2. basic immunologic procedures terry kotrla, ms, mt(ascp)bb fall 2005
TRANSCRIPT
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
Standard Curve
Precipitin Rings A B C
a b c
Standards Samples
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://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://web.indstate.edu/thcme/PSP/labtests/precip.htm http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibo
dy.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