. Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to be sensitized with IgG or complement. In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. Because antibodies are gamma globulins, an antibody to gamma globulin can form bridges between red cells sensitized with antibody and cause them to agglutinate. The two types of Coombs tests are: Direct Coombs test and Indirect Coombs test Direct Coombs test: The direct Coombs test (also known as the direct antiglobulin test or DAT) is used to detect if antibodies or complement system factors have bound to RBC surface antigens in vivo. A blood sample is taken and the RBCs are washed and then incubated with antihuman globulin. If this produces agglutination of RBCs, the direct Coombs test is positive, a visual indication that antibodies are bound to the surface of red blood cells. This is the test that is done on the newborn’s blood sample, usually in the setting of a newborn with jaundice. The test is looking for “foreign” antibodies that are already adhered to the infant’s RBCs, a potential cause of hemolysis. The indirect Coombs test (also known as the indirect antiglobulin test or IAT) is used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient’s plasma/serum prior to a blood transfusion. In antenatal care, this test is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, RBC phenotyping, and titration studies.

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Experiment 11: The complement fixation test

Aim: Principle: The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum, based on whether complement fixation occurs. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as ELISA and by DNA-based methods of pathogen detection, particularly PCR. It is the nature of the complement to be activated when there is formation of antigen-antibody complex. The first step is to heat the serum at 56°C to destroy patient’s complement. A measured amount of complement and antigen are then added to the serum. If there is presence of antibody in the serum, the complement is fixed due to the formation of Ag-Ab complex. If no antibody is present then the complement remains free. To determine whether the complement has been fixed, sheep RBCs and antibodies against sheep RBCs are added. Materials Required: Test Serum, antigen of interest, standard complement proteins, Sheep red blood cells (sRBCs), anti-sRBC antibodies, Test tubes, Pipettes, Centrifuge Procedure:

The complement system is a system of serum proteins that react with antigen-antibody complexes. If this reaction occurs on a cell surface, it will result in the formation of trans- membrane pores and therefore destruction of the cell. The basic steps of a complement fixation test are as follows:

1. Serum is separated from the patient. 2. Patients naturally have different levels of complement proteins in their serum. To

negate any effects this might have on the test, the complement proteins in the patient's serum must be destroyed and replaced by a known amount of standardized complement proteins.

1. The serum is heated in such a way that all of the complement proteins—but

none of the antibodies—within it are destroyed. (This is possible because complement proteins are much more susceptible to destruction by heat than antibodies.)

2. A known amount of standard complement proteins are added to the serum.

(These proteins are frequently obtained from guinea pig serum)

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3. The antigen of interest is added to the serum. 4. Sheep red blood cells (sRBCs) which have been pre-bound to anti-sRBC antibodies

are added to the serum. The test is considered negative if the solution turns pink at this point and positive otherwise.

Interpretation:

If the patient's serum contains antibodies against the antigen of interest, they will bind to the antigen in step 3 to form antigen-antibody complexes. The complement proteins will react with these complexes and be depleted. Thus when the sRBC-antibody complexes are added in step 4, there will be no complement left in the serum. However, if no antibodies against the antigen of interest are present, the complement will not be depleted and it will react with the sRBC-antibody complexes added in step 4, lysing the sRBCs and spilling their contents into the solution, thereby turning the solution pink.

Result: Inference: Clinical Applications:

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o Antigen Complement Serum with antibody against antigen Complement fixation Sheep RBC Antibody to sheep RBC No hemolysis (complement tied up in antigen—antibody reaction) o Antigen Complement Serum without antibody No complement fixation Sheep RBC Antibody to sheep RBC Hemolysis (uncombined complement available) (a) Positive test. All available complement is fixed by the antigen—antibody reaction; no hemolysis occurs, so the test is positive for the presence of antibodies. (b) Negative test. No antigen—antibody reaction occurs. The complement remains, and the red blood cells are lysed in the indicator stage, so the test is negative.

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Experiment 14: Western blotting

Aim: Principle: Western blotting (protein or immunoblotting) is a rapid and sensitive assay for detection and characterisation of proteins. Western Blotting technique exploits the inherent specificity of antigen-antibody interaction to identify specific antigens by polyclonal or monoclonal antibodies. It involves the following steps:

• Electrophoresis of protein (SDS PAGE).

• Electro transfer of protein onto nitrocellulose membrane (Western Blotting).

• Immunodetection of transferred protein (Blot development).

