ag-ab reactions 1.ppt - انجمن علمی دکترای علوم آزمایشگاهی...
TRANSCRIPT
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Ag-Ab reactionsTests for Ag-Ab reactions
EISA SALEHI PhD. Immunology Dept.
TUMS
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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
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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
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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
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Calculation of Affinityy
Ag + Ab ↔Ag-Ab
Applying the Law of Mass Action:
[Ag-Ab]Keq =
[ g ]
[Ag] x [Ab]
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Avidity• The overall strength of binding between an Ag
with many determinants and multivalent Absy
Keq = 104
Affinity106
Avidity1010
AvidityAffinity Avidity Avidity
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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.
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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
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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
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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
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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
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What you want
• Accuracy • PrecisionPrecision• Speed
S i i i• Sensitivity• Specificity
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Spectrophotometry: Luminescence• Can you tell the difference between how many marks are in each box?
Spectrophotometry: Luminescence
Sensitivity of Absorbance Measurements
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Spectrophotometry: Luminescence• Can you tell the difference between how many marks are in each box?
Spectrophotometry: Luminescence
0 40
Sensitivity of Luminescence Measurements
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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
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Antigen antibody testsAntigen antibody tests
• QualitativeQualitative• Semi-quantitative
Q tit ti
• In-vivo• In-vitro
• Quantitative
• End point• Kinetic• Kinetic
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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
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Tube testingTube testing
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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
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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
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Rapid Agglutination tests
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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
+
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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
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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
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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
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Coombs (Antiglobulin)Tests
•Direct Coombs Test– Detects antibodies on erythrocytes
+
Patient’s RBCs Coombs Reagent(Antiglobulin)( g )
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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)
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Applications of Coombs (Antiglobulin)Tests
Detection ofDetection of Incomplete AbDetection of
anti-Rh Abanti-Rh Ab
A iAutoimmune hemolytic anemia
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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
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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
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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
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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
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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
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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
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Immuno-preciptation in solution
• Ring test• TurbidimeteryTurbidimetery• PET
N h l• Nephelometery
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Light reflection
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Molecular size and scattering
+- -
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Nephelometry vs. Turbidimetry
0°-90°
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Flocculation• Ags are lipids
• eg, VDRL, RPR
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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)
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Immunoassays basics
(virus?)
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Indirect versus direct (sandwich) ELISA
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Why ELISA cant be fully automated
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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
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Tests for Cell Associated Antigens
Lattice formation not required
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Phosphorescence
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Chemiluminescence
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BioluminescenceBioluminescence
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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
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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
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Fluorescence
FITC + UV light
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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
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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
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Assays Based on ComplementAssays Based on Complement
Lattice formation not required
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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
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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