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