immunology practical file, Exams of Immunology

Download immunology practical file and more Exams Immunology in PDF only on Docsity!

Practical File

Name- Charvi Nayyar

Subject- Immunology

Class Roll Number- 21

Examination Roll Number- 19030553007

Section- B

Semester- 6

Year- 3

Contents

S.

No.

Experiment Title Date Teacher’s

Signature

1. To study the technique of Ouchterlony^ double

diffusion (ODD) to measure the titre of

antiserum (antibody).

2. To study the reaction pattern of an antigen with

a set of antibodies with Ouchterlony Double

Diffusion Method ODD).

3. To learn the technique of radial-

immunodiffusion

4. To^ perform sandwich Dot ELISA test for

antigen.

5. To perform sandwich ELISA (enzyme linked

immunosorbent assay)

6. To isolate peripheral blood mononuclear cells

(PBMC) from whole blood using Ficoll and

calculate cell viability.

7. To perform assay based on agglutination

reactions active blood typing and passive

agglutination.

8. To purify immunoglobulins from a given serum

sample using Ion Exchange Chromatography.

of Ab. As there is not enough Ag to produce visible lattice formation, this is called the antibody excess zone (Prozone phenomenon). The Ag and Ab concentrations are relatively higher near their respective wells. As they diffuse further from the wells, their concentration decreases. An antigen will react with its specific antibody to form an Ag-Ab complex. As more antigens are added, the amount of protein precipitated increases until the Ag/Ab molecules are at an optimal ratio, where maximal precipitation occurs. This is known as the equivalence zone or equivalence point. When the amount of Ag in solution exceeds the amount of Ab, the amount of precipitation will decrease. This is known as the antigen excess zone. Fig. antigen-antibody reactions Procedure:

1. Prepare 10mL of 1% agarose (0.1g/10mL) in 1x assay buffer by heating slowly till the agarose is dissolved completely. 2. Pour 6mL of the agarose solution onto a clean glass plate placed on a horizontal surface and allow the gel to set. This will take approximately 20- 30 minutes. 3. Place the glass plate on the template provided and punch wells in the gel with the help of the gel puncher provided, corresponding it with the markings on the template. Use gentle suction to avoid forming rugged wells. 4. Serially dilute the test antiserum up to 1:32 dilution as follows: - Add 20μL of 1x assay buffer in each of the five vials. - Add 20μL of the re-constituted test antiserum into the first vial and mixed. The dilution of antiserum in this vial was 1:2. - Transfer 20μL of 1:2 diluted antiserum from the first vial into the second vial and mix well. The dilution of antiserum in that vial is 1:4.

5. Add 10μL of the antigen to the well in the centre of the gel and 10μL each of undiluted, 1:2, 1:4, 1:8, 1:16, 1:32 of test antiserum into each of the surrounding wells. 6. Place the plate in a moist chamber (box with a wet absorbing sheet with distilled water and formaldehyde) and incubate overnight at room temperature. 7. After incubation, observe for opaque precipitin line between the antigen and antisera wells. 8. Note down the highest dilution at which the precipitin line is formed. This is the titre value of the antiserum. Observation: Result: Discussion:

Experiment 2 Aim: To study the reaction pattern of an antigen with a set of antibodies with Ouchterlony Double Diffusion Method (ODD). Materials required : alcohol, distilled water, buffer, agarose gel, micropipettes, spatula, moist chamber, template, glass plates, gel puncher with syringe, measuring cylinder, 3 different antiserum, 3 different antigens. Principle: Ouchterlony Double Diffusion is also known as agar gel immunodiffusion or passive double immunodiffusion. Immunodiffusion in gel encompasses a variety of techniques, which are useful for analysis of antigens and antibodies. An antigen reacts with a specific antibody to form an antigen-antibody complex or immune complex, the composition of which depends on the nature, concentration and proportion of the initial reactants. The immune complex precipitates in the gel to give a thin white line which is a visual signature of antigen recognition. Immunodiffusion in gels are classified as single diffusion and double diffusion. In ODD, both the antigen and the antibody are allowed to diffuse into the gel. This array is frequently used for comparing different antigen preparations, each containing single antigenic species are allowed to diffuse from separate wells against the antiserum. Depending on the similarity between the antigen, different geometrical patterns are produced between the antigen and antiserum wells. The pattern of lines can be interpreted to determine whether the antigens are same or different. Pattern of Identity: A (given two antigens are identical)

• Both the antigens share all the epitopes.
• Precipitation line will be formed at the zone of equivalence. Since the antigens are identical two precipitation lines will be formed but they will fuse and result in an arc shaped precipitation band as the antigens are identical and antibody fails to distinguish them so at the zone of equivalence, they will Overlap each other and antigen concentration will increase at the overlapping area.
• this pattern of precipitation line is known as the pattern of Identity.

