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SI OPD LECTURE NOTESIMMUNOLOGICAL TECHNIQUES
ANTIGEN substances or molecule that trigger the
production of antibody
ANTIBODY
SENSITIZATION basic reaction of antigen and antibody binding measure antigen or antibody
FACTORS THAT AFFECT Ag-Ab REACTION:1. Concentration of reactants2. Temperature3. Length of incubation4. pH of the system
3 DISTINCT PHASES (Ag-Ab Combination):1. PRIMARY PHENOMENON
Sensitization Initial Ag-Ab binding Single Ab to single Ag site Test to detect these reactions are
difficult, complex, expensive, require special equipment and time consuming
i.e. immunoflourescence, RIA, EIA
2. SECONDARY PHENOMENON Aggregation of complexes Lattice formation to create large
molecules that are easily detectable Combination of antibody and
multivalent antigen to produce a stable network that results in a visible reaction
Methods are quick and easy to perform, less expensive, less time-consuming, do not require special equipment, less specific, less sensitive, more interferences
Precipitation, agglutination, complement fixation
3. TERTIARY PHENOMENON Antigen-antibody reaction is not visible
but is detected by the effect of the reaction on tissues or on cells
Inflammation, phagocytosis, deposition of immune complexes, chemotaxis, immune adherence
METHOD OF CHOICE: SECONDARY PHENOMENON
PRECIPITATION Soluble antigen and soluble antibody Produce insoluble complexes
AGGLUTINATION Particulate antigen such as cells are aggregated
to form large visible aggregates if the specific antibody is present
COMPLEMENT FIXATION Triggering of the classical complement pathway
due to the combination of antigen with specific antibody
PRIMARY UNION OF AG AND AB DEPENDS ON 2 CHARACTERISTICS:
1. AVIDITY2. AFFINITY
Weak Initial forces of attraction that an antibody
has for a specific epitope or determinant
EPITOPE Determinant size Key portion of ag against which immune
response is directed
BONDS1. IONIC BOND
Between oppositely charged particles2. HYDROGEN BOND
Involve an attraction between polar molecules that have a slight charge separation and in which a positive charge resides on a hydrogen atom
3. HYDROPHOBIC BONDS Occur between non-polar molecules
that associate with one another and excludes molecules of water as they do so
4. VAN DER WAALS Interaction between electron clouds of
associating dipoles
Weak bonds Dissociation easily occur Strength- specificity of ab to the ag
AVIDITY
Sum of all attractive forces between antigen and antibody
Force that stabilizes the ag-ab reaction keeping the molecules together
The stronger the chemical bonds that form between ag and ab, the less likely that the reaction will reverse
LAW OF MASS ACTION Governs the reversibility of ag-ab reactions Free reactants are in equilibrium with bound
reactants Equilibrium constant Rate of ag-ab binding and dissociating Measures the goodness of fit Avidity (up), rate of dissociation (down)
PRECIPITATION Simplest method of detecting ag-ab ; initial turbidity
is followed by precipitation
PRO-ZONE Excess antibody False negative results
POSTZONE Excess antigen False negative reaction
ZONE OF EQUIVALENCE Optimum precipitation occurs where the
number of multivalent sites of ag and ab are approximately equal
NEPHELOMETRY Measures light scattered at a particular angle
from the incident beam as it passes through the suspension
Amount of light scattered is dependent on the number and size of particles scattered in the light beam
Several automated instruments are based on these principles
Light scatter < 10o to about 70o
2 KINDS:1.ENDPOINT
Reaction is allowed to run to completion but particles tend to fall out of solution and decrease light scatter
2.KINETIC
Rate of increase in scattering immediately after reagent is added
TURBIDIMETRY Denote turbidity Detection device is placed in a direct line to the
incident light after it has passed through solution
Measure the reduction in light intensity due to reflection, absorption of scatter
Scattering of light occurs in proportional to size, shape, concentration of molecules to the amount of solution
Amount of substance being quantitated is calculated based on results on obtained from standards and control
Very simple but not very sensitive
Rate change is directly proportional to antigen-antibody concentration
This technique is used to quantitate immunoglobulins, complement components, CRP and other acute phase proteins
PASSIVE IMMUNODIFFUSION Precipitation of ag-ab complexes can also be
determined in support medium __> gel (agar, agarose)
Agar= high molecular weight complex; derived from seaweed
