Post on 01-Jan-2017
Ag-Ab reactionsTests for Ag-Ab reactions
Nature of Ag/Ab Reactions
• Lock and Key Concept
• Non-covalent Bonds– Hydrogen bonds– Electrostatic bonds– Van der Waal forces– Hydrophobic bonds
• Reversible
• Multiple Bonds
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
Affinity = ∑ attractive and repulsive forces
Ab
Ag
High Affinity
Ab
Ag
Low Affinity
Affinity• Strength of the reaction between a single antigenic
determinant and a single Ab combining site
Calculation of Affinity
Ag + Ab Ag-Ab
Keq = [Ag-Ab]
[Ag] x [Ab]
Applying the Law of Mass Action:
Avidity• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity106
Avidity1010
Avidity
Specificity
• 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 Reactivity• The ability of an individual Ab combining site to
react with more than one antigenic determinant.• The ability of a population of Ab molecules to
react with more than one Ag
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope
Anti-A Ab
Ag C
Similar epitope
Cross reactions
Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
Tests Based on Ag/Ab Reactions
• 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
Agglutination Tests
Lattice Formation
Agglutination/Hemagglutination
• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin
+ ↔
• Qualitative agglutination test– Ag or Ab
Agglutination/Hemagglutination• Quantitative agglutination test
– Titer– Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
1/10
24
Pos.
Neg
.
Titer
648
512<232
128324
Patient
12345678
Agglutination/Hemagglutination
• Definition • Qualitative test• Quantitative test• Applications
– Blood typing– Bacterial infections
–Fourfold rise in titer
• Practical considerations– Easy– Semi-quantitative
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
Passive Agglutination/Hemagglutination
• Definition - agglutination test done with a soluble antigen coated onto a particle
+ ↔
• Applications– Measurement of antibodies to soluble antigens
Coombs (Antiglobulin)Tests
• Incomplete Ab• Direct Coombs Test
– Detects antibodies on erythrocytes
+ ↔
Patient’s RBCs Coombs Reagent(Antiglobulin)
Coombs (Antiglobulin)Tests • Indirect Coombs Test
– Detects anti-erythrocyte antibodies in serum
Patient’s Serum
TargetRBCs
+ ↔Step 1
+ ↔Coombs Reagent
(Antiglobulin)
Step 2
Coombs (Antiglobulin)Tests • Applications
– Detection of anti-Rh Ab– Autoimmune hemolytic anemia
Agglutination/Hemagglutination Inhibition• Definition - test based on the inhibition of
agglutination due to competition with a soluble Ag
+ ↔
Prior to Test
+ ↔+
Test
Patient’s sample
Agglutination/Hemagglutination Inhibition
• Applications– Measurement of soluble Ag
• Practical considerations– Same as agglutination test
• Definition
Precipitation Tests
Lattice Formation
Radial Immunodiffusion (Mancini)
• Interpretation– Diameter of ring is
proportional to the concentration
• Quantitative– Ig levels
• Method– Ab in gel– Ag in a well
Ag Concentration
Dia
met
er2
AgAgAgAg
Ab in gel
Immunoelectrophoresis• Method
– Ags are separated by electrophoresis
• Interpretation– Precipitin arc represent individual antigens
Ag-+
Ag
Ab
Ag
Ab
– Ab is placed in trough cut in the agar
Immunoelectrophoresis
• Method• Interpretation• Qualitative
– Relative concentration
Countercurrent electrophoresis• Method
– Ag and Ab migrate toward each other by electrophoresis
– Used only when Ag and Ab have opposite charges
• Qualitative–Rapid
Ag Ab- +
Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent
Assays (ELISA)
Lattice formation not required
Competitive RIA/ELISA for Ag • Method
– Determine amount of Ab needed to bind to a known amount of labeled Ag
+ ↔
Prior to Test
Labeled Ag
+ ↔
Test
+Patient’ssample
LabeledAg
+
– Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
Competitive RIA/ELISA for Ag • Method cont.
– Determine amount of labeled Ag bound to Ab
• ↓ NH4SO4
• ↓ anti-Ig • Immobilize the Ab
• Quantitative– Most sensitive test
+ ↔Test
+Patient’ssample
LabeledAg
+
– Concentration determined from a standard curve using known amounts of unlabeled Ag
SolidPhase
SolidPhase
Solid Phase Non-Competitive RIA/ELISA• Ab detection
– Immobilize Ag– Incubate with sample– Add labeled anti-Ig– Amount of labeled Ab
bound is proportional to amount of Ab in the sample
• Quantitative
SolidPhase
AgImmobilized
Ab in Patient’s
sample
LabeledAnti-Ig
Solid Phase Non-Competitive RIA/ELISA
• Ag detection– Immobilize Ab– Incubate with sample– Add labeled antibody– Amount of labeled Ab
bound is proportional to the amount of Ag in the sample
• Quantitative
SolidPhase
AgImmobilized
Ag in Patient’s
sample
LabeledAb
Tests for Cell Associated Antigens
Lattice formation not required
Immunofluorescence
• Direct– Ab to tissue Ag is labeled with fluorochrome
Ag
FluorochromeLabeled Ab
Tissue Section
Immunofluorescence
• Indirect– Ab to tissue Ag is
unlabeled– Fluorochrome-labeled anti-
Ig is used to detect binding of the first Ab.
Ag
FluorochromeLabeled Anti-Ig
Tissue Section
UnlabeledAb
• Qualitative to Semi-Quantitative
Immunofluorescence
• Flow Cytometry– Cells in suspension are labeld with fluorescent tag
• Direct or Indirect Fluorescence– Cells analyzed on a flow cytometer
FlowTip
Laser
FLDetector
LightScatter
Detector
Immunofluorescence
• Flow Cytometry cont.– Data displayed
Green Fluorescence Intensity
Num
ber
of C
ells
Unstained cells
FITC-labeled cells
One Parameter Histogram
Red Fluorescence Intensity
Gre
en F
luor
esce
nce
Inte
nsity
Two Parameter Histogram
Assays Based on Complement
Lattice formation not required
Complement Fixation
– Ag mixed with test serum to be assayed for Ab– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determined
Ag
Patient’sserum
Ag No Ag
Ag
• Methodology