Ag-Ab reactionsTests for Ag-Ab reactions

EISA SALEHI PhD. Immunology Dept.

TUMS

Importance of Ag-Ab Reactions

• Understand the mechanisms of defense• Abs as tools in:Abs as tools in:

– TreatmentDiagnosis– Diagnosis

• As biomarkers• As tools to measure analytesAs tools to measure analytes

Nature of Ag/Ab Reactions

• Lock and Key Concepthttp://www.med.sc.edu:85/chime2/lyso-abfr.htm

Lock and Key Concept

• Non-covalent Bonds– Hydrogen bonds– Electrostatic bonds– Van der Waal forces– Hydrophobic bonds

• Multiple Bonds• Reversible

Multiple Bonds

Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000

Affinity• Strength of the reaction between a single antigenic

determinant and a single Ab combining site

High Affinity Low Affinity

determinant and a single Ab combining site

Ab Ab

Ag Ag

Affinity = attractive and repulsive forces

Calculation of Affinityy

Ag + Ab ↔Ag-Ab

Applying the Law of Mass Action:

[Ag-Ab]Keq =

[ g ]

[Ag] x [Ab]

Avidity• The overall strength of binding between an Ag

with many determinants and multivalent Absy

Keq = 104

Affinity106

Avidity1010

AvidityAffinity Avidity Avidity

S ifi itSpecificity

• The ability of an individual antibody combining site to react with only one antigenic determinant.

• The ability of a population of antibody molecules to react with only one antigen.

Cross Reactivityy• The ability of an individual Ab combining site to

react with more than one antigenic determinant.react with more than one antigenic determinant.• The ability of a population of Ab molecules to

react with more than one Agg

Cross reactions

Anti-A Ab

Anti-A Ab

Anti-A Ab

Ag A Ag B Ag CAg A Ag B

Shared epitope

g

Similar epitope

Factors Affecting Measurement of A /Ab R tiAg/Ab Reactions

• Affinity

• Avidity

A Ab i

Ab excess Ag excess

• Ag:Ab ratio

• Physical form of Agy g

Equivalence – Lattice formation

Do you need to know what happensDo you need to know what happens in Lab.

• Know different sources of random and systematic errorsy

• Know limitations of current techniques• Know which instruments are being used• Know which instruments are being used• Know how reliable the results are in clinical

d i i kidecision making

ErrorsRequesting appropriate testsRequesting appropriate testsWriting prescriptionReading prescriptiong p pSample collectionSampling timesenvironmental factorsDrug interferencesPatient identificationPatient identification Sample transferTechnician errorsTechnician errorsInstrumental errorsMethod limitationsData entry mistakesInterpretation of results How can we trust

What you want

• Accuracy • PrecisionPrecision• Speed

S i i i• Sensitivity• Specificity

Spectrophotometry: Luminescence• Can you tell the difference between how many marks are in each box?

Spectrophotometry: Luminescence

Sensitivity of Absorbance Measurements

Spectrophotometry: Luminescence• Can you tell the difference between how many marks are in each box?

Spectrophotometry: Luminescence

0 40

Sensitivity of Luminescence Measurements

Lab Tests Based on Ag/Ab R tiReactions

• All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes

• All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Antigen antibody testsAntigen antibody tests

• QualitativeQualitative• Semi-quantitative

Q tit ti

• In-vivo• In-vitro

• Quantitative

• End point• Kinetic• Kinetic

Qualitative/quantitative

• Qualitative– determines antigen or antibody is present or

absent• Quantitative

– determines the quantity of the antibody– Titer– The highest dilution of the specimen usually

serum which gives a positive reaction in the testtest

Tube testingTube testing

Classification of Ag-Ab interactions 1.Primary serological tests: (Marker techniques) e.g.

Enzyme linked immuono sorbent assay (ELISA) Immuno florescent antibody technique (IFAT) Radio immuno assay (RIA)

2 Secondary serological tests: e g2.Secondary serological tests: e.g. Agglutination tests Precipitation tests Flocculation testsFlocculation tests Complement fixation tests (CFT) Serum neutralization tests (SNT) Toxin antitoxin testToxin-antitoxin test

3.Tertiary serological test: e.g. Determination of the protective value of an anti serum in an animal. p

Agglutination/hemagglutinationLattice FormationLattice Formation

• Rapid agglutination test• eg Blood group CRP RFeg, Blood group, CRP,RF

• Standard tube agglutination test

• Active• Passive

• Standard tube agglutination test• eg, wright test, Widal test

• Inhibition

Rapid Agglutination tests

Active Agglutination/Hemagglutination

• Definition - tests that have as their endpoint the agglutination of a particulate antigenthe agglutination of a particulate antigen– Agglutinin/hemagglutinin

Q i i i i• Qualitative agglutination test– Ag or Ab

+

Agglutination/Hemagglutination• Quantitative agglutination test

– TiterTiter– Prozone

8 6 2 24

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

2

Pos.

Neg

.

