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2. Basic Immunologic Procedures

2. Basic Immunologic Procedures. Terry Kotrla, MS, MT(ASCP)BB Fall 2005. Introduction. Detection of antigen/antibody reactions difficult Sensitization is the binding of a specific antibody to its’ specific antigen Cannot be visualized

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2. Basic Immunologic Procedures

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  1. 2. Basic Immunologic Procedures Terry Kotrla, MS, MT(ASCP)BB Fall 2005

  2. Introduction • Detection of antigen/antibody reactions difficult • Sensitization is the binding of a specific antibody to its’ specific antigen • Cannot be visualized • Multitude of laboratory methods have been developed to make this visible

  3. Three Distinct Phases of Antigen/Antibody Reactions • Primary Phenomenon – Sensitization • Secondary Phenomenon – Lattice formation • Tertiary Phenomenon – Detected by affect on tissues or cells.

  4. Primary phenomenon • Sensitization – binding of antibody to antigen – not visible

  5. Secondary Phenomenon • Lattice Formation • The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination • If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation • http://www.cehs.siu.edu/fix/medmicro/agabx.htm

  6. Tertiary Phenomenon • Reaction not visible, detected by affect of reaction on tissues or cells. • http://www.cellsalive.com/mac.htm

  7. Phagocytosis

  8. Secondary Phenomena Most Frequently Utilized • Precipitation – soluble antibody reacts with soluble antigen • Agglutination – particulate antigens bound together by antibody • Complement Fixation – antibody binding to antigen triggers activation of complement

  9. Antigen-Antibody Binding • Affinity • Avidity • Law of Mass Action

  10. Affinity • Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. • It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site . • Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated in Figure 3. Most antibodies have a high affinity for their antigens.

  11. Affinity

  12. Avidity • Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. • Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies. • Avidity is influenced by both the valence of the antibody and the valence of the antigen. • Avidity is more than the sum of the individual affinities.

  13. Avidity

  14. Law of Mass Action • Governs the reversibility of the antigen-antibody reaction. • Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation.

  15. Precipitation Curve • Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative • Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative • Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

  16. Precipitation Curve

  17. Precipitation Curve

  18. Measurement of Precipitation by Light • Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. • Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. • Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

  19. Precipitation/Flocculation • When soluble antibody binds to soluble antigen (sensitization) there will come a point where lattice formation will occur resulting in precipitation occurring resulting in a visible reaction • These immune complexes have fallen out of solution. The Ab at the bottom in the illustration at right is still in the soluble phase.

  20. Turbidimetry • Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution. • Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. • The more cloudy the solution, the less light can pass through.

  21. Nephelometry • Measures SCATTERED light bouncing off antigen-antibody complexes.

  22. Nephelometry

  23. Passive Immunodiffusion • Reactions in gels • Migrate towards each other and where they meet in optimal proportions form a precipitate. • http://perso.wanadoo.fr/svt.ronsard/svt.ronsard/travaux/exper/albumine/anim1.gif

  24. Four Methodologies • Single diffusion, single dimension • Single diffusion, double dimension • Double diffusion, single dimension • Double diffusion, double dimension

  25. Ouidin Single Diffusion, Single Dimension

  26. Oudin Precipitation • Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two.  • With time at the interface where the two layers meet, antigen-antibody complexes form a visible precipitate.  The other two tubes are negative controls, containing only antibody or only antigen plus an irrelevant protein in the second layer. 

  27. Radial Immunodiffusion

  28. Standard Curve

  29. RADIAL IMMUNODIFFUSION Precipitin Rings A B C a b c Standards Samples Standard Curve

  30. Ouchterlony Gel Diffusion • Holes punched in agar. • Known antibody or antigen added to center well. • Known sample added to outer well. • Unknown sample added to outer well next to unknown sample. • Wait for bands to form.

  31. Ouchterlony Immunodiffusion

  32. Ouchterlony - Identity • The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity.

  33. Ouchterlony – Partial Identity • FIGURE 2:If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity

  34. Ouchterlony – Non-Identity • If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig. 3

  35. Ouchterlony-Interpret • Determine which interpretation fits for samples 1, 2 and 3.

  36. Electrophoretic Techniques • Immunodiffusion can be combined with electrical current to speed things up.

  37. Rocket Immunoelectrophoresis • Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

  38. Rocket Electrophoresis

  39. Immunoelectrophoresis

  40. Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen. Immunoelectrophoresis

  41. Immunoelectrophoresis-Antivenom • Each antibody molecule can bind two separate sites on an antigen molecule (venom toxin), consequently antibodies have the ability to cross link many antigen molecules simultaneously.    This cross-linking causes the antibody antigen-complex to become insoluble and precipitate out from the solution. • The immunoelectrophoresis technique makes use of this capability of the antibodies to form giant insoluble complexes with their respective antigens.    The antigen-antibody precipitate which forms can be visualised by specific staining techniques, or quantified by various means.

  42. Immunofixation Electrophoresis • Immunofixation Electrophoresis (IFE) combines zone electrophoresis with immunoprecipitation. • This technique may be used to identify and characterise serum proteins. • In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospesific antiserá reactive with specific protein - antigen. • If the antigen is present a characteristic immunoprecipitin band will be formed.

  43. Immunofixation Electrophoresis

  44. Immunofixation Electrophoresis

  45. Enhancement of Agglutination • Additive to neutralize charge • Viscosity • Treatment with enzymes • Agitation and centrifugation • Temperature • pH

  46. Direct Agglutination • Antigen found naturally on particle. • Blood Grouping is an example, antigen on cell

  47. ABO Blood Grouping

  48. Passive Agglutination • Employs particles that are coated with antigens, ie , RBCs, polystyrene latex, bentonite or charcoal.

  49. Reverse Passive Agglutination • Antibody attached to carrier particle instead of antigen. • Serologic Typing of Shigella: Positive Test

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