SDS-PAGE: Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is carried out in a discontinuous buffer system wherein the reservoir buffer is of different pH and ionic strength from the buffer used to cast the gel. The SDS-Polypeptide complexes in the sample applied to the gel are swept along by a moving boundary created when an electric current is passed 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 further electrophoresis, polypeptides get resolved based on their size in the resolving gel. Western Blotting: Blotting is transfer of resolved proteins from gel onto a surface of suitable membrane, done commonly by electrophoresis and referred to as electroblotting. The gel is placed 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 elecrophoretic tank containing blotting buffer. The proteins get transferred to the corresponding position on the membrane as resolved on polyacrylamide gel, forming a mirror image of the gel. Protein of interest on the membrane is further located by immunodetection. Immunodetection: The transferred proteins bound to the surface of nitrocellulose membrane are detected using immunological reagents, by a process called immunodetection. All the unoccupied sites on the membrane are first blocked with inert protein or detergent or any suitable blocking agent. The membrane is then probed with a primary antibody specific to protein of interest. The Ag- Ab complex formed on the membrane is then identified using an enzyme- labelled secondary antibody and substrate to the enzyme, which results in a coloured band on the nitrocellulose membrane, referred to as blot development.

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Procedure: Day1: SDS-PAGE:

1. Assemble the plates for casting the 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 50 µl of APS solution to 5 ml of SDS separating gel mix and pour the gel

solution between the plates till the level is 2 cm below the edge of notched plate.

4. Add 200-250 µl of water to make the surface even. 5. After the gel is set (approximately 20-30 min.), wash the top of the separating gel with

distilled water and drain off the water completely.

6. Add 20 µl of APS solution to 2 ml of stacking gel mix and pour directly onto the

polymerised separating gel.

7. Insert the comb into the gel solution carefully without trapping any bubbles, about 1

cm above separating gel. The stacking gel will set in approximately 10 min.

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 stacking the gel has set, carefully remove the comb and the bottom spacer. Wash

the wells immediately with distilled water to remove non polymerised acrylamide. Fill the bottom reservoir with 1X reservoir buffer and carefully fix the plates to PAGE apparatus. Fill the top reservoir with 1X reservoir buffer.

12. Load 30 µl of protein marker in well 1, 40 µl of protein sample in well 2 and 5 µl of

protein sample in well 4. Note down the order of loading. Connect the cords to the power supply according to convention red: anode, black: cathode.

13. Set voltage to 100 V and switch on 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 60-90 minutes.

15. Remove the gel plates and gently pry the plates apart using spatula. 16. Transfer it to a tray containing water; wash it for 2 minutes at room temperature. 17. Decant water, cut gel along the lane 3. 18. Transfer lane 4 i.e. protein sample in 10 ml of blotting buffer taken in a petridish.

Following incubation of 10 minutes, go for electroblotting (step 22).

19. To lane 1and 2 add minimum of 20 ml of water. 20. Decant the water; add 20 ml of Ezee blue stain. Stain it for 1-2 hours. 21. Decant the staining solution add minimum quantity of water to cover the gel.

Note-Cover the tray and leave it overnight at room temperature.

Electroblotting:

1. Assemble the blotting sandwich within the blotting cassette .Take care to avoid air

bubbles between the gel and NC membrane.

2. Insert the cassette into the apparatus filled with blotting paper and connect blotting

unit to power supply as per the convention, red: anode, black: cathode.

3. Electrophorese the sample at 50 V for 2 hours for blotting to occur. 4. Remove the NC-membrane gently from the cassette and place the membrane in 10 ml

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of freshly prepared blocking buffer taken in petridish. Leave it overnight at 40C.

Day 2: Immunodetection

1. Discard blocking buffer. 2. Immerse blot in 10 ml of primary antibody solution and mix gently for 30 minutes.

Discard the primary antibody solution.

3. Wash the blot by immersing in 10 ml wash buffer for 3-5 minutes (twice). 4. Immerse the blot in 10 ml of 1X HRP labelled antibody. Mix gently for 30-minutes.

Discard the HRP labelled antibody.

5. Wash the blot by immersing 10 ml of substrate solution, mix gently for 10-15

minutes, within this time coloured band will appear.

6. Remove blot, wash with distilled water, discard and dry.

Western Blotting Procedure I. Load and separate protein sam leson SDS-PAGE 6. Incubate the blot with chemiluminescent HRP substrate and expose to film WESTERN BLOT 98 64 52 36 22 2. Electrophoreticallytransfer fractionated proteins onto PVDF membr e MEMBRANE GEL 5. Incubate the membrane with HRP-labeled secondary antibody specificto primary antibody 3. Block the membrane with neutral protein (BSA or milk casein) 4. Incubate the membrane with primary antibody specific to target protein

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Result: Inference:

Clinical Applications: