9. functions of antibodies
• to neutralize
– toxins
– viruses,
• to opsonize microbes
– so they are more easily phagocytosed,
• to activate complement
• to prevent the attachment of microbes to
mucosal surfaces.
• antibodies have a catalytic (enzymatic)
capability
10. • Strength of the reaction between an antigenic
determinant (antigen) and an antigen combining
site (antibody)
High Affinity
Ab
Ag
Affinity
Low Affinity
Ab
Ag
Affinity = attractive and repulsive forces
11. Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity
106
Avidity
1010
Avidity
12. Specificity
• The ability of an antibody combining site to react
with only an antigenic determinant.
• The ability of antibody to combine with an antigen
13. Cross Reactivity
• The ability of an individual Ab combining site to
react with more than one type of antigenic
determinant.
• The ability of a Ab molecules to react with more
than one Ag
Anti-A
Ab
Ag A
Cross reaction
Anti-A
Ab
Ag C
Similar A.D
14. Factors Affecting Happening of
Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
15. Tests Based on Ag/Ab Reactions
• All tests based on Ag/Ab reactions will
have to depend on lattice formation itself or
• we 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
17. Uses of antigens/antibody reactions
• Diagnosis Infectious diseases
• Diagnosis of autoimmune diseases
• Determination of blood type and HLA types
• Determination of chemical levels
18. Diagnosis of infectious
diseases
• When the organism cannot be cultured-or
difficult to culture hep A B C Syphilis
• When the organism is too dangerous to
culture- rickettsial disease
• When the culture technique is not readily
available-HIV,EBV
• When the organism takes too long to grow
e.g Mycoplasma
19. 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
test
20.
21. Antigen and antibody reactions in
the lab
• Precipitation tests
• Agglutination
• Elisa
• Radioimmunoassay
• Immunofluorescence
• Complement Fixation
22. Precipitation tests
• The antigen and antibody are in, soluble
form
• Combine to form a visible precipitate
• Presence of electrolytes
• Positive controls and negative controls
23. Precipitation tests
• Precipitation techniques
– Tube precipitation test
– Gel diffusion
• Double
• Single radial
• precipitation in agar with an electric field
– Immuno electrophoresis
– Countercurrent electrophoresis (CEP),
25. Double Diffusion
• antigen and antibody are placed in different
wells in agar and
• allowed to diffuse and form concentration
gradients.
• Where optimal proportions occur, lines of
precipitate form
26.
27.
28. Double diffusion method (Ouchterlony)
• indicates whether
– antigens are identical,
– Antigens not identical
– Partially identical
30. Radial Immunodiffusion (Mancini)
• Interpretation
– Diameter of ring is
proportional to the
concentration
• Quantitative
– Ig levels
Ag Concentration
Ab in gel
Diameter2
Ag Ag Ag Ag
34. Interpertation
• serum proteins are characterized in terms
of their
• presence,
• absence
• unusual pattern (e.g., human myeloma protein).
35. Counter-
Immunoelectrophoresis
• Method
– Ag and Ab migrate toward each other by
electrophoresis
– Used only when Ag and Ab have opposite charges
- +
• Qualitative
–Rapid
Ag Ab
36. uses
• The meeting of the antigen and antibody is
greatly accelerated
• made visible in 30–60 minutes.
• detection of bacterial and fungal
polysaccharide antigens in cerebrospinal
fluid.
37. Agglutination
• Visible clumping together of particulate matter by
antigen combining with its specific antibody.
• The clumps will be called agglutinates
• Performed
– Slide
– Tube
– Tile
– Micrtitration plates
38.
39. Agglutination
• Used in both directions
• Antigen part of a particulate matter
Salmonella
• Particulate matter
– Latex
– Carbon particles
– cells
– Bacteria
• Stablised staphylococcal cells
40. Agglutination
• Active agglutination
test
• The antigen part of a
particulate matter per
se
• examples
• Salmonella, vibrio,
41. Agglutination
• Passive agglutination test
• Antigen or antibody are not part of
particulate matter but are attached (rided on
inert particles like latex, carbon,)
+ Particulat
matter
43. Prozone phenomenon
• Tube agglutination
• The lower dilutions do not show agglutination
• the tubes (prior/before the optimum zone )
• The tubes in higher dilutions show agglutination
• Reasons/factors
– Antibody excess:High level of antibody
– Non specific inhibitory factors
44. Haemagglutination
• Active Haemagglutination test
– (blood group)
• Passive haemagglutination (TPHA)
– Known antigen coated on to treated RBC,s
– Treated To remove their own antigens
– Turkey,s RBC,s are used
45. Immunofluorescence
• Fluorescent dyes illuminated by ultraviolet
light are used to show combination of
antigen antibody .
• The end point antigen antibody complexes
are seen fluorescing against a dark
background
• Direct
• Indirect
50. Immunofluorescence
• Indirect
– Ab to tissue Ag is
unlabeled
– Fluorochrome-labeled
anti-Ig is used to detect
binding of the first Ab.