Pattern of Partial Identity: B (given two antigens are partially identical)

• The given antigens share one or more epitopes.
• A cross reaction will occur. There seems a pattern of Identity but along with a spur formation known as incomplete cross or an arc with a spur.
• This pattern is known as pattern of Partial Identity Pattern of Non-Identity: C (given two antigens are non identical)
• The given antigens have no common epitopes.
• Two precipitation bands will be formed but they will not fuse, they will cross each other without any interaction.
• This happens because each antigen has its own antibody with which they form precipitation band at their respective zone of equivalence.
• This pattern is known as pattern of Non-Identity. When a soluble antigen conjugates with its specific antibody in the presence of electrolyte, the Ag- Ab complex formed can be seen as an insoluble precipitate. The Ab capable of getting precipitated are termed precipitins. The amount of precipitate is greatly influenced by the Ag/Ab ratio. An excess of any of these can inhibit the form of a precipitate. According to lattice theory of precipitation, precipitate is formed as a result of random, reversible binding wherein each Ab binds to more than one of the Ag and vice versa forming a lattice of Ag and Ab. If Ab are in excess, a proper lattice cannot be formed and hence precipitation is minimal. This zone of graph of precipitation is termed as prozone. As the quantity of Ab is decreased, prozone is followed by zone of equivalence where lattice formation takes place and precipitate is

Interpretation: Precautions:

• Wipe the glass plate with alcohol thoroughly to make it grease free for even spreading of agarose.
• While boiling cells, care should taken that wells are circular with no irregular surface.
• Wells should be made when gel has settled properly.

Experiment 3 Aim: To learn the technique of radial-immunodiffusion. Materials required : Conical flask, measuring cylinder, alcohol, distilled water, micropipette, tips, moist chamber, RID kit Theory : Single Radial immunodiffusion (RID) is used extensively for the quantitative estimation of antigens. The antigen antibody precipitation is made more sensitive by the incorporation of antiserum in agarose. Antigen (Ag) is allowed to diffuse from wells cut in the gel in which the anti-serum is uniformly distributed. Initially, as the antigen diffuses out of the well, its concentration is relatively high and soluble antigen-antibody adducts are formed. However, as antigen diffuse farther from the well, the Ag-Ab complex reacts with more amount of antibody resulting in a lattice that precipitates to form precipitin ring. By loading a range of unknown antigen concentrations on the gel and by measuring the diameters of their precipitin rings, a calibration graph is plotted. Concentrations of unknown antigens can be determined by measuring the diameter of precipitin rings and extrapolating this value on the calibration graph. Procedure:

• Prepare 10ml of 1% agarose (0.1g/10ml) in 1X Assay Buffer by heating slowly till agarose dissolves completely till agarose dissolves completely. Take care not to scorch or froth the solution.
• Allow the molten agarose to cool to 55ᵒC.
• Add 120μl of antiserum to 6 ml of agarose solution. Mix by gentle swirling for uniform distribution of antibody.
• Pour agarose solution containing the antiserum onto a grease free glass plate set on a horizontal surface. Leave it undisturbed for 15 minutes to solidify.
• Cut wells using a Gel Puncher as shown in figure 2, using the template provided.
• Add 20μl of the given standard antigen and test antigens to the wells.
  1. Standard antigen A (0.25 mg/ml)
  2. Standard Antigen B (0.5mg/ml)
  3. Standard Antigen C (1.0mg/ml)
  4. Standard Antigen D (2.0mg/ml)
  5. Test Antigen- 1
  6. Test Antigen- 2
• Keep the gel plates in moist chamber (box containing wet cotton) and incubate overnight at RT.
• Mark the edges of the circle and measure the diameter of the ring. Note down your observations.
• Plot a graph on a semi-log graph sheet and determine the concentration of unknowns.

