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

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 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.

Affinity

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.

Avidity

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

Precipitation Curve

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.

Nephelometry

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

Ouidin 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

Standard Curve

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 Immunodiffusion

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.

Rocket Electrophoresis

Immunoelectrophoresis

Immunoelectrophoresis 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-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.

Immunofixation Electrophoresis

Immunofixation Electrophoresis

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

ABO Blood Grouping

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

Agglutination Inhibition Based on competition between particulate and soluble antigens for limited antibody combining sites. Patient sample added to reagent antibody specific for antigen being tested, if antigen is present it binds to reagent antibody. Reagent particles (latex or RBCs) coated with the same antigen are added, if antigen was present in the sample all reagent antibody binds to it so no antibody is present to react with antigens coating the particles

Agglutination Inhibition In row A wells 1-8 are positive.

Coagglutination Name given to systems using bacteria as the inert particles to which antibody is attached.

Labeled Immunoassays Some antigen/antibody reactions not detected by precipitation or agglutination. Measured indirectly using a labeled reactant. Referred to as receptor-ligand assays. Ligand is the substance to be measured and is defined as a molecule that binds to nother molecule of a complementary configuration, usually it binds to the substance the test is trying to detect. The receptor is what binds the specific target molecule.

Labeled Immunoassays Ligand – substance to be measured. Receptor – binds the specific target molecule Sandwich technique Next slide - example

“Sandwich” Technique ELISA

Labels Used to detect reaction which has occurred. Radioactive, fluorescent, chemiluminescent and enzymes. Competitive or non-competitive Heterogenous or homogeneous

Standards or Calibrators Substance of exact known concentration. Usually run for each new lot number Based on results create standard curve. Standard curve used to “read” results or built into machine to provide results.

Competitive Binding Add known labeled antigen Add unknown antigen Will compete with each other for sites on bound antibody molecule. Must wash off unreacted substances. Type of label on known antigen will determine method of detection.

Competitive Binding

Radioimmunoassay http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/ria/ria.htm

Radioimmunassay (RIA) Competitive binding Uses Iodine 125 (I 125) as label Radioactive label competes with patient for sites High radioactivity, small amount of patient substance Low radioactivity high amount of patient substance Textbook page 159 figure 11-2

Radioimmunassay

Radioimmunoassay Competitive A plasma (or urine) sample that contains ADH is mixed with the antibodies. A known amount of "standard" ADH that has been tagged with radioactivity is added to the antibody mixture. The "standard" ADH is something that has already been measured and that can be used to compare the unknown quantity of natural ADH. A radioactive tag is added to the standard ADH so that it can be distinguished from the natural ADH that is in the plasma. As the antibodies, the standard radioactive ADH, and the plasma are mixed, one main requirement must be met: there must be too little antibody to bind completely to both the radioactive hormone and the natural hormone in the fluid to be assayed. Therefore, the natural hormone in the plasma and the radioactive standard hormone compete for the binding sites on the antibody (next slide)

Radioimmunoassay Competitive

Immunoradiometric Assay (IRMA) Labeled antibody plus patient antigen Solid phase antigen added to bind excess antibody Spin down to separate Labeled antibody/antigen remain in solution. Measure radioactivity Textbook page 160 fig 11-3

Enzyme Immunoassay Advantages Reason for choosing enzyme as label Enzymes used Two classifications: Heterogenous – separation of reactants must be done Homogeneous – no separation step Two types: Competitive – known and unknown compete Noncompetitive – only unknown reacts

Enzyme Immunoassay http://tinyurl.com/ckdaz http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/elisa/elisa.htm http://tinyurl.com/ckdaz

Heterogenous Enzyme Assays Competitive ELISA Noncompetetive ELISA Immunoenzymometric Assay Sandwich or capture assays

Competitive ELISA Unknown antigen competes with labeled known antigen Enzyme produces color reaction

Noncompetitive ELISA Referred to as “Indirect” ELISA Antigen bound to solid phase Add patient, antibody will bind if present Add known labeled antibody Measure enzyme label Textbook page 161 figure 11-4 Disadvantage more manipulation of test

Immunoenzymometric Assay Noncompetitive ELISA Detects unknown antigen by means of excess labeled antibody Textbook page 162 figure 11-5

Sandwich or Capture Assays Used for antigens with multiple epitopes, ie, HCG Antibody to one epitope fluid, antibody to second epitope fixed. Enzyme label used to detect reaction Textbook page 163 figure 11-6

Homogeneous Enzyme Assay Labeled reagent antigen and patient sample added. Compete for bound antibody Antibody binds to patient antigen causing inactivation of enzyme Competitive Assay Enzyme activity proportional to concentration of patient antigen

Fluorescence Immunoassay

Markers Fluorophores or fluorochromes Ability to absorb energy and emit light Two most commonly used: Fluorescein – green Tetramethylrhodamine – red

Fluorescence

Fluorescence Antibodies and bacteria are fixed on a glass-plate. The surplus i.e. non-bounded antibodies are washed out, antibody-bacteria-complexes ("sandwiches") remain. The "sandwich" becomes visible by adding fluorescent anti bovine immunoglobulin which can be seen as green light in the fluorescence microscope.

Fluorescent Immunoassay Direct immunofluorescence Tagged antibody added to unknown antigen fixed to slide Indirect immunofluorescence Patient plus known fixed antigen Add tagged anti-antibody Fluorescence

Positive

Heterogeneous Fluorescent Immunoassays

Homogenous Assays

Fluorescence Polarization Immunoassay http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/fpia/fpia.htm

References http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html http://web.indstate.edu/thcme/PSP/labtests/precip.htm http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibody.htm http://www.cellsalive.com/mac.htm http://jeeves.mmg.uci.edu/immunology/Assays/Assays.htm http://www.medschool.lsuhsc.edu/microbiology/DMIP/dmex03.htm http://www.tulipgroup.com/Common/html/TurbidTech.pdf http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm http://www.mercodia.se/global/mainpage.asp?page_id=41 ELISA http://www.clinprointl.com/technical.htm ELISA http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm molecular diagnostics

References (Continued) http://www.liv.ac.uk/~agmclen/Medpracs/practical_5/theory_5.html http://www.fao.org/docrep/W0049E/w0049e06.htm