Basic Immunologic Procedures Part 1 November 11, 2014

Introduction Detection of antigen/antibody reactions difficult Can measure EITHER antigen or antibody. Sensitization is the binding of a specific antibody to its’ specific antigen Sensitization cannot be visualized Multitude of laboratory methods have been developed to make this reaction visible

Factors Which Affect Reactions Concentrations of reactants Temperature Length of incubation pH of test system

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 single antibody to single antigen site These tests are Difficult Complex Expensive Require special equipment Time consuming

Primary phenomenon Sensitization – binding of antibody to antigen – not visible

Primary Phenomenon Tests Techniques include: Immunofluorescence Radioimmunoassay Enzyme immunoassay

Secondary Phenomenon Sensitization taken a step further called lattice formation Antibody molecule binds to two separate antigens on adjacent antigens If antigen on large structures such as RBCs causes agglutination. If both antibody and antigen are soluble results in precipitation precipitation agglutination

Secondary Phenomenon These tests are: Downside is Easy to perform Less expensive Less time consuming Do not require special equipment Downside is Less sensitive Less specific More interference

Secondary Phenomenon Examples of tests: Precipitation Agglutination Complement Fixation

Tertiary Phenomenon Reaction not visible, detected by affect of reaction on tissues or cells. Tests include: Inflammation Phagocytosis Deposition of immune complexes Immune adherence Chemotaxis

Phagocytosis

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-The higher the affinity of the antibody for the antigen, more stable will be the interaction Avidity-Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect Law of Mass Action

Antigen-Antibody Binding Union of antigen and antibody requires Affinity Avidity Affinity and avidity determined by Law of Mass Action

Antibody Affinity Describes the strength of a single Ag-Ab bond. When Ag and Ab come close together a chemical bond forms which is weak and can dissociate. How well the Ab fits the Ag will determine stability of bond, “lock and key” fit has strongest affinity. Ab may react with structurally similar Ags, results in cross reactivity. Most antibodies have a high affinity for their antigens.

Affinity

Avidity Describes the combined strength of multiple Ag-Ab bonds. Initially bond is easily broken, but multiple bindings at the same time the dissociation is overcome by the sheer number of bonds remaining. Avidity is influenced by both the valence of the antibody and the valence of the antigen.

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

Antibody Excess Antigen Excess

Precipitation Curve

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.

Four Methodologies Single diffusion, single dimension Single diffusion, double dimension Double diffusion, single dimension Double diffusion, double dimension

Oudin Single Diffusion, Single 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 (The Mancini method) In radial immunodiffusion, an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum. The antigen diffuses in all directions from the well, and accordingly the region of equivalence is established and a ring of precipitation (precipitin ring) forms around the well. The area of the precipitin ring is proportional to the concentration of antigen. The diameter of the area of precipitation (including the well diameter) is measured to determine the concentration of antigen.

Double immunodiffusion (The Ouchterlony method) In double immunodiffusion , if antigen to be detected, a known reagent antibody is placed in the center well and the unknown samples are placed in the surrounding well. If antibody is to be detected, unknown antigen is placed in the center. After each of the samples and reagents have been added to the appropriate wells, diffusion occurs and both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. A line of precipitation forms at the zone of equivalence.

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 Immunodiffusion

Ouchterlony - Identity The antibodies in the antiserum react with both the antigens resulting in a smooth line of precipitate. The antibodies cannot distinguish between the two antigens. i.e., the two antigens are immunologically identical.

Ouchterlony – Partial Identity In the ‘pattern of partial identity’, the antibodies in the antiserum react more with one of the antigens than the other. The ‘spur’ is thought to result from the determinants present in one antigen but lacking in the other antigen

Ouchterlony – Non-Identity In the ‘pattern of non-identity’, none of the antibodies in the antiserum react with antigenic determinants that may be present in both the antigens, i.e., the two antigens are immunologically unrelated as far as that antiserum is concerned.

End of Part 1