880 likes | 896 Views
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
E N D
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 • Multitude of laboratory methods have been developed to make this visible
Three Distinct Phases of Antigen/Antibody Reactions • Primary Phenomenon – Sensitization • Secondary Phenomenon – Lattice formation • Tertiary Phenomenon – Detected by affect on tissues or cells.
Primary phenomenon • Sensitization – binding of antibody to antigen – not visible
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
Tertiary Phenomenon • Reaction not visible, detected by affect of reaction on tissues or cells. • http://www.cellsalive.com/mac.htm
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
Antigen-Antibody Binding • Affinity • Avidity • Law of Mass Action
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.
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.
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.
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
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.
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.
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.
Nephelometry • Measures SCATTERED light bouncing off antigen-antibody complexes.
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
Four Methodologies • Single diffusion, single dimension • Single diffusion, double dimension • Double diffusion, single dimension • Double diffusion, double dimension
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.
RADIAL IMMUNODIFFUSION Precipitin Rings A B C a b c Standards Samples Standard Curve
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.
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.
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
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
Ouchterlony-Interpret • Determine which interpretation fits for samples 1, 2 and 3.
Electrophoretic Techniques • Immunodiffusion can be combined with electrical current to speed things up.
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.
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
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.
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.
Enhancement of Agglutination • Additive to neutralize charge • Viscosity • Treatment with enzymes • Agitation and centrifugation • Temperature • pH
Direct Agglutination • Antigen found naturally on particle. • Blood Grouping is an example, antigen on cell
Passive Agglutination • Employs particles that are coated with antigens, ie , RBCs, polystyrene latex, bentonite or charcoal.
Reverse Passive Agglutination • Antibody attached to carrier particle instead of antigen. • Serologic Typing of Shigella: Positive Test