Agarose= modified agar Reactant is added Ag-ab reaction __> diffusion When no electric current is used to speed up
the complex __> passive immunodiffusion
Rate is affected by:a. Gel viscosityb. Size of particlesc. Temperaturesd. Amount of hydratione. Interaction between matrix and reactants
*Between agarose and agar, agarose is often preferred since agar has a strong negative charge; interaction between gel and reagent are minimized in agarose
CLASSIFICATIONS:1.SINGLE DIFFUSION, SINGLE DIMENSION
1 reactant moving, either up or down
2.SINGLE DIFFUSION, DOUBLE DIMENSION
1 reactant moving, moving out radially from a well
3.DOUBLE DIFFUSION, SINGLE DIMENSION Both reactants moving, either up or down
4.DOUBLE DIFFUSION, DOUBLE DIMENSION Both reactants moving, moving our radially
Examples: OUDIN SINGLE DIFFUSION
Ab is added to agarose (test tube) Ag is layered over gelled ab and diffuses
down in agarose Precipitin bond will form in the gel
RADIAL IMMUNODIFFUSION Single diffusion, double dimension Ab is incorporated in gel (plate or petri dish) Amount of precipitate is directly related to
amount of ag present C3
2 METHODS:1. ENDPOINT
Allows reaction to go to completion2. KINETIC
Employs measurement taken before zone of equivalence is reacted
TECHNICAL SOURCES OF ERROR:a.Overfilling or underfilling of the wellb.Spilling sample outside the wellc. Nicking the welld.Improper incubation time or temperaturee.Plate unleveledf. Bumping the plate g.Plate dries out
DOUBLE DIFFUSION, DOUBLE DIMENSION OUCHTERLONY Both ag and ab diffuse from wells and travel
toward each other Comparison of antigens
PATTERN:1.NON-IDENTITY
Cross lines2.PARTIAL IDENTITY
Fusion of 2 lines with a spur 2 lines- common determinant but some ab
molecules are not captured by ag and travel to the initial precipitin line combined to the additional determinant found in more complex ag
Spur- always point to the simpler ab
3.IDENTITY Arc
FACTORS AFFECTING ACCURACY Drying out of gel Inadequate time for diffusion Bacterial or fungal contamination of gel
ELECTROPHORETIC TECHNIQUEIMMUNODIFFUSION AND ELECTRIC CURRENT
Separates molecules according to differences in electrical charge when exposed to electrical current
Densitometer Agar once stained; densitometer reads the
patterns For protein and Hb electrophoresis
Direct current to gel Ag-ab migrate Diffusion Precipitin bonds are formed Maybe single or double diffusion
ROCKET IMMUNOELECTROPHORESIS As concentration of ag changes, there is
dissolution or the formation of precipitate for every increasing intervals
Conical shape__> rocket Height measured Directly proportional to concentration of ag
ADVANTAGE OVER RID:1.Results obtained in a few hours2.Quantitate immunoglobulins
Too low for nephelometry Too high for RID
IMMUNOFIXATION ELECTROPHORESIS After electrophoresis has taken place, ab is placed
directly to gel Precipitation in shorter time Bonds with higher resolution
WESTERN BLOT- HIV
IMMUNOELECTROPHORESIS Screening test to differentiate serum proteins and
detect abnormalities Lymphomas Other lymphoproliferative disorders
SOURCES OF ERROR:1.Current applied in wrong direction
2.Incorrect buffer pH3.Incorrect timing4.Amount of current applied
AGGLUTINATION Particulate ag (cells) are coated by ag in such a
way that large complexes
STEPS:1.Sensitization2.Antibody production3.Lattice formation4.Enhancement of agglutination
TYPES:1.DIRECT2.PASSIVE3.COAGGLUTINATION4.REVERSE PASSIVE5.AGGLUTINATION INHIBITION
FACTORS THAT GOVERN LATTICE FORMATION Lattice formation- combination of ab and
multivalent ag to1.pH2.ionic strength3.temperature
ENHANCEMENT OF LATTICE FORMATION LISS- low ionic strength of buffer ALBUMIN (5-30%)- neutralize surface charges and
facilitate agglutination AGITATION/CENTRIFUGATION ENZYMES-increase the viscosity
Dextran Polvinyl pyrrdidone Serum albumin
Papain- most frequently used enzyme Bromylene Ficin- reduce surface charge of RBCs through
cleaving chemical groups, decreasing migration
DIRECT Antigens found naturally on the particle __>
identification of bacterial types If RBCs are involved: hemagglutination ABO typing
PASSIVE Antigens are artificially attached to a particle Polystyrene latex Bentonite
Charcoal
REVERSE Antibody rather than antigen is attached to carrier
particle
AGGLUTINATION INHIBITION Based on competition of particulate insoluble
antigen for limited antibody binding site __> lack of agglutination: +
COAGGLUTINATION Utilizes bacteria from carrier particle to which ab
is attached
Staphylococcus aureus Most frequently used Protein A on its outer surface
ANTIGLOBULIN MEDIATED Coomb’s Test
Determine non-agglutinating antibody by means of coupling with 2nd antibody