Experiment 4 Aim: To perform sandwich Dot ELISA test for antigen. Materials required: Dot ELISA strips, 10x Assay buffer, 1x Assay buffer, Antigen HRP conjugate, 10x TMB/H 2 O 2 , test serum sample, test tubes, dH 2 O, micropipettes. Principle: Dot-ELISA (Enzyme Linked Immunosorbent Assay) is an extensively used immunological tool in research as well as analytical/diagnostic laboratories. In sandwich Dot-ELISA, the antigen is sandwiched directly between two antibodies which react with two different epitopes on the same antigen. Here one of the antibodies is immobilized onto a solid support and the second antibody is linked to an enzyme. Antigen in the test sample first reacts with the immobilized antibody and then with the second enzyme-linked antibody. The amount of enzyme linked antibody bound is assayed by incubating the strip with an appropriate chromogenic substrate, which is converted to a coloured, insoluble product. The latter precipitates onto the strip in the area of enzyme activity, hence the name Dot-ELISA. The enzyme activity is indicated by intensity of the spot, which is directly proportional to the antigen concentration. ELISA strips have three well defined zones:

• Negative Control Zone- It is blocked with an inert protein.
• Test Zone- It has an antibody immobilized on it and then blocked with an inert protein.
• Positive Control Zone- It has an antibody immobilized on it, blocked with an inert protein and has a specific antigen bound to the immobilized antibody. These strips will be used to detect the antigen in the test serum samples supplied, by using a secondary antibody conjugated to Horse Radish Peroxidase (HRP). HRP is then detected using Tetramethylbenzidine (TMB) as a chromogen. Substrate HRP acts on Hydrogen peroxide to release oxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited wherever the enzyme is present and appears as a blue spot. H 2 O 2 → H 2 O + [O] (This reaction occurs in presence of HRP) TMB + [O] → TMBO (Blue) If the test sample does not contain the antigen specific to the antibody, there will be no enzyme reaction and no spot develops. The Reaction Sequence
• Negative Control Zone: In this zone immobilized antibody is not present and hence, there is no reaction when the reagents are added.
• Positive Control Zone: In this zone antigen is bound to immobilized antibody. The antigen binds to antibody enzyme conjugate and develops a spot.

• Test Zone: Spot in test zone indicates presence of specific Ag. The substrate binds to the Ab-Enzyme conjugate. The enzyme oxidises the substrate to give a blue spot. Procedure:
• In a test tube/vial, took 1mL of 1x assay buffer and 50μL serum sample. Mixed thoroughly. Inserted a Dot-ELISA strip.
• Incubated at room temperature for 20 minutes.
• Washed the strip by dipping in 1mL of 1x assay buffer for 5 mins. Repeated thrice and replaced the buffer each time.
• Take 1mL of assay buffer in a fresh tube/vial, added 10μL of HRP conjugate antibody. Mixed thoroughly. Dipped the strip in it and incubated for 20 mins.
• Washed the strip by dipping in 1mL assay buffer for 5 mins. Repeated thrice and replaced the buffer each time.
• In a fresh vial, took 0.1mL of 10x TMB/ H 2 O 2 solution and added 0.9mL of distilled water.
• Observed the strip after 10 - 20minutes for the appearance of a blue/grey spot.
• Rinsed the strip with distilled water to stop the reaction. Observations: Result and Inference: Precautions:
• Prepare the buffers precisely.
• Wash the Dot ELISA strip carefully.
• Use clean and fresh vials for dipping the strip.
• Do not touch the lower end of the strip consisting of negative, test and positive control zones.

Sandwich ELISA: Antigens can be detected by sandwich ELISA. In the technique antibody is coated on the microtiter well. A sample containing antigen is added to well and allowed to react with antibody attached to well forming and antigen-antibody complex. After the well is washed, a second enzyme-linked antibody specific for specific epitope on antigen is added and allowed to react with bound antigen. After unbound secondary antibody is washed or removed. Finally, substrate is added to plate which is hydrolysed by enzyme to form coloured product. Advantages

• High specificity, since two antibodies are used. The antigen is specifically captured and detected.
• Suitable for complex samples, since the antigen does not require purification prior to measurements.
• Flexibility and sensitivity, since both direct and indirect detection methods can be used. Competitive ELISA: This test is used to measure concentration of antigen in a sample. In this test, antibody is first incubated in a solution with sample containing antigen. The more the antigen present in the sample, less free antibody will be available to bind to antigen-coated cells. After well is washed, enzyme-conjugated secondary antibody specific for isotype of primary antibody is added to determine the amount of primary antibody bound to well. The higher the concentration of antigen, lower is absorbance. Advantages
• High specificity, since two antibodies are used. The antigen is specifically captured and detected.
• Flexibility and sensitivity, since both direct and indirect detection methods can be used.
• Suitable for complex samples, since the antigen does not require purification prior to measurements. Applications of ELISA:
• Presence of antigen or antibody in sample can be evaluated.
• Determination of serum antibody concentration in a virus test.
• Used in food industry when detecting potential food allergens
• Applied in disease outbreak- tracking spread of disease. e.g., HIV, bird flu, common cold, cholera, etc. Procedure: Day 1: Coating of wells with capture antibody
• Dilute 60 μl of reconstituted capture antibody with 5.94 ml of coating buffer in a test tube. The concentration of the diluted capture antibody is 10 μg/ml.

