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Chap. 6Chap. 6 AntigenAntigen--AntibodyAntibody

interactionsinteractionsCharacterized asCharacterized as::

NonNon--covalent interaction (similar to lock andcovalent interaction (similar to lock andkey fit of enzymekey fit of enzyme--substrate)substrate)

Does not lead to irreversible alteration of AgDoes not lead to irreversible alteration of Agor Abor Ab

This exact and specific interaction has led toThis exact and specific interaction has led tomany immunological assays used to:many immunological assays used to:

detectdetect AgAg oror AbAb

diagnosediagnose diseasedisease

measure magnitude ofmeasure magnitude ofhumoral IRhumoral IR

identify molecules ofidentify molecules ofbiobio andand medmed interestinterest

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Ag-Ab interactions

Bonds:

Hydrogen

Ionic

Hydrophobic interactions

Van der Waals forces

Each bond is weak; many arestrong

To hold they must be close requiring high amts of

complementarity!

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Affinity-

strength of interaction between one

Ag-binding site and one epitope

Measured by various methods including

equilibrium dialysis, competition assays.

Avidity

Multiple interactions between antibody and

antigen

IgM (with 10 antigen binding sites per molecule)

tends to have low affinity but high avidity

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Figure 18.4

Precipitation ReactionsPrecipitation Reactions

Reaction of soluble antigens withReaction of soluble antigens withantibodiesantibodies

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Figure 18.3

A Precipitation CurveA Precipitation Curve

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Immunologic testsImmunologic tests::

3)3) Agglutination reactionsAgglutination reactions simple,simple,

inexpensive,inexpensive, but sensitivebut sensitive!!

Several types exist:Several types exist:

a) Hemagglutination ofRBCsa) Hemagglutination ofRBCs

b) Bacterial Agglutinationb) Bacterial Agglutination

c) Passive Agglutinationc) Passive Agglutinationd) Agglutination Inhibitiond) Agglutination Inhibition

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Figure 18.5

Agglutination ReactionsAgglutination Reactions

ParticulateParticulateantigens andantigens andantibodiesantibodies

Antigens on aAntigens on acell (cell (directdirectagglutinatioagglutinationn))

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Figure 18.7

Indirect AgglutinationIndirect Agglutination

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Antibody TiterAntibody Titer

ConcentrationConcentrationof antibodiesof antibodiesagainst aagainst a

particularparticularantigenantigen

Figure 18.6

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Figure 18.8

HemagglutinationHemagglutination

HemagglutinationHemagglutination involvesinvolvesagglutination ofRBCsagglutination ofRBCs

Some viruses agglutinate RBCs in vitroSome viruses agglutinate RBCs in vitro

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Figure 18.9b

Viral HemagglutinationViral Hemagglutination--

InhibitionInhibitionHemagglutination involvesHemagglutination involvesagglutination ofRBCsagglutination ofRBCs

Some viruses agglutinate RBCs in vitroSome viruses agglutinate RBCs in vitro

Antibodies prevent hemagglutinationAntibodies prevent hemagglutination

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Figure 18.9a

Neutralization ReactionsNeutralization Reactions

Eliminate the harmful effect of a virusEliminate the harmful effect of a virusor exotoxinor exotoxin

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Complement Fixation TestComplement Fixation Test

Figure 18.10

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Complement Fixation TestComplement Fixation Test

Figure 18.10

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Immunologic testsImmunologic tests::

4)4) RadioimmunoassayRadioimmunoassay (RIA)(RIA) veryverysensitive test; used for measuringsensitive test; used for measuringhormones, serum proteins, drugs, etc. athormones, serum proteins, drugs, etc. atlow [C]s (low [C]s ( 0.001ug/ml) 0.001ug/ml)

measures measures competitive bindingcompetitive binding ofofradiolabelledradiolabelled Ag + unlabelled (test) Ag toAg + unlabelled (test) Ag tohigh affinityhigh affinity AbAb

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Immunologic testsImmunologic tests::

5. ELISA tests: dep on enzyme conugated to 2 Ab reacting with a specific

substrate to produce a color rxn. Most sensitive of tests for Ag/Ab!!

Variations of ELISAs:

Allows for qualitative or quantitative testing.

Each one can be used for qualitative detection of Ag or Ab

Also, a std curve based on known [C]s of Ag/Ab can be prepped and an

unknown [C} can be ascertaineda. Indirect ELISA

b. Sandwich ELISA

c. Competitive ELISA

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Direct ELISADirect ELISA

Figure 18.14a

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Indirect ELISAIndirect ELISA

Figure 18.14b

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Immunologic testsImmunologic tests:: Types of ELISAsTypes of ELISAs

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Immunologic tests:Immunologic tests: 8)8)ImmunofluorescenceImmunofluorescence

Provides a quick method for the id ofProvides a quick method for the id of

pathogens and lymphocytespathogens and lymphocytes

AbsAbs are conjugated with a fluorescent dyeare conjugated with a fluorescent dye((fluoresceinfluorescein,, rhodaminerhodamine,, phycoerythrinphycoerythrin))

If If AbsAbs bind to specific Ags, they can bebind to specific Ags, they can be illumillum

w/ UV light and emit bright colorsw/ UV light and emit bright colors

There are currently 2 methods employed:There are currently 2 methods employed:Direct stainingDirect staining

Indirect stainingIndirect staining

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Figure 18.11a

FluorescentFluorescent--Antibody (FA)Antibody (FA)

TechniquesTechniquesDirect FADirect FA

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Figure 18.11b

Indirect FluorescentIndirect Fluorescent--AntibodyAntibody

TestTest

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Figure 18.12

FluorescenceFluorescence--Activated CellActivated Cell

Sorter (FACS)Sorter (FACS)

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Serological TestsSerological Tests

Figure 18.13

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Serological TestsSerological Tests

PrecipitationPrecipitation: Soluble antigens: Soluble antigens

AgglutinationAgglutination: Particulate antigens: Particulate antigens

HemagglutinationHemagglutination: Agglutination of RBCs: Agglutination of RBCs

NeutralizationNeutralization: Inactivates toxin or virus: Inactivates toxin or virus

FluorescentFluorescent--antibody techniqueantibody technique: Antibodies: Antibodies

linked to fluorescent dyelinked to fluorescent dye

Complement fixationComplement fixation: RBCs are indicator: RBCs are indicator

ELISAELISA: Peroxidase enzyme is the indicator: Peroxidase enzyme is the indicator