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