Titer

64

Patient

18

512<2

234 <2

32128

456

324

78

Agglutination/Hemagglutinationgg gg

2 4 8 16 32 64 128

256

512

1/2

1/4

1/8

1/1

1/3

1/6

1/1

1/2

1/5

• Applications Blood typing– Blood typing

– Bacterial infections–Fourfold rise in titer

• Practical considerations– EasyEasy– Semi-quantitative

Passive Agglutination/Hemagglutinationgg gg

• Definition - agglutination test done with aDefinition agglutination test done with a soluble antigen coated onto a particle

+CRP

• ApplicationsApplications– Measurement of antibodies to soluble antigens

Coombs (Antiglobulin)Tests

•Direct Coombs Test– Detects antibodies on erythrocytes

+

Patient’s RBCs Coombs Reagent(Antiglobulin)( g )

Coombs (Antiglobulin)Tests

• Indirect Coombs TestDetects anti erythrocyte antibodies in serum– Detects anti-erythrocyte antibodies in serum

St 1

Patient’s S

TargetRBC

+Step 1

Serum RBCs

Step 2

+

Coombs Reagent(Antiglobulin)

Applications of Coombs (Antiglobulin)Tests

Detection ofDetection of Incomplete AbDetection of

anti-Rh Abanti-Rh Ab

A iAutoimmune hemolytic anemia

Agglutination/Hemagglutination Inhibition• Definition - test based on the inhibition of

agglutination due to competition with a soluble Agagglutination due to competition with a soluble Ag

Prior to Test

+

T t

+ +

Test

Patient’s sampleHCG

Precipitation testsPrecipitation testsLattice Formation

Th i d ib d i l bl• The antigen and antibody are in soluble form

• Combine to form a visible precipitate• Presence of electrolytesy• Positive controls and negative controls

LAB tests based on Precipitation

• In gel or solid supportg pp• eg, Immunodiffusion (Single radial, Double) ,

Immunochromatography, immunofixation l t h i I l t h ielectrophoresis, Immunoelectrophoresis,

Countercurrent electrophoresis (CCEP),

• In solutionIn solution • eg, Ring test, Turbidimetry, Nephelometry

Radial Immunodiffusion (Mancini)

• Method Ab in gelMethod– Ab in gel– Ag in a well

AgAgAgAg

• Interpretation– Diameter of ring is

g

proportional to the concentration

• Quantitative Dia

met

er2

Quantitative– Ig levels

Ag Concentration

D

Ag Concentration

Immunoelectrophoresis• Method

– Ags are separated by electrophoresis

-+

– Ab is placed in trough cut in the agar

Ag+

Ag

Ab

Ag

i

Ab

• Interpretation– Precipitin arc represent individual antigens

Countercurrent electrophoresis• Method

– Ag and Ab migrate toward each other by g g yelectrophoresis

– Used only when Ag and Ab have opposite charges

- +Ag Ab

• Qualitative–Rapid

Immuno-preciptation in solution

• Ring test• TurbidimeteryTurbidimetery• PET

N h l• Nephelometery

Light reflection

Molecular size and scattering

+- -

Nephelometry vs. Turbidimetry

0°-90°

Flocculation• Ags are lipids

• eg, VDRL, RPR

Test based on Primary Ag-Ab reation• Lattice formation not required• How can be visualized??

Detection of cell associated analytes:ImmunofluorescenceFlowcytometry

Detection of soluble Analytes:Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA)(ELISA)

Immunoassays basics

(virus?)

Indirect versus direct (sandwich) ELISA

Why ELISA cant be fully automated

Competitive RIA/ELISA for AgM h d• Method cont.– Determine amount

of labeled Ag boundof labeled Ag bound to Ab

TestSolidSolid

+ +Patient’sLabeled

+SolidPhase

SolidPhase

sampleAg

C i d i d f d d– Concentration determined from a standard curve using known amounts of unlabeled Ag

• Quantitative– Most sensitive test

Tests for Cell Associated Antigens

Lattice formation not required

Phosphorescence

Chemiluminescence

BioluminescenceBioluminescence

Immunofluorescence

• DirectAb i A i l b l d i h fl h– Ab to tissue Ag is labeled with fluorochrome

FluorochromeLabeled Ab

AgTissue Section

Immunofluorescence

• Indirect FluorochromeLabeled Anti-Ig

– Ab to tissue Ag is unlabeledFl h l b l d i

Labeled Anti-IgUnlabeled

Ab

– Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab.

AgTissue Sectionof the first Ab.

• Qualitative to Semi-Q tit tiQuantitative

Fluorescence

FITC + UV light

Immunofluorescence

• Flow CytometryCells in suspension are labeld with fluorescent tag– Cells in suspension are labeld with fluorescent tag

• Direct or Indirect Fluorescence– Cells analyzed on a flow cytometery y

FlowTip FLTip

Detector

LightScatter

Detector

Laser

Immunofluorescence

• Flow Cytometry cont.Data displayed– Data displayed

One Parameter Histogram Two Parameter HistogramOne Parameter Histogram

nten

sity

Two Parameter Histogram

of C

ells

Unstained cells

ores

cenc

e In

Num

ber

o FITC-labeled cellsG

reen

Flu

o

Green Fluorescence Intensity Red Fluorescence Intensity

Assays Based on ComplementAssays Based on Complement

Lattice formation not required

Complement Fixation

– Ag mixed with test serum to be assayed for AbStandard amount of complement is added

• Methodology

– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determinedAmount of erythrocyte lysis is determined

Ag No Ag

Patient’s

g g

Ag

Agserum

Relative sensitivities of tests (approx)

Usual operating range [Ab] or [Ag]

precipitationimmunoelectrophoresis 10 μg/ml - 1 mg/mlimmunoelectrophoresisdouble/radial diffusion

10 μg/ml 1 mg/ml

immunofluorescence 0.1 - 10 μg/ml

ELISA (colour) 0.1 - 10 ng/ml ELISA (colour) 0.1 10 ng/ml (chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0 01 10 ng/ml radioimmunoassay 0.01 - 10 ng/ml