• Pipette 200 μl of diluted capture antibody into each of microtiter well. Tap or shake the plate gently to ensure that the capture antibody is evenly distributed over the bottom of each well.
• Cover with aluminium foil and incubate the microtiter well overnight at 4˚C. Day 2: Blocking the residual binding site
• Discard the unbound capture antibody. Rinse the well with distilled water 3 times by draining out water after each rinse.
• Add 200 μl of blocking buffer to each well and incubate at room temperature for 1 hr.
• Rinse wells 3 times with distilled water. Drain the water completely by inverting wells on blotting paper. Preparation of dilution of standard antigen
• Concentration of reconstituted standard antigen is 0.4mg/ml. Dilute this to get range of std. conc. using sample diluent. Preparation of working concentration of test samples
• After reconstitution, mix 2 μl of each of test sample (1:2) individually with 2 ml of sample diluent.
• Add 200 μl of standard antigen, diluted test sample and 1x PBST to coated cells.
• Incubate at RT for 30 mins.
• Discard the unbound antigen. Wash wells with 1x PBST for 3 mins each. Addition of antibody- HRP conjugate
• Add 200 μl of 1x antibody HRP conjugate to each well.
• Incubate at RT for 20 mins.
• Discard the unbound conjugate and rinse the wells 3 times with 1x PBST. Addition of substrate and measurement of absorbance
• Add 200 μl of 1x TMB/ H 2 O 2 to each well.
• Incubate at RT for 10 mins.
• Add 100 μl of 1x stop solution to each well.
• Transfer the contents of each well to individual tubes containing 2ml of 1x stop solution.

Result: Precautions:

• Wells shouldn’t be dry at any point during assay.
• Each test sample should be reconstituted with 50μl of diluted water and stored at 4˚C.

Experiment 6 Aim : To isolate peripheral blood mononuclear cells (PBMC) from whole blood using Ficoll and calculate cell viability. Materials required : Heparinised blood, Ficoll-Paque separating solution, Trypan blue staining solution 0.1 %, Phosphate buffer saline (pH=7.2), hemocytometer, centrifuge tubes, centrifuge, auto pipettes, tips, cover slips, light microscope. Theory: A PBMC is any blood cell having a round nucleus, e.g. a lymphocyte, a monocyte, a macrophage etc. These blood cells are critical component in immune system and hence fights infection and adapt to intruder. The lymphocyte population consists of T-cells CD4+ and CD8+^ that is approximately 75%. B-cells and Nk cells (approx. 25% combined). These cells can be extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates blood into a top layer of plasma then PBMC and a bottom layer of polymorphonuclear cells and erythrocytes. The polymorphonuclear cells can be further isolated by using the RBCs. Ficoll: Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin. A sterile aqueous medium containing ficoll and sodium diazotriate at a predetermined density of 1.077g/ml at 25˚C is used for preparation of PBMC by density gradient centrifugation. Differential migration during centrifugation results in formation of layers containing different cell types. The bottom layer has erythrocytes which have aggregates by ficoll and therefore sediment completely through the ficoll-paque. Above erythrocytes is layer containing granulocyte which at the osmotic pressure attain a density gradient enough to migrate through the ficoll-paque layer. Because of their lower density lymphocytes are found at interphase between plasma and ficoll phase with other slowly sedimenting particles (platelets and monocytes) Lymphocytes: It is one of the subtypes of WBC in a vertebrate immune system. Lymphocytes include natural killer cells, T cells, B cells. They are the cells found in lymph. In the circulatory system they move from the lymph nodes while the macrophages remain stationery in the area. T cell and B cell or bursa of fabricus is the major cellular components of the adaptive immunity. T cells are involved in cell mediated while B cells are involved in humoral immunity. Nk cells are a part of innate immunity and protect host from tumor and virally infected cells. They are activated in response to a family of cytokines called interferon. They release cytotoxic molecules which then destroy the altered cells. Cell counting viability: Cells can be counted in Neubauer chamber with trypan blue dye (10μl supernatant + 10μl trypan blue dye). The dye is not permeable to interact through intact cell membrane and thus