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ouchterlony double diffusion method and immunoblotting
- 1. Diagnosis of diseaseusing immunological methods- Ouchterlony double diffusion, Immunoblotting UNIT-5 R.T.VINODHINI
- 2. Immunodiffusion 🞅 Study reactionbetween antigens and antibodies 🞅 Observing precipitates formed 🞅 Combination of specific antigens and antibodies 🞅 Diffused in a gel 🞅 Separately placed
- 3. Ouchterlony double diffusion 🞅Detection, identification and quantification of antibodies and antigens. 🞅 Antibodies and Ag form lattice structures (visible precipitate). 🞅 Formation of Ab-Ag lattice depends on, • Ab must be bivalent. • Ag must be bivalent or polyvalent.
- 4. Principle 🞅 Both antigenand antibody - diffuse into the gel 🞅 Comparing different antigen preparations 🞅 Antigen and antibody are allowed to migrate towards each other in a gel 🞅 Line of precipitation - two reactants are meet 🞅 Precipitation - highly specific 🞅 Diagnosisand protein detection technique
- 5. Required materials 🞅 Chemicals: 🞅Agarose 🞅 Assay Buffer(PBS) 🞅 Anti-serum 🞅 T est antigens 🞅 pH - 7.2
- 6. Required materials 🞅 Equipments: 🞅I ncubator (37°C) 🞅 Micro pipette 🞅 Tips 🞅 Weighing balance 🞅 Gel punch with syringe
- 7. Required materials 🞅 Glassware's: 🞅Glass plate 🞅 Conical flask 🞅 Measuring cylinder 🞅 Reagents: 🞅 Alcohol 🞅 Distilled water
- 8. Procedure 🞅 25 mlof 1.2 % agarose (0.3g/25ml) in assay buffer 🞅 Boiling to dissolve the agarose completely 🞅 Cool - 55 to 60°C 🞅 Pour 4ml/plate 🞅 Gel to set - 30 minutes 🞅 Punch wells
- 9. Procedure 🞅 Fill thecentre well 10 micro liter of anti-serum 🞅 Upper T wo wells with 10 ml of each antigen 🞅 Glass plate - moist chamber over night at 37°C 🞅 Opaque precipitin lines - Antigen and anti-sera wells
- 10. Specific Objective 🞅To interpret the results obtained in Ouchterlony double diffusion method (U/C)
- 11. Interpretation 🞅 Pattern Aor pattern of identity -between antigen and anti-serum -antigens are immunologically identical 🞅 Pattern B–no cross reaction between the antigen (immunological unrelated) 🞅 Pattern C - antigen are partially similar or cross reaction
- 14. Applications 🞅 Identification ofFungal antigen. Eg - Coccidiomycosis, Histoplasma. 🞅 Measuring hormones serum proteins, drugs, etc. 🞅 Clinical laboratory - determination of Immunoglobulin levels in patient sample.
- 16. Immunoblotting/Western blotting 🞅 Viralantigens - detected with a polyclonal or a MAb onto nitrocellulose paper. 🞅 After incubation - protein bands (immune complexes) 🞅 Visualized - peroxidase-conjugated protein and a colour reagent 🞅 Colour - bands where antibody binds to the antigen 🞅 Principe - I mmunochromatography
- 17. Procedure 🞅 T issue preparation 🞅Gel electrophoresis 🞅 T ransfer/blotting 🞅 Blocking 🞅 Hybridization 🞅 Detection
- 18. Tissue preparation 🞅 Wholetissue, cell culture, bacteria, virus or environmental samples 🞅 Solid tissues 🞅 Mechanical 🞅 Chemical 🞅 Enzymatic 🞅 Protection –anti protease & phosphatase 🞅 Cold temperatures
- 19. Gel electrophoresis 🞅 SDSPAGE 🞅Separation - molecularweight, electric charge or a combination
- 20. Transfer/blotting 🞅 Nitrocellulose orpolyvinylidene difluoride (PVDF) –non specific protein binding 🞅 Membrane - on top of the gel & stack of filter papers placed on top of that 🞅 Placed in a buffer solution 🞅 Moves up the paper by capillary action bringing the proteins with it 🞅 Electrobloting - electric current to pull proteins from the gel into membrane
- 21. Blocking 🞅 Prevent interactionsbetween the membrane and the antibody used 🞅 Bovin Serum Albumin (BSA) or non-fat dry milk with Tween20 (detergent) 🞅 The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached 🞅 Reduces "noise" in the final product 🞅 Leading to clearer results & eliminates false positives
- 22. Detection 🞅 Probed -Protein of interest with antibody linked to a reporter enzyme 🞅 Appropriate substrate - colorimetric reaction and produces a color 🞅 Primary antibody (protein of interest) 🞅 I ncubated with membrane - 30 minutes to overnight at different temperatures
- 23. Detection 🞅 Secondary antibody{ After rinsing the membrane to remove unbound primary antibody) 🞅 Secondary antibody +biotin/reporter enzymes (alkalin phosphatase/horseradish peroxidase) 🞅 Horseradish peroxidase-linked secondary antibody - cleave a chemiluminescent agent 🞅 Luminescence in proportion to the amount of protein
- 24. Analysis 🞅 Colorimetric detection 🞅Chemiluminescent detection 🞅 Radioactive detection 🞅 Fluorescent detection
- 25. Colorimetric detection 🞅 Incubationwith substrate -reporter enzyme (such as peroxidase) that is bound to the secondary antibody 🞅 Converts soluble dye - insoluble form of a different color 🞅 Precipitates next to the enzyme & stains the membrane 🞅 Densitometry or spectrophotometry
- 26. Chemiluminescent detection 🞅 Substrate(luminesce) - reporter on secondary antibody. 🞅 The light is then detected by photographic film, and more recently by CCD cameras. 🞅 Densitometry. 🞅 Newer software - molecularweight analysis
- 27. Radioactive detection 🞅 Radioactivelabels - no enzyme substrates 🞅 Placement of medical X-ray film directly against the western blot 🞅 Develops as it isexposed to the label and creates dark regions 🞅 Corresponds - protein bands of interest 🞅 Very expensive, health and safety risks are high
- 28. Fluorescent detection 🞅 Fluorescentlylabeled probe - excited by light 🞅 Emission of excitation -detected by a photosensor such as CCD camera 🞅 Molecular weight analysis & quantitative western blot analysis 🞅 Most sensitive detection methods for blotting analysis
- 29.