• Qualitative to Semi-
Quantitative
51. Fluorescence-Activated Cell Sorting (Flow
Cytometry)
• the patient's cells are labeled with monoclonal
antibody to the protein specific to the cell of
interest, e.g., CD4 protein
• The monoclonal antibody is tagged with a
fluorescent dye, such as fluorescein or rhodamine.
• Single cells are passed through a laser light beam,
• the number of cells that fluoresce is counted by
use of a machine called a fluorescence-activated
cell sorter (FACS).
52. Immunofluorescence
• Flow Cytometry
– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
54. CFT
• The CFT is used to detect antibodies or
antigens
• inference is made by the ability of the
antibodies to fix the complement
• The fixation of the complement is measured
by adding the indicator system
• Used in diagnosis of viral parasitic and
rickettsial diseases
55.
56.
57.
58.
59.
60.
61. Complement Fixation
• Methodology
– Antibody known mixed with test material to be assayed for Ag
– Standard amount of complement is added
– Erythrocytes coated with Abs is added
– Amount of erythrocyte lysis is determined
Ag NO Ag
Ag
Ag
62. RadioImmunoassay
• The radioactivity of the specific labeled
antibody or antigen is used to quantify the
antigen or antibody in patient ,s serum
• HbsAg
• Hav IGM
63.
64. ELISA
• Uses an enzyme system to show the specific
combination of antigen antibody
• An enzyme labeled or linked to a specific antigen
• A substrate
• A color reader
• Double antibody technique to detect and assay
antigen
• Indirect technique to Assay antibody
65. Direct Elisa
• Ag detection
– Add labeled antibody
– Amount of labeled Ab
bound is proportional
to the amount of Ag in
the sample
66. Indirect 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
Immobilized Ag
Solid
Phase
Ab in
Patient’s
sample
Labeled
Anti-Ig
67. Western blot
• HIV proteins are separated electrophoretically in a gel,
• discrete bands of viral protein.
• These proteins are then transferred from the gel, i.e., blotted,
onto filter paper, and
• the person's serum is added.
• If antibodies are present, they bind to the viral proteins
(primarily gp41 and p24) and can be detected by adding
antibody to human IgG labeled an enzyme,
• produces a visible color change when the enzyme substrate is
added.
68.
69. Neutralization Tests
• These use the ability of
• antibodies to block the effect of toxins or
the infectivity of viruses.
• They can be used in cell culture ( inhibition
of cytopathic effect
• in host animals ( mouse protection tests).
76. Immunofluorescence
• Flow Cytometry cont.
– Data displayed
One Parameter Histogram
Unstained cells
Green Fluorescence Intensity
Number of Cells
FITC-labeled cells
Red Fluorescence Intensity
Green Fluorescence Intensity
Two Parameter Histogram
81. Passive Agglutination/Hemagglutination
• Definition - agglutination test done with a
soluble antigen coated onto a particle
• Applications
– Measurement of antibodies to soluble antigens
85. Competitive ELISA for antigen
• Method
– Determine amount
of Ab needed to bind
to a known amount
of labeled Ag
+
Prior to Test
Labeled
Ag
+
Test
+
Patient’s
sample
Labeled
Ag
+
– Use predetermined
amounts of labeled
Ag and Ab and add a
sample containing
unlabeled Ag as a
competitor
86. Competitive 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’s
sample
Labeled
Ag
+
Solid
Phase
Solid
Phase
– Concentration determined from a standard curve
using known amounts of unlabeled Ag
88. Solid Phase RIA for antigen/direct
• 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
Ag
Solid
Phase
Ag in
Patient’s
sample
Immobilized
Labeled
Ab
89. Solid Phase RIA for antibody/indirect
• 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
Immobilized Ag
Solid
Phase
Ab in
Patient’s
sample
Labeled
Anti-Ig
90.
91. Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
92. Cross Reactivity
• The ability of an individual Ab combining site to
react with more than one type of antigenic
determinant.
• The ability of a Ab molecules to react with more
than one Ag
Anti-A
Ab
Ag A
Cross reactions
Anti-A
Ab
Ag B
Shared A.D
Anti-A
Ab
Ag C
Similar A.D
93.
94. Immunoelectrophoresis
• Method
– Ags are separated by electrophoresis
– Ab is placed in trough cut in the agar
+ -
Ag
• Interpretation
Ag
Ab
Ag
Ab
– Precipitin arc represent individual antigens
95.
96.
97. Haemagglutination inhibiotion test
• Certain viruses measles and influenza
arboviruses agglutinate RBC,s
• If specific antibodies are included in the
system a virus is identified
98. Tests for Cell Associated
Antigens
Lattice formation not required
99. Immunofluorescence
• Flow Cytometry
– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
100.
101. Double Antibody 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
Ag
Solid
Phase
Ag in
Patient’s
sample
Immobilized
Labeled
Ab
